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Novel Host Proteins and Signaling Pathways in Enteropathogenic E. S2B). This supports the
FIG. 2. Phosphoproteomic analysis of EPEC-infected HeLa cells.
Collectively, these results reveal that EPEC's impact on the host phosphoproteome is much more extensive than previously thought.
Several Kinase Families Modulate Host Phosphorylation during EPEC Infection-Kinases are pivotal regulators of phosphorylation dynamics in cellular signaling and in turn are often regulated by phosphorylation. Therefore, we examined the data for kinases that might play a key role in mediating some of the observed changes seen in the host phosphoproteome during EPEC infection. A total of 36 kinases were identified and quantified in the phosphoproteome analysis Table S8). Our research identified six kinases with modified phosphorylation following EPEC infection, including the non-T3SS-regulated protein kinase C alpha (PKC␣) and the T3SS-regulated calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2␦), ephrin type-A receptor 2 (EPHA2), and mitogen-activated protein kinase 1 (MAPK1/ERK2) (Fig. 3A).
Phosphorylation of Thr-497 in PKC␣ is critical for kinase activity (45). Its respective phosphopeptide decreased in abundance in infected cells, suggesting that EPEC may reduce PKC␣ activity upon infection. Consistent with this observation is a previous report showing that cytosolic PKC␣ activity was decreased in HeLa cells following EPEC infection at early time points (46). However the same study also showed that membrane-bound PKC␣ activity increased upon EPEC infection, suggesting that the respective membraneassociated PKC␣ could not be observed in our experimental setup.
The identification of changes within multiple kinases and phosphorylation sites prompted us to assess the kinases most likely to be responsible for the observed regulations within EPEC-infection. Using complementary kinase enrichment analysis (32), kinases with an observed regulated phosphosite, such as MAPK1 (p ϭ 0.0000067), CAMK2 (p ϭ 0.0056), and PKC (p ϭ 0.02), were significantly enriched. In addition to these kinases the CDK, RSK, GSK, PIKK, CK1, and CK2 families were also enriched within the regulated phosphoproteome (Fig. 3B), indicating that widespread changes in the kinome regulation occur in response to EPEC infection.
To further focus on the affected phosphosites and the host kinases directly responsible for these phosphorylation events during EPEC infection, we identified consensus kinase phosphorylation motifs among phosphosites that were upregulated both in a T3SS-dependent and -independent manner (Fig. 3C). Motif-based analysis extracted common MAPK and CDK (-S/T-P-), CAMK (-R-X-X-S/T-), and CK2 (-S/T-X-E-E-) consensus phosphorylation sequences. In addition, we pre- T3SS.-dep. up-regulated P-sites ) and not regulated (in gray) kinases based on a total number of 36 kinases that were detected in the phosphoproteome analysis. All six kinases with regulated phosphosites were depicted in detail with respective phosphosites and time points of regulation in parenthesis. The asterisk indicates T3SS-regulated phosphosites (arrows indicate an increase or decrease of phosphorylation). We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine<|endoftext|>Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells
Background EDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. The secondary anti-rabbit HRP and anti-mouse HRP antibodies, were obtained from Sigma-Aldrich, whereas anti-rabbit Alexa555 and anti-mouse Cy-3 were obtained from Jackson laboratories (Bar Harbour, MA).
cDNA encoding the mouse EDEM1 fused to an hemagglutinin (HA) tag in the pCMV-SPORT2 vector was a kind gift from Prof. Kazuhiro Nagata and Prof. Nobuko Hosokawa (Institute for Frontier Medical Sciences, Kyoto University, Japan). For details of the cloning refer to [8]. cDNA encoding the mouse EDEM2 fused to an hemagglutinin (HA) tag in the pRK7 vector was a generous gift from Prof. Maurizio Molinari (Institute for Research in Biomedicine, Basel, Switzerland). For details of the cloning refer to [12].
Purification of ricin A-chain proteins and reconstitution with ricin B-chain
RTA His-tag, modified RTA DHF His-tag and RTA IHF His-tag were expressed in E. coli Rosetta cells (Merck) and purified using Ni-NTA agarose beads (Qiagen, Germantown, MD) according to the manufacturer's manual. The eluate was finally dialysed overnight in PBS.
Ricin A-chain sulf-1 and modified RTA IHF and RTA DHF sulf-1 fused to maltose binding protein (MBP) were applied to a column with amylose resin and purified as previously described [22]. Free wild-type RTA, RTA DHF or RTA IHF were cleaved off with factor Xa (New England Biolabs, Ipswich, MA). For further purification, wild-type RTA, RTA DHF or RTA IHF proteins were applied on a MonoS column (GE Healthcare) and purified using GE Pharmacia Acta Purifier (GE Healthcare). 25 mM phosphate buffer, pH 6.5 was used as the column equilibrating buffer and the wash buffer, proteins were eluted with 0-500 mM NaCl gradient, and the fractions containing ricin A-chain were identified by Coomassie-stained SDS/ PAGE. Purified wild-type RTA, RTA DHF or RTA IHF were mixed with the ricin B-chain and dialyzed extensively against PBS to remove reducing agents.
Protease digestion assays 500 ng of RTA, RTA DHF or RTA IHF were incubated with increasing concentrations of trypsin (0-100 μg/ml) in NaCl/P i (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) at 37°C for 15 min and then visualized by SDS/PAGE and Coomassie Blue staining. Pronase digestion was performed at 40°C for 20 min in a buffer containing 0.1 M Tris-HCl, pH 6.5 and 0.5% SDS with increasing concentrations of the protease (0-1.5 μg/ml), inactivation of the protease was done at 80°C for 5 min. Proteins were visualized by SDS/PAGE and Coomassie Blue staining. Denatured forms of RTA were prepared by incubation of RTA at 85°C for 30 min in 25 mM phosphate buffer, pH 6.5.
The interchain disulfide bond stability
100 ng reassociated wild-type RTA:RTB, RTA DHF :RTB or RTA IHF :RTB were incubated with increasing concentrations (0.1-10 mM) of dithiothreitol (DTT) at 37°C for 30 min. The reaction was quenched with iodoacetamide (final concentration 20 mM) at 37°C for 10 min and then visualized by SDS/PAGE and Coomassie Blue staining. Denatured forms of ricin were prepared by incubation of RTA at 85°C for 30 min in 25 mM phosphate buffer, pH 6.5.
Far-UV CD was measured on Jasco J-815 spectrapolarimeter (Jasco, Tokyo, Japan). Experiments were performed in 25 mM phosphate buffer, pH 6.5 using a 1-mm-path length cuvette. Proteins were detected by chemiluminescence reagent SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific).
Immunofluorescence microscopy
HEK293 cells transfected with cDNA encoding BACE457 , BACE457 DHF or BACE457Δ were grown on coverslips.
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Domain: Biology Medicine<|endoftext|>Coevolution Trumps Pleiotropy: Carbon Assimilation Traits Are Independent of Metabolic Network Structure in Budding Yeast Phenotypic traits may be gained and lost together because of pleiotropy, the involvement of common genes and networks, or because of simultaneous selection for multiple traits across environments (multiple-trait coevolution). cerevisiae and 28 strains of S. paradoxus and found an enrichment of duplicates for genes with catalytic activity and sugar transport. Furthermore, they demonstrated that certain sets of over-and underrepresented duplicates correlate with adaptation to different environments.
Our results provide further support for how network structure can be impacted by the environment, suggesting that a wide metabolic breadth requires larger numbers of nodes, in the form of unique assemblages of specialized enzymes. Such networks will also be more expansive since most carbon sources are not funneled through a single pathway. These two factors suggest that metabolic networks change as a result of variation in metabolic breadth. The recent emphasis on molecular networks has received few rigorous tests about the impact of network structures on evolutionary processes [25,26]. Our results indicate that metabolic network topology may not impose severe constraints on the evolution of carbon utilization phenotypes. Instead, our observation that traits are gained and lost independently of known metabolic network structure suggests that the networks themselves vary and evolve. Figure 6. The substrate a strain was isolated from can be predicted based on the set of carbon sources a strain can utilize. Dark gray bars represent the proportion of time that a substrate was correctly predicted. Lighter gray bars represent the background prediction rate and associated confidence intervals based on shuffled data. doi:10.1371/journal.pone.0054403.g006
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Domain: Biology Medicine
|
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DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice
Background Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. Survival time was monitored daily after lethal challenge with 2 × 10 4 tachyzoites of virulent T. gondii RH strain, 2 weeks after the last immunization. The mice immunized with pTgDPA were dead from day 11 to day 20, showing an increased survival time compared with mice in the control groups (pVAX1, PBS, blanking controls), which died within 8-9 days after challenge (P < 0.05).
the immune group displayed significantly low concentrations of TGF-β1 after the booster immunization compared to the control groups. It indicated that immunization with TgDPA down-regulated T reg cells response. This character of TgDPA will be beneficial to its potential as a vaccine candidate.
Resistance against T. gondii parasite is characterized by the induction of specific CD4 + and CD8 + T cells, which eventually lead to the killing of the parasite [61,62]. In murine models, CD4 + T cells were crucial regulators of the immune response during resistance against toxoplasmosis, while in humans CD4 + displayed cytotoxic activity against T. gondii infected cells [15,63,64]. On the other hand, CD8 + subtype were considered to be the major effector cytotoxic T lymphocyte (CTL) cells mediating lysis of T. gondii infected host cells [65][66][67]. In this investigation, our data demonstrated that both cell subtypes were significantly accumulated in response to immunization with TgDPA. This result corresponds with reports regarding immunological responses to T. gondii antigens [16,[68][69][70][71][72].
As a result of these significant immunological changes, pTgDPA vaccinated mice survived for a longer time compared to the control groups in this research. However, due to uncontrolled parasite replication, pTgDPA mice ultimately succumb during late acute infection. It indicated that the DNA vaccine of pTgDPA did not provide complete protection. However, investigations concerning this protein should further be conducted.
One of the prominent advantages of DNA vaccine application is their induction of CTL cells. CTL cells kill the pathogen infected cells mainly by inducing apoptosis [74]. T. gondii maintains its survival and replication by interfering with infected cells apoptosis, blocking an important pathway known as caspase cascade [75][76][77][78]. This finding may encourage more investigations in evaluating the immunogenicity of DPA based vaccines against Toxoplasmosis.
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Domain: Biology Medicine<|endoftext|>Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. 5D shows that in the 30-min interval, the amount of virion gD st associated to cells decreased; gB decreased in parallel. We did not detect any gH/gL, likely because of limits of detection. The results indicate that virion gD st , and the complexed gB, tended to decrease rather than increase during virus fusion to 293T cells.
Characterization of gD Mutants Carrying Substitutions, Deletions, or Mutations across Hawkins Inc Airport objective of the next series of experiments was to preliminarily define the gD regions involved in complex assembly with gB and gH/gL. We employed a panel of gD mutants carrying substitutions, deletions, or mutations across the entire gD ectodomain (linear maps shown in Fig. 6A). Some were previously described, and some were generated for the purpose of this study. As mentioned in the Introduction, gD ectodomain can be schematically subdivided into two regions; the N-terminal region up to aa 240/260 includes the receptorbinding sites; the downstream region, spanning from aa 240/ 260 to 310 carries the profusion domain. In gD ⌺260 -310 (previously named gD ⌬PFD ) (10) and in gD ⌺240 -310 , the indicated sequences were substituted with the CD8 sequences corresponding to the pretransmembrane region. CD8 was initially chosen as donator of heterologous sequence because it is a transmembrane protein, totally unrelated to HSV attachment entry (9,53). gD ⌺240 -260 carries an 18-aa-long Ser-Gly linker in place of the endogenous 240 -260 aa sequence. The gD P291L-P292A and gD T304A-P305L mutants, herein and elsewhere referred as gD PP and gD TP , respectively, carry the indicated substitutions in the profusion domain. Both are partially impaired in infection (9). In gD ⌺218 -240 , the 218 -240-aa segment, carrying ␣-helix3, was substituted with an 18-mer Ser-Gly linker. gD ⌬ 61-218 was generated in this study following the discovery that, in a very peculiar form of gD split into two fragments, one of which carried the Kringle domain from urokinase plasminogen activator, the 61-218-aa region was dispensable (54). Studies from our laboratory confirm that this sequence can be substituted with heterologous sequences. 4 gD ⌬6 -60 was designed during the course of the study; of note, it is known that the gD N terminus can be deleted at least up to aa 38 (55) and leave a functional gD that cannot interact with HVEM any longer.
The panel of gD mutants was preliminarily characterized with respect to ability to be transported to the cell surface and to mediate cell-cell fusion and virus infection. Cell surface expression was measured in transfected 293T cells and expressed as a percentage relative to wt gD-transfected cells. Fig. 6B shows that the gD ⌺260 -310 , gD ⌺240 -260 , and gD TP , gD TP mutants were expressed at the cell surface in amounts ranging from 50 to 80% of wt gD; the remaining mutants were severely impaired. Increasing the amount of transfected plasmid DNA by 3-fold did not result in substantially increased cell surface expression (data not shown). The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. As additional target for glioma progression was identified in the GRIA2 transcript which is edited by ADAR2 [71], where less editing at the Q/R site enhances glioblastoma invasion through activation of the Akt pathway [141]. Overall, these data indicate the essential role of ADAR2 editing in glioblastoma, acting on multiple targets (CDC14B, pri-miR-221/222, pri-miR-21, miR-376a-5p, GRIA2) that together contribute to varying extents to cancer progression.
Conclusions
In the last 25 years a lot has been learned about the ADAR family of proteins; however, many intriguing questions remain. Despite considerable effort, there is no co-crystal structure of an ADAR enzyme bound to dsRNA. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine
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14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
To further explore the potential signaling pathways associated with mammalian Eag, we set forth to identify novel binding partners of rEag1 channels in the brain. The gradient was centrifuged at 65,0006g for 2 hrs in a Beckman Instruments SW-28 rotor and the synaptosomal fraction was recovered from the 1.0-1.2 M sucrose interface. The synaptosomal fraction was Co-immunoprecipitation of 14-3-3h and rEag1. Detergent solubilized proteins from the lysates of rat forebrain were immunoprecipitated (IP) with the anti-14-3-3h (upper panel) or the anti-rEag1 antibody (lower panel), followed by immunoblotting (WB) analyses with the anti-14-3-3h or the anti-rEag1 antibody. The non-immune mouse or rabbit IgG was used in parallel as negative control. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. The arrowhead and arrow refers to the protein bands of 14-3-3h and rEag1, respectively. (B) Immunofluorescence staining of rEag1 (left panels) and 14-3-3h (middle panels) in cultured hippocampal neurons. The area highlighted in the white boxes is viewed under a higher magnification (I, II). Arrows label the sites of co-localization of 14-3-3h and rEag1 (right panels), which displayed significant punctuate patterns over a wide region along the neurites. Scale bar, 25 mm. These coimmunoprecipitation and immunofluorescence data are representative of four to seven independent experiments. doi:10.1371/journal.pone.0041203.g005 14-3-3h Interacts with rEag1 PLoS ONE | www.plosone.org extracted in ice-cold 0.5% Triton X-100/50 mM Tris-HCl (pH 7.9) for 15 min and centrifuged at 32,0006g for 45 min to obtain the PSD I pellet. The pellet was resuspended and further extracted a second time with 0.5% Triton X-100/50 mM Tris-HCl (pH 7.9), followed by centrifugation at 200,0006g for 45 min to obtain the PSD II pellet. Protein concentration was determined by the Bio-Rad protein assay kit (Bio-Rad).
Electrophysiology
Conventional whole-cell patch clamp technique was used to record rEag1 K + currents as described previously [17]. In brief, whole-cell patch clamp was performed 24-48 hrs post-transfection of HEK293 or HEK293T cells. Patch electrodes were filled with a solution containing (in mM) 140 KCl, 1 MgCl 2 , 10 EGTA, 10 HEPES, pH 7.2. External recording solution comprised (in mM) 140 NaCl, 5 KCl, 1 CaCl 2 , and 10 HEPES, pH 7.2. Data were acquired with an Axopatch 200A amplifier (Molecular Devices) and digitized with the Digidata 1322A system and the pCLAMP 9.0 software (Molecular Devices). Cells with large currents in which voltage clamp errors might appear were excluded from data analyses. cRNA injection and conventional two-electrode voltage clamp recording of rEag1 K + currents in Xenopus oocytes were performed as described previously [17]. (TIF)
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. There are two different isoforms of ADAR1; a 150 kDa protein (ADAR1 p150) that is induced by interferon (IFN) and a 110 kDa protein (ADAR1 p110) that is constitutively expressed [19]. The two isoforms have distinct translation initiation sites so that ADAR1p150 starts translation in exon 1A (AUG1) whilst the ADAR1p110 protein initiates from exon two (AUG296) [19,20]. The generation of different ADAR1 isoforms is due to the alternative splicing of the exon one. An IFN-inducible promoter located in exon 1A contains a 12-bp IFN-stimulated response element (ISRE), thereby generating the transcript coding for the longer ADAR1p150 isoform [21]. However, transcripts encoding ADAR1p110 are generated from multiple promoters located within exon 1B, 1C and exon two [21,22]. As neither exon 1B nor exon 1C contain translation initiation sites, translation starts at the AUG296 codon within exon two. Additional alternative splicing events have been identified within exon 7 to generate variants that contain either exon 7a or 7b [20]. Exon 7b is missing 26 amino acids that are present in exon 7a [20]. Furthermore, exon 7b is associated with ADAR1p150 transcripts whereas exon 7a is present in the constitutively-expressed ADAR1p110 transcripts [23].
A unique feature of ADAR1 when compared to other members of the ADAR family, is the presence of Z-DNA binding domains (ZBDs) at the N-terminus. The Z-alpha (Zα) domain is exclusive to the ADAR1p150 isoform whereas both ADAR1p150 and ADAR1p110 contain a Zβ domain [24]. The ZBDs bind DNA/RNA that is in a left handed conformation [25]; however, only the Zα domain has Z-DNA/RNA binding capacity [26]. Although the functional significance of the ZBDs in ADAR1 is unclear, all proteins containing Zα domains have been implicated in the type I IFN response pathway [27]. Furthermore, the Zα domain is essential for the localization of ADAR1p150 to cytoplasmic stress granules following activation of type I IFN-induced stress [28].
In vitro analysis of recombinant ADAR3 demonstrated that it can bind both dsRNA and ssRNA [29]. Site-directed mutagenesis of ADAR3 revealed that it binds ssRNA via a unique arginine/lysine rich domain (R-domain) located in the N-terminus. This R-domain is important for determining the subcellular localization of ADAR3 and acts as a nuclear localization signal (NLS) [30]. The importin alpha-1 factor, KPNA2 recognizes and interacts with this R-domain to promote the nuclear import of ADAR3.
dsRNA Binding Domains of ADARs
All proteins that bind to dsRNA contain an evolutionarily-conserved 65-68 amino acid homologous sequence that is known as dsRBD [31]. It was originally identified in Drosophila Staufen and Xenopus Xlrbpa proteins [31]. Nuclear magnetic resonance (NMR) studies have characterized the canonical dsRBD fold with a topology of: α-helix 1, β-strand 1, β-strand 2, β strand 3, and α-helix 2 (αβββα) [32][33][34]. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Activation of RIG-I and MDA5 results in the recruitment of MAVS via CARD domain interactions. MAVS dependent signaling causes the translocation of the transcription factors IRF3 and NF-κB for the induction of type I IFN and pro-inflammatory cytokines respectively. The inflammasome is activated by dsRNA via NALP3-dependent signaling pathway. NALP3 together with ASC recruit pro-caspase-1 which in turn undergoes auto-cleavage to produce caspase-1. Activation of caspase-1 is required for the cleavage of pro-IL-1β and pro-IL-18, and subsequent secretion of the proinflammatory cytokines IL-1β and IL-18, respectively. ADAR1p150 edits dsRNA within the cytoplasm and is induced by IFN.
Investigations into the biological role of ADAR1 have found that it is a negative regulator of the type I IFN response. The Adar1 null mouse have defects in hematopoiesis and liver disintegration as well as elevated levels of type I IFN and widespread apoptosis [107][108][109]. Mouse embryonic fibroblasts (MEFs) deficient in ADAR1p150 are highly susceptible to infection and have enhanced viral induced cytotoxicity effects [105], implicating ADAR1 as being involved in type I IFN response.
The Adar1 null mice are dead by stage E12.5 and for over 10 years investigators endeavored to elucidate what caused this early lethality [66]. They presumed that these mutant mice lacked an essential editing event that would be analogous to ADAR2 editing the Q/R site of GRIA2. A major breakthrough was achieved by taking a genetic approach and generating a double mutant with Adar1 [66]. When Adar1 heterozygous mice were crossed with mice that were homozygous for Mavs, the embryos that were double-homozygous survived to birth. The Adar1 mutant embryos had a heightened IFN response whereas the double mutants had a response that was similar to their Mavs parents. This places Adar1 upstream of RIG-I and MDA5 in the innate immune pathway. As MEFs from Adar1 were not viable, MEFs were generated from embryos that were Adar1:p53 double mutants. When these MEFs were stressed they had a heightened immune response however the addition of inosine containing dsRNA alleviated the heightened immune response [66,67]. The explanation for these results is that the cell uses inosine to help discriminate between self and non-self dsRNA. If inosine is present in dsRNA it binds to -RLRs and prevents activation of the innate immune response. However, if no inosine is present, then the cell recognizes the dsRNA as being of viral origin and mounts a type 1 IFN response.
ADARs in Human Disease
Aicardi-Goutié res syndrome (AGS) is an autoimmune disorder where patients develop severe inflammatory encephalopathy due to chronic activation of the type I IFN [110] (Table 1). AGS patients present with symptoms that are similar to an acquired infection. However, there is no infection and mutations in genes involved in the regulation of nucleic acid metabolism have been identified in patients with this disorder [111]. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
==
Domain: Biology Medicine
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Coevolution Trumps Pleiotropy: Carbon Assimilation Traits Are Independent of Metabolic Network Structure in Budding Yeast Phenotypic traits may be gained and lost together because of pleiotropy, the involvement of common genes and networks, or because of simultaneous selection for multiple traits across environments (multiple-trait coevolution). Dark gray bars represent the proportion of time that a substrate was correctly predicted. Lighter gray bars represent the background prediction rate and associated confidence intervals based on shuffled data. doi:10.1371/journal.pone.0054403.g006
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Domain: Biology Medicine<|endoftext|>This below document has 3 paragraphs that start with 'Effects of various ratios of'. It has approximately 764 words, 27 sentences, and 11 paragraph(s).
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Protective effects of various ratios of DHA/EPA supplementation on high-fat diet-induced liver damage in mice
Background A sedentary lifestyle and poor diet are risk factors for the progression of non-alcoholic fatty liver disease. The forward and reverse primers for the target genes are listed in Table 2. Relative gene expression was normalized (by means of the C T method) to that of the endogenous control β-actin, and the final results were calculated using the formula 2 −ΔΔCt .
Analysis of western blots
Protein expression levels of c-Jun, c-Fos, and Fra1 were analyzed by western blotting. Briefly, the total protein was extracted in PMSF:RIPA = 1:99 (v/v), a homogenizing and lysis buffer. Equal amounts of protein extracts, which were normalized to the total amount of protein, were mixed (1:3, v/v) with the loading buffer for electrophoresis using 10% SDS-PAGE gels kits (Boster Bio-engineering, Ltd., Wuhan, China). The mixture was subsequently electroblotted to a nitrocellulose transfer membrane (Millipore, USA) by a Trans-Blot SD semi-dry transfer cell (Bio-Rad, USA) as per the manufacturer's instructions. The target protein levels were detected with specific primary antibodies (Bioss Biological Technology, Ltd., Wuhan, China) against the target protein, and then incubated with the species-specific secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG antibody (Boster Bio-engineering), as recommended by the manufacturer. The chemiluminescence intensity of the bands was quantified by an ECL developer (Millipore, USA) using a Western Blotting Detection System (Bio-Rad, USA), and the optical densities of the bands were detected using Image Lab
Statistical analysis
All data were recorded with Excel. After confirming the homogeneity of variances, differences among the groups were tested by one-way ANOVA. Means ± SEM were calculated for each group, and the figures were prepared using GraphPad Prism v6.0.1 software. The significant differences were shown by the LSD multiple comparison test using SPSS statistics 17.0 software, and any two groups without the same lowercase and uppercase letters, as marked in the figures, indicate a significant difference of P value <0.05 and <0.01, respectively.
Results
Effects of various ratios of DHA/EPA with an n-6/n-3 ratio of 4:1 on steatosis and liver cell histopathological lesions At 12 weeks post-administration, the histopathological lesions of livers in all groups were assessed. The oil red O staining patterns showed orange lipid droplets and blue nuclei, which were smaller and fewer in number in the DHA/EPA (2:1, 1:1, and 1:2) groups; while obvious numerous orange-red lipid droplets were observed in HFC-treated mice, nearly none were observed in the NC group (Fig. 1). Moreover, fewer lipid droplets were observed in mouse groups treated with DHA/EPA (ratios of 2:1, 1:1, and 1:2) compared to the group treated with DHA.
Effects of various ratios of DHA/EPA with an n-6/n-3 ratio of 4:1 on body weight, serum, and liver lipids Compared to the HFC group, significant decreases (p < 0.05) in serum TC, TG, LDL-C levels, and liver TC and TG levels were observed in 12-week n-3 PUFA supplementation groups ( Table 3). The growth curve of mice revealed that the body weight increased slower with n-3 PUFA supplementation than with HFC treatment (Fig. 2), indicating the inhibitory effect of n-3 PUFA supplementation with DHA and different DHA/ EPA ratios (2:1, 1:1, and 1:2) in the mice. Moreover, mice treated with a DHA/EPA ratio of 2:1 and 1:2 showed a reduced hepatic organ coefficient with a significant difference of p < 0.01, while the statistical differences of reduction in DHA and DHA/EPA 1:1 groups were p < 0.05 compared to those in the HFC group (Table 3).
Mice treated with a DHA/EPA ratio of 1:2 showed a more appreciable decrease in serum TC, TG, and LDL-C levels, whereas mice treated with the DHA/EPA ratio of 2:1 showed lowered liver TC and TG levels to a greater degree. However, mice treated with various ratios of DHA/EPA (2:1, 1:1, and 1:2) revealed no reduction in serum TC, TG, or LDL-C levels or liver TC and TG levels in comparison with those in the DHA group. Nevertheless, as compared to the levels in the HFC group, serum HDL-C levels in DHA and DHA/EPA (2:1, 1:1, and 1:2) groups were significantly higher (p < 0.01). Furthermore, serum HDL-C levels increased more significantly in the DHA/EPA 1:2 group than in other DHA/EPA groups (p < 0.01), but the increase was not remarkable in comparison to that in the DHA group.
Effects of various ratios of DHA/EPA with an n-6/n-3 ratio of 4:1 on liver damage Levels of serum ALT, AST, GSH, SOD, and MDA, and liver GSH, SOD, and MDA are presented in Fig. 3. Further research is required to explain this discrepancy.
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Domain: Biology Medicine<|endoftext|>
Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data.
Laboratory methods. Whole cellular DNA was extracted from ethanol-preserved muscle (primarily) or liver tissue samples following Lyra et al. [32] or using a DNeasy Qiagen kit following manufacturer's protocols. PCR amplification was carried out using Taq Table 1). The standard PCR program consisted in an initial denaturing step of 2 minutes at 94˚C, 35-40 cycles of 30 seconds at 94˚C, 30 seconds at 48-56˚C, and 2 minutes at 72˚C, followed by a final extension step of 10 minutes at 72˚C. PCR non-purified products were sent to Macrogen Inc. (South Korea) where they conducted purification and sequencing in an ABI 3730XL sequencer.
Alignment, partition schemes, and model selection. We performed alignment using MAFFT v. 7.273 [41] with the FFT-NS-i algorithm, except for the 16S gene fragment, for which we used the E-INS-i algorithm. This strategy is applicable for regions with conserved domains surrounded by nonalignable regions, such as rDNA. For both algorithms we used 1000 as a maximum for iterative refinements. We conducted the search for the best partition scheme and best fitting molecular model using PartitionFinder 1.1.1 [42] with the Corrected Akaike Information Criterion (AICc; [43]) and considering each codon as a separate partition.
Phylogenetic analyses, haplotype network, and genetic distance. We used both Maximum Likelihood (ML) and Bayesian Inference (BI, concatenated locus approach) for constructing the phylogenetic trees. We conducted ML analysis in RAxML v. 8.2.10 [44], with 100 runs for tree search and 1000 non-parametric bootstrap replicates. We constructed BI trees in MrBayes [45] using two independent runs of 2.0 x 10 7 generations, starting with random trees and four Markov chains (one cold), sampled every 2000 generations. We discarded 25% of generations and trees as burn-in and performed the run with unlinked character state frequencies, substitution rates of GTR model, gamma shape parameters, and proportion of invariable sites between partitions.
We examined if candidate species display signs of reproductive isolation by analyzing nuclear DNA (nDNA), allele sharing, and network cohesion. To determine the most probable alleles for individuals heterozygous for nDNA sequences we used gametic phases reconstructed through the algorithm implemented in phaSe 2.1.1 software [46], which were interconverted to fasta format using SeqPHASE web tool [47]. Using these alignments we computed statistical parsimony networks using TCS [48] method.
We computed uncorrected pairwise p-distances using R version 3.6.0 [49] with the packages APE version 5.0 [50] and SPIDER version 1.5.0 [51]. In order to avoid alignment effect, we ignored sites within missing data (pairwise.deletion = TRUE).
We genotyped all collected and recorded specimens. (DOCX)
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Domain: Biology Medicine
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Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex
The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. [6,[8][9][10][11][12][13][14]). The TMs of TASK-1 each contain hydrophilic amino acids that might potentially compromise membrane insertion (Supplementary Figure S1 at [URL]), and we have used single cysteine residues to probe their proximity to specific components of the ER translocon.
To estimate a 'window of proximity' to the Sec61 complex for different TMs of TASK-1, we built a series of singlecysteine mutants and generated ribosome-bound integration intermediates for each one of increasing chain length ( Figure 1A). All intermediates included a C-terminal V5 tag to identify authentically truncated polypeptides and encoding mRNAs lacked stop codons to favour the formation of ribosome-bound ER translocon-associated integration intermediates [10,13]. The treatment of 74-residue-long TASK-1 integration intermediates, containing single cysteine probes at different locations, with BMH resulted in discrete cross-linking products ( Figure 1B). Among these products, adducts with subunits of the Sec61 complex could be identified by immunoprecipitation using validated antibodies known to recognize specific components of the ER translocon [10,11,13,31,34]. The most prominent cross-linking partner that could be identified in this fashion was the Sec61α subunit ( Figure 1B), although we also observed a trimeric adduct of TASK-1-Sec61α-Sec61β when the cysteine probe was located at residue 28 ( Figure 1B, A28C panel). A TASK-1 Cys-null variant generated no such products ( Figure 1B, Cys null panel), confirming the site-specific nature of these adducts. Furthermore, adduct formation depended on a stable ribosome-bound nascent chain, hence puromycin treatment before BMH addition abolished cross-linking to components of the ER translocon (Supplementary Figure S2 at [URL]). Taken together, these data suggest that TM1 of a 74-residue-long TASK-1 integration intermediate engages the Sec61 complex upon its delivery to the ER membrane.
Interestingly, at this shortest chain length, we also observed rather weak cross-linking products formed between a single cysteine probe located at both the N-and C-terminal side of TM1 and the single cytoplasmically located cysteine residue of Sec61β ( Figure 1B, A10C and L35C panels, and Supplementary Figure S2, lane 3). The adduct with L35C was unexpected, and suggested that either TM1 is in a loop conformation at an early stage of biogenesis, or at least a proportion of nascent TASK-1 chains might assume an inverted topology during membrane integration. When the cross-linking partners of a longer integration intermediate were analysed, in addition to adducts with Sec61α, the L35C-mediated adduct with Sec61β was still present and, if anything, became more apparent ( Figure 1C, L35C panel, and Supplementary Figure S2, lane 6). We extended these studies to look at probes at or near the first pore loop, P1, and TMs 2, 3 and 4 ( Figure 1A, and Supplementary Figure S1) and could identify further adducts with Sec61α and Sec61β ( Figures 1C-1E, Table 1 and Supplementary Figure S2). Although these cross-linking products became more diffuse with longer integration intermediates, as observed previously [11], a clear pattern emerged (Figure 1, Table 1 and results not shown). In short, these data indicate that: (i) TM1 remains in close proximity to the Sec61 complex after the P1 loop and TM2 are synthesized; (ii) P1 remains adjacent to Sec61 after TM2 synthesis; (iii) TM2 remains close to Sec61 when the nascent chain is extended to include TM3 and P2; and (iv) TM3 is adjacent Sec61α after TM4 synthesis. Taken together, these data are consistent with models suggesting that the ER translocon may provide an environment that facilitates the folding of polytopic membrane proteins by orchestrating the assembly of their TMs [11,12]. Furthermore, since Sec61β has only a single cysteine residue located in its cytoplasmic domain, its capacity to cross-link probe L35C from integration intermediates of 74 and 172 residues (Figures 1B and 1C, and Supplementary Figure S2) suggests that TASK-1 biogenesis may not simply involve the sequential threading of TMs in their native orientation into the ER translocon.
Truncated TASK-1 chains assume a mixed topology in the ER membrane
Table 1 Summary of TASK-1 cross-linking analysis
Schematic representations of the truncation lengths are shown for each column, together with the number of residues in the chain (including the 14-residue C-terminal V5 tag). These fragments are named after the last residue before the chain was truncated. Each row summarizes data from one or more cysteine residues located within either the TM indicated, or the first P-loop (P1). Cysteine mutants that generated specific adducts with subunits of the Sec61 complex (as specified in the Experimental section) are indicated with α, β or αβ where a single adduct of the nascent with both components was detected. Some cysteine probes could not be tested with short integration intermediates (NA), some combinations were not tested (NT), and a number of combinations of probe location and longer chain lengths resulted in no discrete cross-linking products being observed (NDC). 1 kcal = 4.184 kJ.
Figure S4 Cysteine-dependent adducts with Kcv membrane integration intermediates
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Domain: Biology Medicine<|endoftext|>This below document has 2 sentences that end with 'Archipelago) 5E1 and gL 5E1'. It has approximately 557 words, 25 sentences, and 6 paragraph(s).
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Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. The specificity of co-immunoprecipitation was assessed by the absence of gC from the co-immunoprecipitated proteins (Fig. 4D). The results indicate that complexes formed in transfected cells closely mirror those formed in the infected cells and imply that no viral protein other than those that are recruited to the complexes is required for complex formation. Because the complexes formed in transfected cells did not differ from those in infected cells, most of the subsequent experiments were carried out in transfected cells.
FIGURE 2. gD co-immunoprecipitates gB and gH/gL, and not gC, from infected cells.
A-D, 293T cells were infected with wt HSV-5(F) or deletion ⌬gD, ⌬gB, ⌬gH, or ⌬gL mutant viruses (5 PFU/cell). To enable gH and gL detection, 6 h prior to infection, the cells to be infected with wt HSV, ⌬gD, or ⌬gB viruses were transfected with British Indian Ocean Territory (Chagos Archipelago) 5E1 and gL 5E1. Her . E and F, 293 T cells were transfected with HVEM plasmid (ϩR) or no HVEM (ϪR) at the time of British Indian Ocean Territory (Chagos Archipelago) 5E1 and gL 5E1. Her transfection. All of the cells were harvested 18 h after infection and immediately lysed with EA1 buffer. gD was immunoprecipitated with pAb R8 (IP-gD) and harvested with protein A-Sepharose beads. The beads from one sample were split into two portions and run in two separate gels: one for detection of gB and gD and one for detection of gH and gL; gC was detected in the same blot as gH and gL, after stripping. The gD co-immunoprecipitated proteins were detected by WB with mAb H170 to gD (A and E), mAb H1817 to gB (B and E), mAb5E1 to tagged gH and gL (C and F), and mAb H633 to gC (D). In D, the only positive lane is the one loaded with an aliquot of the infected cells lysate. The secondary antibody used for WB detected the pAb R8 Ig, whose electrophoretic mobility is faster than that of gD.
Genetic Engineering of HSV-5 Carrying One-strep-tagged gD (HSV1(BAC)-gD st )-
Glycoprotein Complexes Are Present in Virions-HSV1(BAC)-gD st was employed to ask whether glycoproteins already interact with each other in virions independent of entry into the cells and whether complexes formed at virus entry into the cell. Partially purified extracellular HSV1(BAC)-gD st virions were lysed, and gD st was absorbed to Strep-Tactin resin; complexed glycoproteins were analyzed by WB. The results in Fig. 5C show the WB reactivity of virion lysate (lane 1) and furthermore that small amounts of gB, gH, and gL were complexed to gD st in resting HSV(BAC)-gD st virions (lanes 2 and 3). The amounts of pulled down gB and gH/gL was small, and to visualize them the gB-gD and gH/gL lanes (Fig. 5C, lanes 2 and 3, respectively) were loaded with material pulled down from 2.8 ϫ 10 2 PFU (lane 2) and 1.4 ϫ 10 3 PFU (lane 3), respectively; for comparison, Fig. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine
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Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. Stephen Tomlinson (Medical University of South Carolina, Charleston, SC). Serotonin, ketanserin, epidermal growth factor, and all other chemicals were from Sigma.
Cell Culture-HEK293 cells were maintained in minimal essential medium supplemented with 10% (v/v) fetal bovine serum in a 5% CO 2 incubator at 37°C. Cells were passaged every 3-4 days to maintain cells in logarithmic growth. C6 glioma cells were grown in high glucose Dulbecco's modified Eagle's medium and maintained as above.
Plasmids-All recombinant DNA procedures were carried out following standard protocols. The wild type pBK-CMV-GFP-PKCII was described previously (19). PKCII-mCherry was described previously (11). The 5-HT 2A R-yellow fluorescent protein (YFP) receptor construct was generated by cloning the cDNA sequence of human 5-HT 2A R into XhoI and BamHI sites of EYFP-N1 vector (Clontech, BD Biosciences). The HA-PAR1 was a gift from Dr. JoAnn Trejo (University of California, San Diego, CA). The cells with stable expression of AT1AR-GFP were a gift from Dr. Thomas A. Morinelli (Medical University of South Carolina, Charleston, SC). The GFP-tagged Rab11 was kindly provided by Dr. Marino Zerial (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). The HA-tagged PLD1 and PLD2 wild type (WT) and mutants of PLD1 (K898R) and PLD2 (K758R) were a gift from Drs. Michael Frohman and Guangwei Du (Stony Brook University, New York, NY).
Transient Transfection, Indirect Immunofluorescence, and Confocal Microscopy-Cells were plated onto 35-mm confocal dishes (MatTek, Ashland, MA) at a density of 5 ϫ 10 5 cells/dish and grown for 24 h. Transient transfections of DNA (0.5-1 g/dish) were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Transfected cells were grown for 24 h in 10% fetal bovine serumcontaining medium. Transfection efficiencies of 40 -60% were obtained in these experiments. Cells expressing GFP or YFP fusions alone were viewed after fixation with 3.7% formaldehyde (10 min) followed by cold methanol (5 min). Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. The symbols are as explained in legend to Fig. 7. An arrowhead points to heavy IgG band.
complexed with gB and with gH/gL in resting virions; the amount of complexed gB and gH/gL was indeed low. When HSV1(BAC)-gD st virions were allowed to enter cells for a 30-min time interval, we did not obtain evidence of de novo gD-gB complex formation; rather, the amount of gD-gB complex decreased. This finding is in agreement with an earlier report showing that gE from input virions first associate with and then disappear from cell surfaces (56).
The Significance of the Glycoprotein Complexes and Interactions Identified in These and Preceding Studies-A key question raised by the results presented in this report and elsewhere (29,30) relates to the significance of the diverse complexes involving members of the glycoprotein quartet. There are two fundamental possibilities. Foremost, current evidence indicates that resting virions harbor small amounts of preformed complexes; these complexes did not increase during virus entry; rather, they tended to decrease. In principle, the process of HSV-mediated fusion must entail activation of gB and/or gH/gL from fusion-inactive to fusionactive conformations. A possibility compatible with the current results is that conformational changes in gD may be signaled from gD to the precomplexed gB or gH/gL and in this way induce conformational changes to the fusion executors. A nonexclusive possibility is that complexes made by the glycoproteins, or one of the complexes, either is disassembled or is triggered to become fusogenic, once the fusion executors encounter cellular proteins (e.g. receptors), without any appreciable biochemical modification to the glycoproteins themselves.
Second, the possibility exists that members of the glycoprotein quartet, like most HSV proteins analyzed to date, perform multiple functions. In support of this view is the observation that gB is targeted to the multivesicular body compartment and plays a role in HSV envelopment and exit (57), gD and gB traffic to inner nuclear membrane to enable primary envelopment (58), and gD encodes an anti-apoptotic function (47). Conceivably, the formation of these complexes requires the involvement (e.g. direct binding, posttranslational modifications, etc) of cellular proteins that enable the execution of these functions. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine<|endoftext|>14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
To further explore the potential signaling pathways associated with mammalian Eag, we set forth to identify novel binding partners of rEag1 channels in the brain. All statistical analyses and curve fitting were performed with the Origin 7.0 software (Microcal Software).
Results
Interaction of 14-3-3h with the N-and C-termini of rEag1 in vitro We carried out the yeast two-hybrid screening of a rat brain cDNA library by using the N-terminus (amino acids 1-207) of the rEag1 protein (rEag1-N207) as the bait (Fig. 1A). One of the positive clones isolated by the screening was 14-3-3h. 14-3-3 proteins are ubiquitously expressed in all eukaryotes [19]. In HEK293T cells were co-transfected with the cDNAs for rEag1 and myc-vector or myc-14-3-3h in the molar ratio of 1:5. The holding potential was 290 mV. The pulse protocol comprised 300-ms depolarizing test pulses ranging from 290 to +50 mV, with 10-mV increments. (Right panel) Normalized mean K + current density (at +40 mV) of rEag1 channels in the absence or presence of myc-14-3-3h. The numbers in the parentheses refer to the number of cells analyzed, and the asterisk denotes significant difference from the rEag1 control mammals, there are seven 14-3-3 isoforms: a/b, e, g, c, t/h, f/d, and s [20]. Except for the s isoform, 14-3-3 proteins are abundantly expressed in the brain, and have been implicated in the modulation of neurotransmission, brain development, and learning and memory [21,22,23].
The potential interaction of 14-3-3h with the N-terminus of the rEag1 protein was further validated by our yeast two-hybrid assay result showing that yeasts co-transformed with 14-3-3h and the plasmid encoding the rEag1-N207 segment were capable of growing in synthetic leucine-lacking media (Fig. 1B). Furthermore, yeasts co-transformed with 14-3-3h and the plasmid encoding the cytoplasmic C-terminus (amino acids 493-962) of the rEag1 protein (rEag1-C0) were also growing in the leucine-lacking medium (Fig. 1B), suggesting that 14-3-3h may interact with the C-terminus of rEag1 as well. To address this hypothesis, we employed GST pull-down assay with GST fusion proteins encoding the N-or C-terminus of the rEag1 protein (GST-N207 or GST-C0). As depicted in Figure 1C, in vitro translated 14-3-3h was efficiently retained by both GST-N207 and GST-C0 fusion proteins, but not by the GST protein per se (Fig. 1C), indicating a direct interaction between 14-3-3h and the GST fusion proteins. We also performed GST pull-down assay with the cell lysates prepared from HEK293T cells transfected with myc-tagged 14-3-3h (myc-14-3-3h). Figures 1D and 1E demonstrate that myc-14-3-3h was proficiently precipitated by the fusion proteins GST-N207 and GST-C0, respectively, further supporting the idea that multiple 14-3-3h-interacting sites may exist within the rEag1 protein.
To further address the structural basis of these novel interactions, we generated additional GST fusion proteins encoding specific protein domains of rEag1. The cytoplasmic N-terminus of rEag1 protein contains a Per-Arnt-Sim (PAS) (amino acids 14-145) domain that constitutes parts of the common structural features for the EAG K + channel family [25]. (TIF)
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Domain: Biology Medicine
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Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells
Background EDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. In these models, it is assumed that degradation and retrotranslocation are tightly coupled and occur at the ER membrane. On the other hand, some integral membrane ERAD substrates, such as MHCI [52] and cystic fibrosis transmembrane conductance regulator (CFTR) [53], have been observed to reside in the cytoplasm when proteasome function is compromised. These data suggested that transmembrane hydrophobic segments might be solubilized from the lipid bilayer of the ER prior to proteasome-mediated degradation. Indeed, it has been demonstrated that the transmembrane domain of the ERAD substrate, Ste6p, is released into the cytosol in a Cdc48/p97-and ATP-dependent manner [44]. Candidates for factors that could maintain the solubility of transmembrane domains include cytoplasmic chaperones (such as Cdc48p), proteasome associated factors such as Rad23p/Dsk2p [54], and the 19S particle. Interestingly, it has been recently demonstrated that the proteasome 19S subunit can act as chaperone protein also for ricin A-chain, preventing aggregation of unfolded RTA [55]. Moreover, it was suggested that misfolding status of the cytoplasmatic domains of membrane ERAD substrates might influence the assembly of the intramembrane domain, which is recognized by Hrd1p, an E3 ubiquitin ligase complex [44]. Some reports suggest that an Hsp70 family member-containing or putative lectin-containing complex in the ER helps to recruit a misfolded substrate to the Hrd1p complex [56][57][58]. The mannosidase-like domain of EDEM1 does not appear to be required for ERAD substrate binding, but this domain is involved in binding to SEL1L-containing ER membrane dislocation and ubiquitination complex [19]. It is possible that this domain also recognizes hydrophobic patches of aberrant proteins. We cannot exclude the possibility that recognition of hydrophobic transmembrane domains by EDEM1 and EDEM2 is necessary for solubilization from the lipid bilayer or that EDEMs recognize already solubilized transmembrane domains. Importantly, it has been demonstrated that several less-hydrophobic transmembrane sequences derived from multimeric transmembrane protein complexes can enter the ER lumen completely, where they are recognized as substrates of the chaperone BiP, which in turn initiate degradation of the unassembled subunit [59]. This new mechanism suggests a general link between proper integration of transmembrane segments and the ER luminal chaperone machinery. Interactions between hydrophobic transmembrane domain of BACE457 and EDEMs might explain differences between ERAD of membrane BACE457 and its luminal form BACE457Δ. Degradation of BACE457Δ is faster than that of membrane BACE457; half-life of BACE457Δ is about 40 min, while that of BACE457 is 4 h [47]. Importantly, the lag phase for the soluble variant of BACE457 is only 15 min, whereas for membrane bound BACE457 it is 90 min [47]. These differences might be connected with necessity of extraction of transmembrane domain of BACE457 out of the ER membrane. Hebert and colleagues suggested that EDEM1 serves as a quality control receptor that acts as a molecular link between misfolded proteins and SEL1L [19]. Recently published results show that degradation of BACE457 did not require SEL1L complex [60]. However, ricin A-chain transport to the cytosol depends on SEL1L [32]. Proteins were detected by chemiluminescence reagent SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific).
Immunofluorescence microscopy
HEK293 cells transfected with cDNA encoding BACE457 , BACE457 DHF or BACE457Δ were grown on coverslips.
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Domain: Biology Medicine<|endoftext|>DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice
Background Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. The infection is mainly acquired either by using water contaminated with oocysts released by the final host or handling intermediate host tissues infested with the asexual cysts [2,3]. In humans, there are two types of the infection according to symptoms; the first type is the asymptomatic form, resulting in a latent infection with tissue cysts. This form is less frequently seen in immunologically intact individuals. However, the infection could be severe in specific groups of patients, such as immunologically impaired individuals (AIDS or organ transplants) or congenitally infected fetuses and newborns [4,5].
Currently, the strategies of toxoplasmosis control mainly rely on the application of chemotherapeutics targeting the acute phase of the infection, however, some drawbacks were found to be associated with drug application, e.g; rapid re-infection besides toxic effects of the drugs [6,7]. Such issues 'blew the whistle', shifting the research directions into the area of vaccine development as an alternative control strategy for toxoplasmosis, with DNA vaccines receiving considerable attention [6].
Recent important progress has been made identifying anti-toxoplasma vaccine candidates that can stimulate an immunological response, with most of the work focusing on tachyzoite surface antigens, namely SAG1, SAG2 and SAG3, and SAG1 was recognized to be the most promising candidate in this group [8][9][10][11]. In the same context, T. gondii excretory secretory antigens like GRA molecules, have also been reported to demonstrate significant immunogenic capabilities [12][13][14]. Vaccination with DNA vaccines has been found to induce effective humoral and cellular immune responses, with both CD4 + T helper cells and CD8 + cytotoxic T cells included in these responses [15]. Such elements are important for understanding the mechanisms through which the parasite modulates the host immune response during both acute and chronic phases of the disease [16].
Blocking the parasite from invading the cell and consequently preventing the parasite form multiplying may help in reducing the parasitic burden and leave the parasite exposed to other immunological elements, thus in this study we demonstrated the immunological changes after vaccination of mice with a DNA vaccine encoding TgDPA followed by challenge with virulent T. gondii RH strain.
Animals and parasite
Six to eight week-old female Swiss Webster (SW) mice were purchased from The Center of Comparative Medicine, Yangzhou University (Yangzhou, China) and maintained under specific-pathogen-free standard conditions. All animal experiments were approved by the Animal Ethics Committee of Nanjing Agricultural University (Approval number 200709005). Toxoplasma gondii strain RH (Type I), was provided by The Laboratory of Veterinary Molecular and Immunological Parasitology, Nanjing Agricultural University, China. To maintain the parasite, as described by [21], intraperitoneally injected SW mice were infected with the parasite tachyzoites. Every 3 days, the tachyzoites were harvested and recovered from peritoneal washings of infected mice to be used for re-infection.
Construction of the prokaryotic plasmid
According to the manufacturer's protocol Trizol reagent (Takara, Life Technologies), total RNA of T. gondii was extracted from T. gondii tachyzoites, followed by construction of the cDNA. The open reading frame (ORF) of Deoxyribose Phosphate Aldolase (TgDPA) gene (XM_002365690.1) was obtained from T. This finding may encourage more investigations in evaluating the immunogenicity of DPA based vaccines against Toxoplasmosis.
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Domain: Biology Medicine
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This below document has 2 sentences that end with 'steatosis and liver lesions'. It has approximately 711 words, 26 sentences, and 9 paragraph(s).
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Protective effects of various ratios of DHA/EPA supplementation on high-fat diet-induced liver damage in mice
Background A sedentary lifestyle and poor diet are risk factors for the progression of non-alcoholic fatty liver disease. Before the mice were randomized into six groups (n = 15), they were acclimated to the laboratory for 7 days with basic feed, which was purchased from WQJX Bio-technology (Wuhan, China). High-fat diet I (HFD I) was composed of 700 g of basic feed, 150 g of lard, 100 g of yolk powder, 40 g of casein, and 10 g of cholesterol per kg. In high-fat diet II (HFD II), the 150 g/kg lard was reduced to 100 g/kg. The normal control group (NC) was fed with basic feed; the high-fat control group (HFC) was fed with HFD I; the DHA group received HFD II plus algal oils (containing 100 mg of DHA per 260 mg) and sunflower seed oil (containing n-6 PUFA at 60% but low amount of n-3 PUFA, adjusting the proportion of n-6/n-3 to 4:1); and the DHA/EPA group was fed HFD II as well as fish oils (containing 200 mg DHA and 400 mg EPA per 1900 mg) and algal oils to achieve various DHA/EPA ratios (2:1, 1:1, and 1:2), and sunflower seed oils to adjust the n-6/n-3 ratio to 4:1. The total caloric intakes of mice in all groups were equivalent except for mice in the NC group. The fatty acid compositions of oils, basic feed, control diet, and high fat diets are described in Table 1. The PUFAs were administered orally (0.75 mg/g body weight) every day for 11 weeks, and NC as well as HFC mice were given the same dose of distilled water via intragastric administration as control groups. The mice were individually housed in a humidityand temperature-controlled room (relative humidity 60% and temperature 20-22°C) with a 12-h light/dark cycle environment and were provided ad libitum access to water and their group-specific diet. Measurements of body weight and food intake were taken weekly. After 11 weeks, five mice from each group were anesthetized with isoflurane, and blood was drawn through the retro-orbital sinus puncture prior to killing the mice by decapitation after an overnight fast and used for analyses to ensure the establishment of models of steatosis and liver lesions. The rest of the mice were euthanized in the same way. Serum was collected from blood, subsequently centrifuged (4000×g, 4°C, 10 min) after incubation at room temperature for 2 h, and then stored at −80°C until analysis. Fresh liver tissue samples were collected for histopathology analysis or were rapidly frozen and stored at −80°C before use.
Analysis of steatosis and liver lesions
The liver was removed and weighed immediately after the mice were euthanized. Liver fragments were sectioned at a thickness of 5 μm, and fixed with 4% paraformaldehyde overnight. Then the slices were stained with oil red O, in order to analyze the steatosis and liver lesions. Images were acquired by means of an inverted fluorescence microscope (Olympus Co., Japan) equipped with a SONY DXC-970MD color video camera, and analyzed using the Image-Pro plus program.
Measurement of serum and liver lipid profiles
The serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), and lowdensity lipoprotein-cholesterol (LDL-C) levels were measured using similar commercial kits (Biosino Biotechnology Co., Ltd., Beijing, China) with spectrophotometric methods in accordance with the manufacturer's instructions.
To analyze the hepatic levels of GSH, SOD, and MDA, 50 mg of liver tissues were homogenized with 450 μl of normal saline. After centrifugation at 4000×g and 4°C for 10 min, the supernatant of the homogenate was used for analysis with the corresponding commercial kits (Nanjing Jiancheng Corporation) as specified by the manufacturer. In addition, protein content was measured using commercial bicinchoninic acid (BCA) kits (Cloud-Clone Corp, Wuhan, China).
Real-time PCR
The total RNA was extracted from liver tissues using the RNAiso Plus reagents (TaKaRa BIO Inc., Dalian, China). Then, the cDNA was extracted using a Prime Script RT Reagent Kit (TaKaRa BIO Inc.). The mRNA was quantified with a real-time PCR machine (IQ5, Bio-Rad, USA) using SYBR Premix Ex Taq (TaKaRa BIO Inc., Dalian, China) and specific primers (BGI Tech Solutions Co., Ltd., Shenzhen, China) in accordance with the manufacturer's recommendations. Further research is required to explain this discrepancy.
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Domain: Biology Medicine<|endoftext|>Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. receptors), without any appreciable biochemical modification to the glycoproteins themselves.
Second, the possibility exists that members of the glycoprotein quartet, like most HSV proteins analyzed to date, perform multiple functions. In support of this view is the observation that gB is targeted to the multivesicular body compartment and plays a role in HSV envelopment and exit (57), gD and gB traffic to inner nuclear membrane to enable primary envelopment (58), and gD encodes an anti-apoptotic function (47). Conceivably, the formation of these complexes requires the involvement (e.g. direct binding, posttranslational modifications, etc) of cellular proteins that enable the execution of these functions. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine
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Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex
The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. Hence at least this fraction of molecules appears to undergo a complete inversion of TM1 at some stage during biogenesis. No evidence for triply Nglycosylated chains was apparent with the longer OPG-TASK-1 chains studied ( Figure 3B). Taken as a whole, the N-glycosylation profile of these OPG-TASK-1 derivatives suggest a model where TM1 can assume at least two quite distinct topologies within the ER membrane, and suggests that the topology of TM1 may be influenced by the synthesis of additional regions of polypeptide to its C-terminus. It should be noted that this approach provides no information regarding the topology of any membrane integrated TASK-1 fragments that are not N-glycosylated (see Figure 5).
To confirm that the BMH-mediated cross-linking to Sec61β from TASK-1:L35C, where the probe is located C-terminal to TM1, and N-glycosylation of an OPG tag placed N-terminal to TM1 both reflect the same population of nascent chains, OPG-TASK-1:L35C was created and its cross-linking to components of the Sec61 translocon was analysed, paying particular attention to whether Sec61β adducts were EndoH-sensitive ( Figure 3E). For the three chain lengths analysed, a substantial proportion of the Sec61β adducts are formed with doubly N-glycosylated TASK-1 nascent chains ( Figure 3E, compare lanes 5, 6, 11, 12, 17 and 18). Together, these data suggest that a proportion of nascent TASK-1 polypeptides, with chain lengths up to and including 172 residues, are oriented with their N-terminus in the ER lumen and residue 35 at or close to the cytoplasmic side of the ER translocon, and hence able to cross-link Sec61β.
TM1 of the small potassium channel Kcv displays a mixed topology
Although previous studies have shown an initial 'head-first', and hence inverted, insertion of signal-anchor sequences [6,22], the suggestion that TM1 of TASK-1 can assume a completely inverted topology was nevertheless unanticipated. To establish whether this process reflected a specialized pathway or a more general feature of membrane protein biogenesis, we analysed an even simpler potassium channel subunit with only two TMs separated by a putative pore loop, the viral Kcv protein [28] ( Figure 4A, and Supplementary Figure S3 at [URL]). Preliminary studies revealed that a number of BMH-dependent adducts could be detected using a ribosome-bound integration intermediate of Kcv that contained a single cysteine probe, but not with the equivalent Cys-null version of Kcv (Supplementary Figure S4 at [URL]). This 89-amino-acid-long integration intermediate, denoted Kcv:N37C, lacks TM2, and consists of the first 75 residues of the coding region followed by a V5 epitope tag (cf. Figure 4A). When these BMH-dependent adducts were analysed further by immunoprecipitation using antibodies recognizing well-defined components of the ER translocon, Sec61α and Sec61β were found to be two of the most prominent cross-linking partners that could be identified ( Figure 4B, lanes 9 and 10, and Supplementary Figure S4). Although this is consistent with the insertion of the Kcv monomer via an ER translocon-mediated process, the crosslinking of this nascent chain to Sec61β is especially relevant, since if TM1 attains its native topology, Asn 37 would be on the ER-luminal side of the membrane and hence incapable of forming such a product when replaced with a cysteine residue [13] (cf. Figure 4A).
The strength of the Sec61β adduct obtained with the Kcv integration intermediate suggested that a substantial proportion of these nascent chains may have an 'inverted' topology, and therefore we used N-glycosylation reporters to further explore Kcv biogenesis (cf. Figure 3A). To this end, OPG-Kcv, an N-terminally tagged Kcv derivative, and Kcv:K47N, a variant with a single novel site for N-glycosylation in the loop region joining TM1 and TM2, were created (see Figures 4C and 4D respectively). OPG-Kcv was synthesized in the presence of ERderived microsomes, either in its full-length form or as a truncated chain that lacked TM2, in both cases using an mRNA containing a stop codon to terminate translation (cf. Figure 3B). For each of these OPG-Kcv polypeptides, both singly and, especially, doubly N-glycosylated species were observed at a level comparable with the non-glycosylated Kcv chains observed ( Figure 4E, compare lanes 1-4). This pattern of N-glycosylation is consistent with a substantial proportion of nascent Kcv chains assuming an inverted topology at the ER translocon and maintaining this topology upon termination of translation. With full-length OPG-Kcv, some of the protein forms an SDS-resistant complex, probably a tetramer [29]. 1 kcal = 4.184 kJ.
Figure S4 Cysteine-dependent adducts with Kcv membrane integration intermediates
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Domain: Biology Medicine<|endoftext|>Coevolution Trumps Pleiotropy: Carbon Assimilation Traits Are Independent of Metabolic Network Structure in Budding Yeast Phenotypic traits may be gained and lost together because of pleiotropy, the involvement of common genes and networks, or because of simultaneous selection for multiple traits across environments (multiple-trait coevolution). However, if there was a bias for growth phenotype, the observed data would deviate from similar numbers of weak and strong growth phenotypes.
Carbon source utilization cluster analyses by pathway and enzyme
We assessed whether gains and losses of carbon sources cluster by strain using multiscale bootstrap resampling, with 1000 permutations (R v2.14 package pvclust v1.2-2). We produced a matrix between carbon sources reflecting ability to be utilized by the same strains. Each carbon source was then assigned a cluster and similar clusters were joined together until there was only a single cluster remaining. To assess if these patterns were driven by overlapping metabolic pathways, pathway data for each carbon source was acquired from the Kyoto Encyclopedia of Genes and Genomes (KEGG) v62.0 [13]. Carbon sources were clustered by Ward's method (also with pvclust in R) according to their presence
Isolation predicted by carbon sources
We assessed whether carbon source use patterns were driven by a common environment by predicting strain isolations based on carbon source sets. We used a k-nearest neighbor classification (k = 3) with a leave-one-out cross validation scheme to determine if carbon source sets could be used to predict strain isolation (MATLAB 2012b).
Carbon source utilization diversity
Carbon utilization is diverse within the genus Saccharomyces. On average, strains can grow on approximately 8 carbon sources ( Table 1); however strains can use a range of 1 to 37 carbon sources ( Figure 1).
Saccharomyces strains differ in their growth rate on most carbon sources. In the data analyzed here, strains display either a normal or weak growth phenotype on each carbon source. On average, strains grow normally on 7 carbon sources and grow weakly on an additional 1.88 carbon sources (Table 1). All strains grow normally on glucose. To test whether some carbon sources are more likely to result in a slow versus normal growth phenotype, we examined the association between carbon sources and growth rate phenotype. Out of the 45 tested carbon sources, 11 carbon sources are overrepresented for normal growth across strains, relative to a weak growth phenotype (x 2 -test, p,0.001, indicated with a light gray boxes in Figure 2). For example, all 488 strains display a normal growth phenotype on glucose, indicating that glucose is overrepresented for the normal growth phenotype (p = 1.96610 299 ). This over-representation is expected, as glucose is the preferentially used carbon source of S. cerevisiae and other species in the genus [14]. Additional carbon sources which display an overrepresentation for the normal growth phenotype include sucrose (p = 3.45610 283 ), D-galactose (p = 3.68610 275 ), a,atrehalose (p = 1.53610 228 ), and maltose (p = 1.38610 258 ).
In contrast, 4 carbon sources show an overrepresentation for a weak growth phenotype, relative to normal growth phenotype: starch (p = 7.48610 24 ), succinate (p = 9.41610 24 ), ribitol (3.21610 25 ), and propane 1,2 diol (p = 9.67610 24 ) (dark gray boxes in Figure 2). For example, of the 55 strains that can use starch, 40 display a weak growth phenotype. This over-representation of weak growth is consistent with previous work showing that while starch can be used by S. cerevisiae and other species, they are inefficient at hydrolyzing starch [15,16].
Patterns of gain and loss in carbon utilization
The diversity found for carbon use traits raises the question -is there covariance for gain and loss in carbon utilization? In other words, are strains that have gained or lost the ability to use a particular carbon source more or less likely to have gained or lost the ability to use other carbon sources? There are two mechanisms by which the ability to use particular carbon sources may be gained and lost. doi:10.1371/journal.pone.0054403.g006
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Domain: Biology Medicine
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In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. However, at 8.5dpc, only the effects of maternal ICRs were focused on biological pathways related to the fetal-maternal interface. In contrast, paternal ICRs elicited, in terms of biological processes, a broad and shallow effect.
We previously hypothesized that the different methylation histories of the two parental germlines may underlie the numerical imbalance between maternal and paternal ICRs [5,6]. Deamination of 5-methylcytosine occurs at a 10-fold higher rate than other transitions, leading to frequent CpG to TpG/CpA mutations in mammalian genomes despite a dedicated repair pathway [14][15][16][17].
Here, we test this hypothesis by a systematic assessment of the sequence evolution of ICRs in different mammalian lineages and in comparison to other sequence categories. In doing so, we provide evidence that paternal ICRs have lost CpG sites and therefore their methylation targets at a significantly higher rate than maternal ICRs, while the latter in fact exhibit a relative gain of CpG motifs compared to similar but non-imprinted genomic regions. We propose that a combination of high mutational pressures at paternal ICRs together with functional selective pressure reinforcing methylation-dependent repression of ICRs, has led to the oocyte dominating the control of the fetal-maternal interface through genomic imprinting in eutherian mammals. Our results provide the first comprehensive view of the forces acting upon the regulatory sequences governing genomic imprinting in mammals.
Developmental and epigenetic characterization of imprint-free embryos
The impact of imprinted gene expression on development prior to mid-gestation has never been investigated on a genome-wide scale. To understand which biological pathways are regulated by maternal and paternal ICRs, respectively, we compared the developmental potential and transcription profiles of 8.5dpc embryos that differ in their imprinting status but have an otherwise normal genome. Three different imprinting states were investigated: fully-imprinted (MP) embryos, maternal imprint-free (0P) embryos and completely imprint-free (00) embryos. Here, M and P denote a normally imprinted set of respectively maternal and paternal chromosomes, and 0 denotes a chromosome set without imprints.
Author Summary
In mammals, a subset of genes is expressed from only one chromosomal copy, depending on its parental origin. This process, known as genomic imprinting, results from DNA methylation marks deposited in gametes at regulatory sequences called imprinting control regions (ICRs). Most of the DNA methylation controlling imprinting is established in the oocyte, while very few ICRs are methylated in the sperm. We provided insight into the impact and origins of the parental imbalance in genomic imprinting control. We defined the transcriptome-wide effect of imprinting, during the transition period when the embryo becomes dependent upon maternal resources. We found that maternal ICRs have a vital effect on developmental pathways related to the mother-to-fetus exchanges, while paternal ICRs have a dispersed and non-significant effect at that stage. We evidenced that paternal ICRs are lost at a much faster rate than maternal ICRs during mammalian evolution, probably as a mechanistic consequence of different kinetics of the parental germlines. Our results support the notion that two independent evolutionary forces have led to the numerical and functional dominance of maternal ICRs: a selective advantage of parent-specific regulation of genes important for the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.
were not affected. In particular, retrotransposons of the IAP and LINE-1 classes showed similar methylation levels in MP, 0P and 00 embryos ( Figure 1B). Microarray analysis of imprinted gene expression showed that, as expected, genes controlled by maternal ICRs were significantly misexpressed in 0P and 00 embryos compared to MP embryos, while genes under the control of paternal ICRs were specifically misexpressed in 00 embryos compared to MP and 0P embryos ( Figure 2 and Figure S1). In addition, the 0P versus MP comparison revealed a number of paternally imprinted genes significantly affected by the lack of maternal imprints ( Figure 2A). In the maternal case for example, 470 probes sets detected a decrease of expression in the 0P and 00 samples to 80% (-0.322 log2-ratio) or less relative to the MP sample.
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Domain: Biology Medicine<|endoftext|>Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions*
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. We evaluated the role of ERK1/2 on the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene, a master regulator of mitochondrial function. The potent and specific MEK1/2 inhibitor trametinib rapidly blocked ERK1/2 phosphorylation, decreased cytosolic and nuclear FOXO3a/1 phosphorylation, and increased PGC-1α gene expression and its downstream mitochondrial biogenesis (MB) targets under physiological conditions in the kidney cortex and in primary renal cell cultures. The epidermal growth factor receptor (EGFR) inhibitor erlotinib blocked ERK1/2 phosphorylation and increased PGC-1α gene expression similar to treatment with trametinib, linking EGFR activation and FOXO3a/1 inactivation to the down-regulation of PGC-1α and MB through ERK1/2. Pretreatment with trametinib blocked early ERK1/2 phosphorylation following ischemia/reperfusion kidney injury and attenuated the down-regulation of PGC-1α and downstream target genes. These results demonstrate that ERK1/2 rapidly regulates mitochondrial function through a novel pathway, EGFR/ERK1/2/FOXO3a/1/PGC-1α, under physiological and pathological conditions. As such, ERK1/2 down-regulates mitochondrial function directly by phosphorylation of upstream regulators of PGC-1α and subsequently decreasing MB.
ERK1/2 is a major player in various cell signaling pathways, including proliferation, differentiation, senescence, cell injury and recovery, and apoptosis. ERK1/2 becomes activated through a variety of extracellular stimuli, including receptor tyrosine kinases (RTK) such as the epidermal growth factor receptor (EGFR), 3 which leads to the activation of mitogenactivated protein kinase kinase (MEK) (1,2). EGFR agonists activate intrinsic tyrosine kinase activity within the cytoplasmic domain of the receptor, initiating the recruitment of Ras, a GTPase, which allows for interaction with downstream effectors, including the Raf protein kinases. Raf phosphorylates MEK1/2, which phosphorylates ERK1/2. ERK1/2 is thought to be the only substrate for MEK1/2 phosphorylation, which has allowed for MEK1/2 inhibitors to be used specifically for ERK1/2 inactivation (3,4). ERK1/2 is also activated by cell stressors, including reactive oxygen species. In a study utilizing H 2 O 2 injury in human renal cells, ERK1/2 inhibition was shown to decrease necrosis and apoptosis (5), whereas in a cisplatin-induced cell injury model, ERK1/2 inhibition reduced caspase 3 activation and apoptosis (6). In renal proximal tubular cells (RPTC), phosphorylated ERK1/2 was shown to reduce mitochondrial respiration and ATP production by decreasing complex I electron transport chain activity in response to tert-butyl hydroperoxide (TBHP), a model oxidant (7). Nowak et al. (7) also showed that expression of a constitutively active MEK1 increased ERK1/2 activation and decreased basal and uncoupled oxygen consumption, a measure of electron transport chain activity. Zhuang et al. (8) demonstrated that H 2 O 2 treatment of RPTC led to a high level of ERK1/2 phosphorylation and loss of mitochondrial membrane potential, which was attenuated by treating with a MEK/ ERK1/2 inhibitor. Finally, Fe 2ϩ -induced mitochondrial swelling was shown to occur through activation of the ERK1/2 pathway (8). These findings illustrate that ERK1/2 decreases mitochondrial function in response to injury.
The kidney is a high energy-consuming organ, and its cells have an abundance of mitochondria to meet ATP demand, especially within the proximal tubules (9). Previous studies demonstrated that rapid and persistent disruption of mitochondria homeostasis is an important contributor to the pathology of renal ischemia/reperfusion (IR) injury (10,11).
Peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1␣) is thought to be the master regulator of mitochondria biogenesis (MB) and is enriched in tissues with high metabolic demand, such as the heart, skeletal muscle, and kidney (12,13). was responsible for the funding acquisition.
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Domain: Biology Medicine
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In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. A fourth intergenic locus undergoing paternal-specific methylation has been recently characterized, but its function as an ICR has not been ascertained yet [9]. It nonetheless has likely been exposed to the evolutionary forces that we describe here, with an obs/exp CpG ratio within the range we defined for paternal ICRs (0.34).
Taken together, our results suggest that the functional dominance of maternal ICRs during early embryonic development is the consequence of two orthogonal evolutionary forces: 1) selection pressure to tightly regulate the expression of genes affecting the fetal-maternal interface once the placenta had evolved, increasing the number of imprinted loci per se and the number of CpG methylation targets, and 2) simultaneous pressure to avoid the deamination-prone environment of the paternal germline, favoring the evolution of maternal ICRs. The resulting numerical dominance of maternal ICRs implies a greater chance of some maternal ICRs to fulfill a vital role earlier in development than any one of the paternal ICRs, explaining the earlier lethality of maternal imprint deficiency and their functional dominance over the fetal-maternal interface at the time of its establishment. These two forces may have been aided by an intrinsic ability of the female germline to methylate CpG-rich regions. Indeed, we previously showed that de novo insertions of CpG-dense sequences are naturally targeted by methylation in the oocyte, provided that the insertion happened in an active transcription unit [42]. Mechanistic reasons for this association were more recently provided, by demonstrating that maternal ICRs need to be traversed by upstream transcripts to be methylated in the oocyte [43]. The exceptionally high transcriptional activity of the growing oocyte related to the necessity to establish a maternal store [44] may therefore have led to a propensity for the oocyte to methylate genes associated with CpG-rich promoters. Oocyte-methylation is then maintained after fertilization at a few loci, for the purpose of controlling expression levels of developmentally important genes and notably related to the vital transition step towards maternalfetal exchanges. (Table S1). The positions of 3,530 validated Low (L) CpG-content promoters and 10,872 High to Intermediate (HI) CpG-content promoters were extracted from [16]. The 12 maternal ICRs that fell into the HI category were ejected from the HI category. Definition of genomic intervals and euarchontoglire species used to retrieve multiple alignment data are presented in Text S1.
Evolutionary model estimation
Strand-symmetric context-dependent substitution rates and branch lengths were estimated using Ambiore and PhyloFit [30,31]. The topology of the phylogenetic tree for euarchontoglires was taken from the 44-species UCSC conservation track of the human genome [45]. Details of the methodology are provided in Text S1.
Generation and epigenotype confirmation of MP, 0P, and 00 embryos Details of the procedure are provided as supplemental information. Conceptuses were dissected at 8.5, 9.5 and 10.5dpc (relative to the foster mother) and VYS were genotyped: MP were Dnmt3L+/+, 0P Dnmt3L2/+, and 00 Dnmt3L2/2. Epigenotypes were confirmed by assessing the methylation status of the H19 and Kcnq1ot1 ICRs by bisulfite sequencing, before inclusion on the microarray.
Microarray creation and analysis
All samples were assayed using Affymetrix Mouse MOE430v2 expression microarrays. Four 8.5dpc embryos with confirmed genotype and epigenotype were pooled per category (MP, 0P and 00) to account for individual biological diversity. Five to seven mg of total RNA was used per sample as input. Probe level summarization was executed utilizing the Affymetrix GCOS/ MAS5 (target value of 500; otherwise default parameters) and GC-RMA (ArrayAssist implementation; default parameters) algorithms [46]. Further details are provided in [29]. In the maternal case for example, 470 probes sets detected a decrease of expression in the 0P and 00 samples to 80% (-0.322 log2-ratio) or less relative to the MP sample.
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Mutagenic analysis of the predicted Zn 2+ coordinating residues in ADAR1; His 910, Cys 966, and Cys1036 resulted in complete loss of ADAR1 activity demonstrating the essential role of Zn 2+ in the catalysis [40]. A highly conserved glutamate residue (E) participates in the proton transfer cascade during deamination. In ADAR2 the glutamate residue (E396) is the acceptor of a proton from the nucleophilic water molecule bound by the zinc atom. This produces a highly-reactive negatively-charged zinc-hydroxide that reacts with carbon 6 (C6) of the adenosine occupying the active site. The removal of a proton from the zinc-hydroxide ion results in the displacement of ammonia (NH3) during the reaction. Replacing this glutamate residue with an alanine results in a loss of catalytic activity of ADAR2 [40].
A unique feature identified in the crystal structure of ADAR2 is the presence of an IP6 molecule in the core site of the deaminase domain [41]. In the cell, the cleavage of phosphatidylinositol biphosphate by phospholipase C (PLC) generates IP3 which is subsequently phosphorylated to produce IP6 [46]. In ADAR2, the IP6 molecule is located in a basic cavity where it is tightly associated to conserved residues via hydrogen bonds and it is thought to contribute to the folding of the protein [41]. Macbeth and colleagues demonstrate that IP6 is an essential ADAR2 cofactor as S. cerevisae that are unable to synthesize IP6 cannot express catalytically-active ADAR2 [41]. Another study also demonstrated the importance of IP6 for ADAR2 activity. Schmauss and colleagues were investigating the regulation of the serotonin receptor (5-HT2C) mRNA and observed that an increase in serotonin (5-HT) levels resulted in increased editing of 5-HT2C pre-mRNA [47]. High levels of 5-HT led to the induction of PLC and the subsequent production of IP6 which could contribute to the production of ADAR2 which edits the 5-HT2C pre-mRNA. Editing of the 5-HT2C pre-mRNA decreases the production of 5-HT implicating that IP6 is a rate limiting factor in this negative feedback regulatory loop [47].
As the edited base lies within the deep and narrow major groove of the A-form RNA helix, it was proposed that the ADARs use a base-flipping mechanism to facilitate the deaminase domain access to the adenine base [48]. The mechanism of recognition of the edited base by ADAR1 and ADAR2 is very similar however ADAR1 is more dependent on the presence of N7 in the edited base [49]. Structural studies of the ADAR2 active site revealed that the architecture of the zinc ion and its coordinating residues would require a conformational change in the RNA before deamination. Stephens and colleagues used a fluorescent RNA probe that is quenched when the RNA helical duplex is naturally stacked and observed a significant increase in fluorescence emission from an RNA substrate bound at the active site of ADAR2 indicating that ADAR2 causes a conformational change at the editing site [50,51]. Another study used molecular dynamics simulations to investigate base-flipping at two sites within the Gria2 transcript, at an edited and a non-edited site [52]. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine
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New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. However, in hindsight, are RNA editing and RNA modification two sides of the same coin?
The cytidine deaminase (CDA) super family of enzymes includes both the ADAR and the APOBEC family of proteins as well as adenosine deaminase acting on the tRNA (ADAT) family. These enzymes catalyze deamination and their catalytic domain is evolutionarily conserved [8]. Following the identification of ADAR1 [9][10][11] the search for other ADAR-like genes began and this led to the discovery of ADAR2 (also known as ADARB1) [12][13][14]. The brain-specific ADAR3 (also known as ADARB2) was also identified based on its homology with ADAR2 [15]. A screen for genes that encode RNA binding proteins led to the identification of the testis nuclear RNA binding protein (TENR) [16]. TENR is testis-specific and found to contain a catalytically-inactive deaminase domain [16]. Due to the annotation of a second testis-specific gene encoding an adenosine deaminase domain containing protein All of the proteins contain N-terminal dsRBDs and C-terminal deaminase domains. An arginine rich (R) region is present at the N-terminus of ADAR3. There are two isoforms of hADAR1, ADAR1 p110 and ADAR1p150, both of which contain a Z-DNA binding domains (ZBD) at the N-terminal.
The ADAR1 gene was mapped to the q21.1-21.2 locus on chromosome one in humans [17]. It extends ~40 kb and contains 17 exons [18]. There are two different isoforms of ADAR1; a 150 kDa protein (ADAR1 p150) that is induced by interferon (IFN) and a 110 kDa protein (ADAR1 p110) that is constitutively expressed [19]. The two isoforms have distinct translation initiation sites so that ADAR1p150 starts translation in exon 1A (AUG1) whilst the ADAR1p110 protein initiates from exon two (AUG296) [19,20]. The generation of different ADAR1 isoforms is due to the alternative splicing of the exon one. An IFN-inducible promoter located in exon 1A contains a 12-bp IFN-stimulated response element (ISRE), thereby generating the transcript coding for the longer ADAR1p150 isoform [21]. However, transcripts encoding ADAR1p110 are generated from multiple promoters located within exon 1B, 1C and exon two [21,22]. As neither exon 1B nor exon 1C contain translation initiation sites, translation starts at the AUG296 codon within exon two. Additional alternative splicing events have been identified within exon 7 to generate variants that contain either exon 7a or 7b [20]. Exon 7b is missing 26 amino acids that are present in exon 7a [20]. Furthermore, exon 7b is associated with ADAR1p150 transcripts whereas exon 7a is present in the constitutively-expressed ADAR1p110 transcripts [23].
A unique feature of ADAR1 when compared to other members of the ADAR family, is the presence of Z-DNA binding domains (ZBDs) at the N-terminus. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine<|endoftext|>Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells
Background EDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. We cannot exclude the possibility that recognition of hydrophobic transmembrane domains by EDEM1 and EDEM2 is necessary for solubilization from the lipid bilayer or that EDEMs recognize already solubilized transmembrane domains. Importantly, it has been demonstrated that several less-hydrophobic transmembrane sequences derived from multimeric transmembrane protein complexes can enter the ER lumen completely, where they are recognized as substrates of the chaperone BiP, which in turn initiate degradation of the unassembled subunit [59]. This new mechanism suggests a general link between proper integration of transmembrane segments and the ER luminal chaperone machinery. Interactions between hydrophobic transmembrane domain of BACE457 and EDEMs might explain differences between ERAD of membrane BACE457 and its luminal form BACE457Δ. Degradation of BACE457Δ is faster than that of membrane BACE457; half-life of BACE457Δ is about 40 min, while that of BACE457 is 4 h [47]. Importantly, the lag phase for the soluble variant of BACE457 is only 15 min, whereas for membrane bound BACE457 it is 90 min [47]. These differences might be connected with necessity of extraction of transmembrane domain of BACE457 out of the ER membrane. Hebert and colleagues suggested that EDEM1 serves as a quality control receptor that acts as a molecular link between misfolded proteins and SEL1L [19]. Recently published results show that degradation of BACE457 did not require SEL1L complex [60]. However, ricin A-chain transport to the cytosol depends on SEL1L [32]. It is possible that the role of EDEM1 and EDEM2 in ERAD in some aspects is common for luminal and membrane substrates: extraction from the calnexin/calreticulin cycle and substrate demannosylation (at least in the case of EDEM1). However, after release from EDEM1 or/and EDEM2, terminal acceptors of misfolded membrane proteins might be different from luminal aberrant glycoproteins.
About one-third of all mammalian genes encode secreted and membrane proteins, including numerous important molecules such as ion channels and cell surface receptors [61]. The high rate of protein synthesis and the large fraction of misfolded and unassembled proteins generated in the ER, indicate that the ERAD plays a central role during active secretion, cell growth, and normal turnover in eukaryotic cells. Understanding the mechanism of ER protein quality control, specifically of how the cell recognizes and discriminates misfolded glycoproteins remains to be one of the central issues in cell biology.
Conclusions
We have demonstrated that ricin A-chain and a model misfolded protein, BACE457 carrying mutations that significantly decreased their hydrophobicity, are poorly recognized by EDEM1 and EDEM2 in contrast to their wild-type counterparts. Moreover, degradation of mutated BACE457 with decreased hydrophobicity is significantly reduced. The data presented in this paper contribute to the general understanding of the mechanism of recognition of misfolded proteins in the ER and the role of EDEM1 and EDEM2 chaperone proteins in this process. (Braunschweig, Germany). The mouse monoclonal anti-HA antibodies were obtained from Covance Research Products (Denver, CO), rabbit anti-BACE from Merck (Whitehouse Station, NJ), rabbit anti-Ricinus Communis-Lectin, and mouse anti-α-tubulin came from Sigma-Aldrich, whereas mouse monoclonal anti-ricin A-chain were purchased from Serotec (Oxford, UK). The mouse anti-calnexin were obtained from BD Biosciences (Palo Alto, CA), anti-calreticulin from BioSite (San Diego, CA). The rabbit anti-His antibodies came from Santa Cruz Biotechnology (Santa Cruz, CA). Proteins were detected by chemiluminescence reagent SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific).
Immunofluorescence microscopy
HEK293 cells transfected with cDNA encoding BACE457 , BACE457 DHF or BACE457Δ were grown on coverslips.
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Domain: Biology Medicine
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This below document has 2 sentences that end with 'HFC group (p < 0.05)',
3 sentences that end with 'the DHA group'. It has approximately 674 words, 24 sentences, and 5 paragraph(s).
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Protective effects of various ratios of DHA/EPA supplementation on high-fat diet-induced liver damage in mice
Background A sedentary lifestyle and poor diet are risk factors for the progression of non-alcoholic fatty liver disease. Nonetheless, the protein expression levels of Fra1 were found to be significantly higher in the DHA and DHA/ EPA treated groups in comparison to the HFC group (p < 0.05). Supplementation with DHA/EPA at the ratio of 1:2 did not result in enhancement in Fra1 expression levels. The mRNA expression levels related to c-Jun, c-Fos, and Fra1 were quantified by real-time PCR (Fig. 5e-g). Supplementation with n-3 PUFA showed a significant decrease in c-Jun and c-Fos mRNA expression and an increase in Fra1 mRNA expression, as compared to those in the HFC group (p < 0.05). Moreover, mice treated with the DHA/EPA ratio of 1:1 and 1:2 showed lowered c-Jun and c-Fos mRNA expression, respectively, in comparison to those in the DHA group. Nevertheless, no significant increase in Fra1 mRNA expression was observed in the best performance group (treated with the DHA/EPA ratio of 1:2) as compared with the DHA group. The concentrations of serum TNF-α, IL-1β, and IL-6 were all significantly lower in the DHA and the DHA/ EPA groups as compared to those in HFC group (p < 0.05) (Fig. 6). The quantitative real-time PCR assay, which roughly identified the inflammatory cytokine levels, revealed significantly decreased expression of TNF-α, IL-1β, and IL-6 in liver tissues of DHA/EPAtreated mice in comparison to those in HFC-treated mice (p < 0.05). Furthermore, mice treated with a DHA/EPA ratio of 2:1 revealed greater decreases in serum TNF-α contents and its mRNA expression levels in contrast to the DHA group. Additionally, supplementation with a DHA/EPA ratio of 1:2 demonstrated a more obvious reduction in serum IL-1β and IL-6 contents, and their mRNA expression as compared to those in the DHA group.
Discussion
The n-3 PUFAs, principally DHA and EPA, have been reported to play an increasingly significant role in the growth and health of the human body as shown by many previous studies [9,10]. The physiological functions of n-3 PUFAs have attracted increasing interest because of the increasing number of people choosing to lead healthy lifestyles [18]. However, there was a tendency among most attempted studies to focus on the effects of an individual n-3 or n-6 PUFA or the ratio of n-6/n-3, which did not take the possible influence of the relative ratio of DHA and EPA on the results into account [15]. A marked increase in the n-6/n-3 ratio is associated with lipogenesis, overweight lipid oxidation and secretion, and therefore promotion of NAFLD [11]; fish oil intake varies in its hepatic fatty acid composition in supplements with n-3 PUFA, and a reduction in the n-6/n-3 ratio has already been revealed in another study [7]. Other reports demonstrated that DHA supplementation led to a greater reduction in specific makers of inflammation than that by an equal dose of EPA [17], but supplementation with varying ratios of EPA/DHA influenced different metabolic syndrome markers [15] and enhanced the positive effects of biomarkers of inflammation [12]. Accordingly, in the present study, we concentrated on the effects of various ratios of DHA/EPA (with an n-6/n-3 ratio of 4:1) on liver damage in C57BL/6 J mice with highfat diet-induced obesity.
High-fat diets enriched in saturated fat may promote an increase in body weight and lead to obesity, liver injury, hepatic insulin resistance, and steatosis [19]. Treatment with increasing amounts of n-3 PUFA, however, seems to reduce the rate of increase in body weight [20]. A study showed that animals on high-fat diets appeared to have an increased ability to store TG, whereas animals fed high fish oil diets (along with consumption of excess calories) did not convert excess lipids into TGs [19]. These data appear to be in agreement with our current study showing that lower TG levels both in serum and in the liver were found in groups that were treated with n-3 PUFAs compared with the HFC group. Further research is required to explain this discrepancy.
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Domain: Biology Medicine<|endoftext|>
Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. Dorsolateral stripe was present in specimens in populations of all three CCSs. Tubercles on the dorsal surface of the tibia were present in CCS Ma and CCS K. Specimens from continental populations of CCS J did not have tubercles on the tibia. Nevertheless, specimens of CCS J from the Alcatrazes Island had tubercles on dorsal surface of the tibia similar to CCS Ma and CCS K (Fig 4). Specimens of CCS J from Alcatrazes Island are also larger (male SVL: 25.6 mm, n = 1; maximum female SVL: 27.3, n = 3) than those of continental populations of CCS J (maximum male SVL: 20.5 mm, n = 5; maximum female SVL: 19.4 mm, n = 9).
We analyzed the advertisement calls of 51 individuals of CCS Ma, J, and K from 10 localities (municipalities of Guapimirim, Itatiaia, Maricá, Petrópolis, and Rio de Janeiro, state of Rio de Janeiro, and Mogi das Cruzes, Nazaré Paulista, Santo André, São Luís do Paraitinga, and Ubatuba, state of São Paulo), totaling 1951 calls. The advertisement calls of the three lineages showed similar structure: a single, nonpulsed note emitted at regular intervals, having or not a frequency upsweep along the note (Fig 5). The dominant frequency coincides with the fundamental harmonic. Although CCS J tended to have calls with shorter durations and higher in pitch than those CCS Ma and CCS K, the acoustic traits analyzed usually overlapped among the them ( Table 4).
Taxonomic decision
Redescription of the holotype. The specimen is completely faded and with both forearms detached from the body, although some traits are still present. (Fig 8). Head longer than wide; HW 34% SVL, HL 39% SVL. Snout rounded in dorsal and ventral views; canthus rostralis indistinct. Loreal region slightly concave. Nostril not protruding, dorsolaterally oriented, rounded. Eye protruding, eye diameter larger than eye to nostril distance. Tympanum distinct, rounded, less than half the eye diameter. Supratympanic fold no apparent. Jaw glands absent. Vocal sac single, internal. Vomerine teeth could not be observed. (DOCX)
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Domain: Biology Medicine
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In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Significant upregulation of genes involved in the regularisation of angiogenesis (Serpinf1, Adamts1 and Spint1) was confirmed in 0P and 00 embryos by real time RT-PCR (data not shown). Global brain development was also preferentially under the control of maternal imprints, although a complementary pattern of parental dependence was observed when specific brain structures were considered ( Figure S3). For example, mid-and hindbrain development and light detection were functional categories more significantly affected by paternal than maternal imprints. These observations correlate with previous reports showing that androgenetic PP cells with a pure paternal contribution tend to preferentially colonize hindbrain regions and in particular the pre-optic area in reconstructed chimeric embryos [27]. Further expression analysis of brain development markers may identify differences in neuroectoderm structures between 0P and 00 embryos. Finally, genes involved in gastrulation, antero/posterior patterning, endoderm development, and later developmental processes (B cell development, forelimb morphogenesis) were not significantly affected by maternal or paternal imprints.
The affected biological processes point to defective placentation as the main consequence of a lack of maternal germline imprints and the cause of death of 0P and 00 embryos at mid-gestation. This complements previous studies that have established the importance of genomic imprinting for placentation on a gene-bygene basis and at later stages of development [28]. Moreover, we show that paternal imprints regulate a large number of transcripts at 8.5dpc, but their cumulative effects do not strongly impact on functions that are vital for the early embryo.
The results of the GO overrepresentation analysis pointed to specific gene families being regulated by imprints of maternal origin. For example, the acid organic transport GO category includes Overrepresentation analysis results for Gene Ontology (GO) categories. The analysis was carried out separately and independently for two different scoring schemes. Essentially, the ''maternal'' scheme assigned a non-zero score if the gene's expression pattern across the MP, 0P and 00 samples was consistent with the gene's expression being affected by maternal but not paternal germline imprints (red bars). Analogously, the ''paternal'' scoring scheme gave non-zero scores to genes that appeared affected by paternal but not maternal germline imprints (blue bars). A, p-value distribution across all GO categories for genes affected by maternal versus paternal imprints. P-values below 0.1 were considered as significant, greater values do numerous solute-linked carrier (Slc) genes. We observed that 100 of 299 of Slc genes present on the microarray were either up-or downregulated in both 00 and 0P embryos. Differential expression of numerous Slc genes was previously observed in a microarray study of non-cultured 0P material including pooled embryos and visceral yolk sacs [29]. Slc transporters modulate soluble molecule availability in a variety of physiological contexts, including the regulation of maternal-fetal transfers, and three Slc genes are in fact known to be maternally imprinted. To determine whether the abnormally expressed Slc genes were directly or indirectly controlled by maternal germline imprints, we analyzed the allelic expression of 25 of these genes that carried informative single nucleotide polymorphisms in reciprocal Mus musculus x Mus musculus castaneus F1 hybrid crosses. None were subject to parent-specific monoallelic expression in 8.5dpc conceptuses (Table S2). This indicates that a third of all Slc genes expressed in early mouse embryos may be downstream targets of maternally imprinted genes.
In summary, these results underline the significant direct and indirect effects that maternal imprints have on the transcriptome of the early embryo, converging towards the vital regulation of genes related to the establishment of the maternal-fetal interface. In the maternal case for example, 470 probes sets detected a decrease of expression in the 0P and 00 samples to 80% (-0.322 log2-ratio) or less relative to the MP sample.
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Domain: Biology Medicine<|endoftext|>Sources of heterogeneity in human monocyte subsets
Highlights • Variation in monocyte phenotype is explored using the markers CD14, CD16, HLA-DR, CX3CR1 and CCR2.• The CD14++CD16+ monocytes exhibit a spectrum of markers dependent on location within the gate.• Monocyte phenotype varies dependent on genetic background and history of exposure to infection.• Processing technique for PBMC purification does not lead to changes in monocyte subsets.• Processing technique for purification can alter intensity but not pattern of marker expression.
Introduction
Peripheral blood monocytes, which represent around 10% of circulating leukocytes in humans, are recognized as the largest pool of circulating progenitor cells and form a vital part of the immune system [1,2]. haematobium
In order to determine if any arising differences between the Caucasian and African participants were due to undetected schistosome or Plasmodium parasite infection (current or previous) or parasite-unrelated mechanisms such as genetic differences, serological assays were conducted to determine parasite exposure history. Enzyme linked immunosorbent assay (ELISA) was used to measure antigen-specific antibodies to malaria schizont (IgG and IgM) and schistosome adult worm (IgG4, IgM, IgE) in the serum. Lyophilized soluble S. haematobium adult worms (SWAP) was obtained from the Theodor Bilharz Institute (Giza, Egypt) and reconstituted as recommended by the manufacturer. Schizont extract was a kind gift from David Cavanagh (University of Edinburgh, UK). ELISAs were performed as reported elsewhere [27,28], and all ELISAs were performed in duplicate on the same day with positive and negative controls on each plate.
Statistical analysis
All statistical analyses were conducted using the statistical package SPSS version 19. Parametric tests were used when assumptions of parametric tests were met, otherwise non-parametric tests were used [29]. When using parametric tests data were transformed using appropriate transformations: surface marker expression (measured as MFI) was log transformed (log 10 (x + 1)), proportions of subsets were arcsine square root transformed, and antibody responses were square root transformed. In parametric models age was taken as a continuous variable, sex (male/female) and donor ethnicity (African/Caucasian) were categorical.
To test the hypothesis that the whole monocyte population is composed of a continuum of 'subsets' consisting of distinct phenotypic profiles, differences in expression of surface markers were analyzed using a one way analysis of variance (ANOVA) with subset as a grouping variable and post hoc tests used to determine significant differences between adjacent subsets. Differences in the proportion of each subset were analyzed using an arcsine square root transformation and a one way ANOVA using type I sequential sums of squares in a similar manner as discussed. When sample size and assumptions did not allow, the Kruskal-Wallis test was used to test for differences between surface marker expression and subset proportion.
In order to investigate the effects of cryopreservation on monocyte phenotype with respect to changes in proportions of subsets and expression of phenotypic markers, the Wilcoxon Signed Ranks Test was used with processing method (fresh PBMCs vs. cryopreserved PBMCs) as grouping variable. Differences in intensity of surface marker expression between subsets were determined by MANOVA, allowing for sex and age using type I sequential sums of squares.
The effects of exposure history to parasitic infection on monocyte subsets were tested by ANOVA, allowing for sex (categorical) and age (continuous) using type I sequential sums of squares. Differences in positive antibody responses to parasite antigen between populations were tested using the Chi-squared test for association after categorizing responses into positive (OD > 0) or negative (OD = 0). Significant p values are reported as p ≤ 0.05 unless otherwise indicated.
Discrimination of MHC II positive monocytes
Monocytes represent a population of MHCII cells that express varying levels of both CD14 and CD16 surface markers. Fig. 1 illustrates the gating strategy used that excludes non-MHCII, CD16 positive NK cells, but includes HLA-DR and CD14 positive monocytes. The commonly observed 'banana' shape that is seen with this cell population and the lack of clustering within the double positive population is demonstrated in Fig. 1C.
Different gating strategies give different phenotypic profile patterns
There is currently no consensus on the best gating strategy of monocytes with at least three different methods published that involve not only different markers of definition [4,30,31] but also different numbers of subsets [32][33][34]. Thus we investigated if there were differences in the basic phenotypic characteristics of monocytes based on different gating strategies according to CD14 and CD16 MFI. Importantly our study indicates that conformity across research groups in gating of these subsets is necessary in order to compare studies.
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Domain: Biology Medicine
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Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. trivittatus as distinct species, maintaining Cochran's [26] and Heyer's [18] decision that Leptodactylus trivittatus is a junior synonym of Adenomera marmorata.
Discussion
We used an integrative approach to test the hypothesis that the CCS Ma, CCS J and CCS K might correspond to different species. We used molecular phylogeny and raised considerably previous sampling of morphology and call characters. Our phenotypic data did not support the three lineages as three different species. Despite the genetic distance and reciprocal monophyly, these candidate species are not acoustically or morphologically diagnosable from each other.
We recovered the same topology recovered by Fouquet et al. [7] regarding CCSs Ma, J and K using an extended sampling. The sole difference is the higher support values of the clade "CCS Ma + CCS J" (0.94 in [7] vs. 1.00 our results). The uncorrected p-distances among the three CCSs were high for both partial 16S and COI (Table 3). Fouquet et al. [68] suggested a mean distance of 3% on 16S to identify Neotropical anuran species and Vences et al. [69] found 10-14% of genetic divergence in mantellid frog species for COI. Even though the genetic divergence among the three lineages of A. marmorata is higher than the genetic distances between A. bokermanni, A. engelsi, and A. nana, it is known that genetic limits within a given genus may be very different (e.g. [69][70][71]). Fouquet et al. [7] tried to address this problem by taking as threshold the genetic distance between the two nominal species recovered as sister taxa in their study (A. engelsi and A. (DOCX)
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Domain: Biology Medicine<|endoftext|>Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions*
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. B. C. and R. M. W. performed the investigation; S. T. E. and R. G. S. reviewed and edited the manuscript; J. B. C. and R. G. S. performed data visualization; and R. G. was responsible for the funding acquisition.
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Domain: Biology Medicine<|endoftext|>Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. 2B, lower panel).
Next the role of cPKC in the agonist-induced 5-HT 2A R sequestration was evaluated. Pretreatment of cells with the cPKC inhibitor Gö6976 prevented sequestration of 5-HT 2A receptors (Fig. 3A, PRE). It should be noted that following treatment with Gö6976 5-HT 2A receptors were detected in vesicular structures close to the plasma membrane that colocalized with the early endosomal marker EEA1 (Fig. 3A). In addition, to quantitate the amount of internalized receptor, a cleavable biotin recycling assay was performed. It was observed that after 2 h of 5-HT treatment, a significant amount of the receptor was internalized. Pretreatment of cells with Gö6976 only moderately decreased the amount of internalized receptor (Fig. 3B).
Thus, cPKC appears to be required for sequestration of receptors and partially involved in internalization but not absolutely required for the latter.
To determine whether activation of PKC is sufficient to induce sequestration of 5-HT 2A R, cells were treated with the phorbol ester PMA. This treatment caused a robust translocation of both PKCII and 5-HT 2A R receptors to the pericentrion (Fig. 3C). Thus, activation of PKC is necessary and sufficient to induce the sequestration of GPCRs.
Next the effects of persistent ligand action on cPKC activity were determined by evaluating the phosphorylation of PKC␣/ II on Thr-638/641. The results demonstrated that treatment of C6 and HEK293 cells with agonist caused an increase in the phosphorylation of Thr-638/641 that persisted for up to 2 h of treatment (Fig. 3D), thus demonstrating sustained activation of cPKC.
Importantly treatment of cells with the cPKC inhibitor following ligand-induced sequestration (Fig. 3A, POST) also caused dispersal of the sequestered 5-HT 2A receptors. Thus, sustained activity of cPKC is required for maintaining sequestration of receptors.
To further elucidate the role of cPKC phosphorylation in 5-HT 2A R sequestration, PKCII autophosphorylation sites (Thr-641 and Ser-660) were mutated to alanine, thus generating a double alanine (DA) mutant of PKCII. 5-HT stimulation of cells co-transfected with 5-HT 2A R and WT PKCII caused perinuclear sequestration of both 5-HT 2A R and PKC (Fig. 3E). However, when cells were co-transfected with the 5-HT 2A receptor and the DA mutant of PKCII, neither PKC nor the receptor was sequestered to the perinuclear region, although the DA mutant did translocate to the plasma membrane. When DA-PKCII was expressed alone it showed a cytoplasmic distribution, typically observed for PKC in unstimulated cells (Fig. Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
To further explore the potential signaling pathways associated with mammalian Eag, we set forth to identify novel binding partners of rEag1 channels in the brain. Input volume was 5% of that of the cell lysates for pull-down. doi:10.1371/journal.pone.0041203.g001 14-3-3h Interacts with rEag1 PLoS ONE | www.plosone.org at 4uC overnight. The bead-protein complexes were then washed with buffer A [(in mM) 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO 3 , 20 Tris-HCl, pH 7.5, plus 1 PMSF, 1 Na 3 VO 4 , 1 NaF, 1 b-glycerophosphate] (with and without 1% Triton X-100), and the proteins were eluted by boiling for 5 min in the Laemmli sample buffer. In vitro protein translation was performed by using the TNT transcription-translation system (Promega).
Cell culture and transient transfection
Dissociated hippocampal culture and HEK293 cells stably expressing rEag1 were prepared as described previously [16]. HEK293 and HEK293T cells were maintained in DMEM (Invitrogen) supplemented with 2 mM L-glutamine, 100 units/ ml penicillin/streptomycin, and 10% (v/v) fetal bovine serum (Hyclone). One day before transfection, HEK cells were grown on poly-lysine-coated coverslips. DNA transfection was performed by using the Lipofectamine 2000 reagent (Invitrogen). (Left panel) rEag1/rEag2 was co-expressed with an empty vector (2) or myc-tagged 14-3-3h (+) in HEK293T cells. Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3h are highlighted with arrow and arrowhead, respectively. (Right panel) Cell lysates from myc-14-3-3h only or co-expression of rEag1 and myc-14-3-3h were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. (B) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3h in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 mM okadaic acid or staurosporine. (Upper panel) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. b-actin was run as a loading control. (Lower panel) Cell lysates were immunoprecipitated (IP) by using the anti-myc antibody, followed by immunoblotting (WB) with the anti-myc or the anti-rEag1 antibody. (C) Quantification of (upper panel) the Akt phosphorylation level (pAkt/Akt) and (lower panel) the co-immunoprecipitation (CO-IP) efficiency of 14-3-3h and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. (TIF)
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Domain: Biology Medicine
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In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. They have also a higher CpG density than L promoters in the human genome, which we show, results from a higher resistance to CpG loss during mammalian evolution. This is coherent with the functional significance of DNA methylation at ICRs in controlling gene expression, while the methylation state of L promoters does not affect the transcription level of associated genes [16]. Although the underlying mechanisms have not been identified, protection against CpG loss at paternal ICRs could result from increased efficiency of T/G mismatch repair, or from reduced deamination frequency of methylated cytosines, entailed for example by local DNA structure. In this regard, replication and transcription generate ssDNA, in which cytosines residues deaminate much more rapidly than in dsDNA [38]: relative localization of replication origins or transcription start sites in intergenic paternal ICRs versus L promoters may result in different CpG loss rate between these two sequence categories. Independently of the parental origin, paternal and maternal ICRs also accumulate new CpG sites during evolution, gaining more CpGs than nonimprinted HI promoters. Imprinted chromosomal regions have unusually high rates of meiotic recombination compared to the rest of the human genome [39,40]. This property could drive the accumulation of CpG sites at ICRs during meiotic repair through biased gene conversion, a process that favors the fixation of AT to GC mutations [41]. Whichever process acts to conserve or create CpG sites in ICRs versus the rest of the genome, it appears to have Ambiore-estimated branch lengths for singleton branches (human, mouse), for the respective sub-tree (euarchonta, glires) or for the entire tree (euarchontoglires). Sequence categories are: P = paternal ICRs, M = maternal ICRs, L = Low CpG-content promoters, HI = High and Intermediate CpG-content promoters. Error bars are 95% confidence intervals. Significant (non-overlapping 95% confidence intervals) changes of interest are labelled with asterisks. Paternal ICRs present with the most evolutionary change, compared to all other sequence types and to maternal ICRs in particular. The values for human and mouse are two orders of magnitude lower than for euarchontoglires, euarchonta and glires. Hence, the use of two y-axis scales (left versus right). B, Rates of substitutions occurring at CpG dinucleotides in euarchontoglires. The estimated substitution rates are relative to each category's overall rate of evolution, e.g., the fact that paternal ICRs are fast evolving intergenic regions, while all other categories are promoter-associated, has been normalized for. Nevertheless, the rate of CpG-loss by deamination at paternal ICRs is higher than for maternal ICRs. Maternal ICRs loose CpGs at the same pace as HI promoters but gain CpGs at a faster rate. doi:10.1371/journal.pgen.1001214.g006 been insufficient in the long term to counteract the hypermutagenic environment of the male germline. Only three functional paternal ICRs have been identified in mouse and genetic manipulation of paternally imprinted expression suggests that this may represent the tally numerate of all developmentally important ICRs controlled by paternal methylation [20]. In the maternal case for example, 470 probes sets detected a decrease of expression in the 0P and 00 samples to 80% (-0.322 log2-ratio) or less relative to the MP sample.
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Domain: Biology Medicine<|endoftext|>DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice
Background Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. Results presented here were from three independent experiments. respectively [27]. Higher IgG 1 levels in this research indicated that TgDPA induced mainly T H 2 responses. Immunoglobulins IgA, IgM and IgE were reported to participate in the immunological responses against T. gondii infection. However, less attention has been placed on these immunoglobulins during vaccination trials against T. gondii [28,29]. IgA is an important immunoglobulin to act on neutralization of toxins and pathogenic microbes, beside regulating interaction between specific receptors and immune mediators [30,31]. With specific relation to T. gondii infection, IgM was reported to enhance the phagocytic capacity of neutrophils and activate the complement cascade which might result in killing of T. gondii as well as reducing the spread of T. gondii by blocking cell invasion [32][33][34]. In our research, high titers of IgA and IgM were detected in the immunized group. This suggested that both IgA and IgM played roles in the protective responses induced by the TgDPA.
IgE was recognized during the infection of toxoplasmosis [35,36]. However, our data revealed no significant traces of this immunoglobulin after vaccination with TgDPA. Our results were consistent with previous studies [37].
IFN-γ is a key cytokine of T H 1 type immune response and is known to play an important role in resistance against T. gondii. This cytokine supports many immunological mechanisms, interferes with survival and multiplication of intracellular pathogenic organisms and leads to the eradication of pathogenic organisms [38][39][40][41][42]. Remarkable levels of IFN-γ were detected in this study. This finding may encourage more investigations in evaluating the immunogenicity of DPA based vaccines against Toxoplasmosis.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. In general, the data suggest that EPEC T3S effectors modulate host signaling via phosphorylation of a wide range of cellular processes. Besides previously unrecognized host components in bacterial pathogenesis such as EPHA2 and TPD52, we also identified signaling hubs that are known targets for EPEC virulence, such as TRAF2 (81,82), but that had not been shown to be impacted by phosphorylation upon EPEC infection.
EPEC-mediated host cytoskeletal reorganization is the best studied process in EPEC pathogenesis (8). However, the role of phosphorylation in this process is not well understood, and research thus far has mainly focused on Tyr-phosphorylation of the bacterial effector Tir and components of the classical NCK-N-WASP-ARP2/3 pathway (10,12). The data presented here provide additional mechanistic insights into serine and threonine phosphorylation-mediated cytoskeletal regulation during EPEC infection. We observed that targets within the cytoskeleton were the cellular components most impacted by T3S effectors. Actin-depolymerization factor and cofilin had increased phosphorylation at Ser-3 early in infection. Cofilin is recruited to pedestals where it presumably modulates actinfilament dynamics (67). Ser-3 phosphorylation by LIMK has been shown to inhibit the actin-filament depolymerizing activity of cofilin suggesting a T3S-effector mediated stabilization of actin filaments via this pathway early during infection (68). An earlier model proposed that cofilin inactivation via the RhoA-ROCK pathway stabilizes the actin microfilaments in transient filopodia that form and disappear within 30 min of infection (83,84). We observed an increased abundance of phosphorylated cofilin up to 1.5 h, which indicates an additional role beyond filopodia stabilization and possibly extends to actin filament stabilization within pedestals. Another regulator of actin filament dynamics, HSP27, showed increased phosphorylation at Ser-82 in a T3SS-dependent manner during EPEC infection. Phosphorylation of Ser-82, one of three sites targeted by MAPKAP2, promotes actin filament formation (85,86). This indicates that, in addition to cofilin phosphorylation, HSP27 phosphorylation might also function as a Role of SEPT9 phosphorylation in EPEC pathogenesis. A, Expression test. Western blot analysis of HeLa cells that were transfected with N-terminally FLAG-tagged SEPT9 constructs including wild-type (WT) and mutant SEPT9 constructs that carried a replacement of Ser-30 with either alanine (S30A) or glutamate (S30E). Twenty-four hour post-transfection, 13 g of HeLa cell lysate was separated using 12% SDS-PAGE and subsequently probed with an antiFLAG M2 antibody (mock, empty vector-transfected control). B, Cell adherence assay of FLAG-SEPT9 construct-transfected and EPEC WT-or ⌬escN-infected HeLa cells to determine the role of SEPT9 phosphorylation in bacterial adherence. HeLa cells were transfected with plasmids encoding the proteins that are indicated in the legend. Twenty-four hour post-transfection, cells were infected for 2 h, washed eight times with PBSϩ/ϩ, and cfu counts were determined. The cfu counts were normalized relative to the average cfu count deriving from EPEC ⌬escN-infected and empty FLAGvector control-transfected HeLa cells (n ϭ 3; ϩS. We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine
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Sources of heterogeneity in human monocyte subsets
Highlights • Variation in monocyte phenotype is explored using the markers CD14, CD16, HLA-DR, CX3CR1 and CCR2.• The CD14++CD16+ monocytes exhibit a spectrum of markers dependent on location within the gate.• Monocyte phenotype varies dependent on genetic background and history of exposure to infection.• Processing technique for PBMC purification does not lead to changes in monocyte subsets.• Processing technique for purification can alter intensity but not pattern of marker expression.
Introduction
Peripheral blood monocytes, which represent around 10% of circulating leukocytes in humans, are recognized as the largest pool of circulating progenitor cells and form a vital part of the immune system [1,2]. The enormous heterogeneity in human monocyte size, morphology, phagocytic function and cell adhesion was first described in 1989 [3] and was quickly followed by multiple attempts to discriminate monocyte subtypes. Recently new nomenclature was suggested by an expert panel in Brescia, Italy to define three subsets according to expression of CD14 and CD16 [4]. The major subset consists of CD14highCD16negative monocytes (CD14++CD16−), while the CD16 expressing monocytes are usually divided into a CD14highCD16low (CD14++CD16+) and a CD14lowCD16high (CD14+CD16++) subset. These groupings can identify monocytes that differ in surface expression of chemokine markers, major histocompatibility complex (MHC) class II expression and in their capacity to produce cytokines and phagocytose microbial particles [1,[5][6][7][8]. However, while there have been some in-depth genetic and proteomic analyses of monocyte functions and cell markers [1,6,9], there is still no universally accepted demarcation of these subsets based on phenotypic markers [9]. Furthermore, there is no visible clustering of the cell subsets based on the CD14 and CD16 surface markers, instead the two markers form a spectrum of expression levels potentially contributing to variation between experiments [4,7,10]. Differential expression of chemokine and scavenger receptors indicates a functional Rural 21 potential in terms of trafficking to sites of infection and inflammation. Indeed, monocyte migration and trafficking has been observed to vary between subsets based on expression of CCR2 and CX3CR1 [11]. Another feature of monocytes is their ubiquitous expression of the MHC class II surface receptor, HLA-DR, which is frequently used to distinguish between CD16 expressing monocytes and CD16 expressing NK cells [12]. As a receptor that is involved in antigen presentation [9], it is often considered an activation marker [13][14][15] and indicates functional differences for the monocyte subsets as well as subset activation status [12]. Thus far, the majority of human monocyte studies have taken place using volunteers of Caucasian background and in high income countries where pathologies arising from non-communicable diseases such as atherosclerosis, liver cirrhosis and asthma dominate [16]. This means that, despite the demonstration of the importance of monocytes in experimental models of parasitic diseases [2,17,18], comparatively little is known about the nature, phenotype and development of monocytes in people exposed to tropical infectious diseases. Furthermore the majority of studies investigating monocyte phenotype and function use whole blood or fresh PBMCs rather than cryopreserved peripheral blood mononuclear cells (PBMCs). Cryopreservation of PBMCs is an indispensable tool for longitudinal clinical studies as well as during fieldwork when samples have to be stored and transported from the collection point to a laboratory. Furthermore, the capability to retrospectively analyze specimens from the same patient allows analysis of large sample populations, monitoring of clinical status over time or after treatment and improves accuracy while reducing within-patient as well as interassay variability [19,20]. To date, studies determining the effects of cryopreservation on PBMCs have focused on cell compartment changes [21] or maturation pathways [22], but no studies have been conducted on the effects of cryopreservation on the cell phenotype which is central to the function of the monocytes.
In this study our aims were (1) to determine changes in expression levels of cell surface markers occurring within the monocyte cell population dependent on CD14 and CD16 expression intensity, (2) to assess the stability of these markers during processes involved in freezing and storage, and finally, (3) to determine if differences occur in the proportion and phenotype of monocytes in the different sub-populations between Caucasian individuals who have been exposed to a typical western lifestyle, and African individuals who are lifelong residents of a rural helminth endemic area.
Ethical statement
Study populations
To address the different questions, three different cohorts were used, which are described in Table 1. For the purposes of phenotyping monocytes for cell surface expression patterns of the markers CCR2, CX3CR1 and HLA-DR, a cohort of 62 individuals living in a rural area where Schistosoma haematobium is prevalent was used. All participants were from the Murehwa district in north eastern Zimbabwe. All individuals recruited into each study were S. haematobium and co-infection negative and had never received anti-helminthic treatment. Importantly our study indicates that conformity across research groups in gating of these subsets is necessary in order to compare studies.
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Domain: Biology Medicine
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Protective effects of various ratios of DHA/EPA supplementation on high-fat diet-induced liver damage in mice
Background A sedentary lifestyle and poor diet are risk factors for the progression of non-alcoholic fatty liver disease. The oil red O staining patterns showed orange lipid droplets and blue nuclei, which were smaller and fewer in number in the DHA/EPA (2:1, 1:1, and 1:2) groups; while obvious numerous orange-red lipid droplets were observed in HFC-treated mice, nearly none were observed in the NC group (Fig. 1). Moreover, fewer lipid droplets were observed in mouse groups treated with DHA/EPA (ratios of 2:1, 1:1, and 1:2) compared to the group treated with DHA.
Effects of various ratios of DHA/EPA with an n-6/n-3 ratio of 4:1 on body weight, serum, and liver lipids Compared to the HFC group, significant decreases (p < 0.05) in serum TC, TG, LDL-C levels, and liver TC and TG levels were observed in 12-week n-3 PUFA supplementation groups ( Table 3). The growth curve of mice revealed that the body weight increased slower with n-3 PUFA supplementation than with HFC treatment (Fig. 2), indicating the inhibitory effect of n-3 PUFA supplementation with DHA and different DHA/ EPA ratios (2:1, 1:1, and 1:2) in the mice. Moreover, mice treated with a DHA/EPA ratio of 2:1 and 1:2 showed a reduced hepatic organ coefficient with a significant difference of p < 0.01, while the statistical differences of reduction in DHA and DHA/EPA 1:1 groups were p < 0.05 compared to those in the HFC group (Table 3).
Mice treated with a DHA/EPA ratio of 1:2 showed a more appreciable decrease in serum TC, TG, and LDL-C levels, whereas mice treated with the DHA/EPA ratio of 2:1 showed lowered liver TC and TG levels to a greater degree. However, mice treated with various ratios of DHA/EPA (2:1, 1:1, and 1:2) revealed no reduction in serum TC, TG, or LDL-C levels or liver TC and TG levels in comparison with those in the DHA group. Nevertheless, as compared to the levels in the HFC group, serum HDL-C levels in DHA and DHA/EPA (2:1, 1:1, and 1:2) groups were significantly higher (p < 0.01). Furthermore, serum HDL-C levels increased more significantly in the DHA/EPA 1:2 group than in other DHA/EPA groups (p < 0.01), but the increase was not remarkable in comparison to that in the DHA group.
Effects of various ratios of DHA/EPA with an n-6/n-3 ratio of 4:1 on liver damage Levels of serum ALT, AST, GSH, SOD, and MDA, and liver GSH, SOD, and MDA are presented in Fig. 3. The results show that treatment with n-3 PUFAs lowered the levels of serum ALT and AST and both serum and liver MDA production, and raised the levels of GSH and SOD both in serum and liver in comparison with those in the HFC group. Nonetheless, no significant differences were found in the levels of any of these biomarkers between the DHA and DHA/EPA groups. Nevertheless, supplementation with a DHA/EPA ratio of 1:2 resulted in greater reduction in serum ALT, AST, and MDA levels and increase of serum GSH levels among the DHA/EPA groups; however, this increase was not higher than that observed in DHA groups. Collectively, supplementation with a DHA/EPA ratio of 2:1 was the most effective among the three DHA/EPA groups at enhancing SOD levels both in serum and liver, but these levels were not higher than those in the DHA group. Particularly, mice treated at a DHA/EPA ratio of 1:1 showed increased liver GSH levels as compared to the DHA group (2.828 ± 0.279 vs. 2.612 ± 0.546 mg/g prot).
Effects of various ratios of DHA/EPA with an n-6/n-3 ratio of 4:1 on lipid metabolism genes Gene expression variation of the enzymes and transcription factors of lipid metabolism was measured by realtime PCR as shown in Fig. 4. Compared to the HFC group, 43%, 27%, 46%, and 27% increases in AMPK Effects of various ratios of DHA/EPA with an n-6/n-3 ratio of 4:1 on the inflammatory response The protein expression levels of the three main subunits of AP-1 (c-Jun, c-Fos, and Fra1) were detected by western blot (Fig. 5a-d). The protein expression levels of c-Jun and c-Fos were significantly lower in the DHA/ EPA groups compared to the HFC group (p < 0.01), meanwhile supplementation with a DHA/EPA ratio of 1:1 revealed greater reductions in the expression levels of these two proteins in contrast to the DHA group. Further research is required to explain this discrepancy.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. As before, no significant differences were noted in EPEC ⌬escN-infected cells.
Collectively, these SEPT9 studies validate and functionally characterize the phosphoproteomic data. These results suggest that SEPT9 is a novel key host player in EPEC pathogenesis controlling the pathogen's attachment to the host cell surface. Furthermore, T3SS-mediated phosphorylation of Ser-30 in SEPT9 is critical for efficient bacterial adherence. DISCUSSION Alterations in host protein phosphorylation following infection can provide substantial insight into the molecular mechanisms by which human pathogens exploit host cell signaling to cause disease. Here we present the first study that examines the global impact of an extracellular diarrheagenic bacterial pathogen on the host phosphoproteome.
Consistent with this hypothesis we observed a less profound impact on the host phosphoproteome by EPEC compared with earlier phosphoproteomic profiling studies of Shigella or Salmonella (19,20). Nevertheless, our unbiased and comprehensive phosphoproteome analysis revealed that EPEC still triggers extensive changes in the host phosphoproteome far beyond what was established from targeted protein studies.
We observed a time-dependent increase of regulated phosphosites, which correlated to increased pathogen exposure. As expected, the impact on host phosphorylation was more profound in EPEC WT-infected cells when compared with T3SS-deficient mutant-infected cells. The observed T3SS-dependent alterations mainly affected cellular components that are known targets of pathogens: e.g. MAPKs have been implicated as an important signaling hub during host infection with EPEC and various other bacterial pathogens, and are often targeted by translocated bacterial virulence factors (16,48,(77)(78)(79)(80). Based on kinase enrichment, motif and T3SSregulated phosphosite analysis as well as the identification of a T3SS-regulated MAPK activation loop phosphopeptide, our results emphasize the central role of MAPK during EPEC infection. In general, the data suggest that EPEC T3S effectors modulate host signaling via phosphorylation of a wide range of cellular processes. Besides previously unrecognized host components in bacterial pathogenesis such as EPHA2 and TPD52, we also identified signaling hubs that are known targets for EPEC virulence, such as TRAF2 (81,82), but that had not been shown to be impacted by phosphorylation upon EPEC infection.
EPEC-mediated host cytoskeletal reorganization is the best studied process in EPEC pathogenesis (8). However, the role of phosphorylation in this process is not well understood, and research thus far has mainly focused on Tyr-phosphorylation of the bacterial effector Tir and components of the classical NCK-N-WASP-ARP2/3 pathway (10,12). The data presented here provide additional mechanistic insights into serine and threonine phosphorylation-mediated cytoskeletal regulation during EPEC infection. We observed that targets within the cytoskeleton were the cellular components most impacted by T3S effectors. Actin-depolymerization factor and cofilin had increased phosphorylation at Ser-3 early in infection. Cofilin is recruited to pedestals where it presumably modulates actinfilament dynamics (67). Ser-3 phosphorylation by LIMK has been shown to inhibit the actin-filament depolymerizing activity of cofilin suggesting a T3S-effector mediated stabilization of actin filaments via this pathway early during infection (68). An earlier model proposed that cofilin inactivation via the RhoA-ROCK pathway stabilizes the actin microfilaments in transient filopodia that form and disappear within 30 min of infection (83,84). We observed an increased abundance of phosphorylated cofilin up to 1.5 h, which indicates an additional role beyond filopodia stabilization and possibly extends to actin filament stabilization within pedestals. We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine
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Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. Choanae moderate, rounded. Tongue elongate; lateral and posterior margins free. Vocal slits elongate, parallel to jaw, extending from posterior portion to the first third of the mouth. Arms poorly preserved, detached from the body. Toes free; toe tips II, III, and IV expanded (toe tips I and V unexpanded). Inner and outer metatarsal tubercles distinct and slightly protruding, ovoid, approximately the same size. Due to the degraded condition of the feet, toe formula could not be observed. Subarticular tubercles rounded and well-developed. Posterior members small and robust; thigh length slightly smaller than shank length (THL 40% SVL and SHL 44% SVL). Metatarsal fold absent. Tarsal fold present, poorly developed, extending from the base of outer metatarsal tubercle to the tibiotarsal junction. Ventral surface of tarsus with few tubercles. Ventral surface of feet with innumerous small and distinct tubercles. Dorsum smooth; anal glands present only on left side of the body, rounded.
Measurements of the Holotype. SVL 22.2 mm; HL 8.7 mm; HW 7.5 mm; ED 2.1 mm; TD 1.0 mm; END 1.3 mm; IND 1.4 mm; THL 8.8 mm; SHL 9.7 mm; TAL 5.3 mm. Measures of anterior members and feet could not be taken due to specimen condition.
Color and Morphological variation. In life, general coloration of body grey; hind limbs with dark brown transversal stripes, posterior surface of the thigh with irregularly distributed melanophores on cream background. Heels with or without orangeade or reddish blotch. Venter cream, gular region with a few melanophore spots concentrated laterally. Lateral surface of the body with dark brown or black scattered blotches. (DOCX)
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Domain: Biology Medicine<|endoftext|>Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions*
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. The role of ERK1/2 activation and subsequent regulation of PGC-1␣ and downstream targets involved in mitochondrial homeostasis and dysfunction remains limited.
Physiological and pathological stimuli, such as exercise, caloric restriction, hypoxia, sepsis, and IR, are known to alter PGC-1␣ expression (9,14). IR-induced renal injury rapidly suppresses MB, PGC-1␣, and its direct downstream targets at the transcriptional and protein level (10,11,13). However, the mechanism(s) by which PGC-1␣ is transcriptionally suppressed after injury has not been determined. Therefore, we examined the role of ERK1/2 in renal PGC-1␣ transcription under physiological and pathophysiological conditions in primary cultures of RPTC and in the renal cortex of mice. We determined that ERK1/2 regulates PGC-1␣ in RPTC and in the renal cortex of mice at a physiological level through phosphorylation of the transcription factors forkhead box O3/1 (FOXO3a/1). EGFR was found to be the upstream activator of ERK1/2 under basal conditions, negatively regulating PGC-1␣ transcription. Finally, under the pathophysiological condition of IR-induced AKI in mice, ERK1/2 activation was responsible for the initial decrease in PGC-1␣ mRNA expression and the decrease in kidney function as measured by serum creatinine. By inhibiting ERK1/2 activation, we attenuated the early decrease in PGC-1␣ and prevented the decrease in kidney function. This research is significant because PGC-1␣ is the key regulator for MB, and by sustaining mitochondrial homeostasis, ERK1/2 inhibition may be a potential therapeutic to prevent further injury and/or increase recovery where early mitochondrial dysfunction is observed. (15,16). RPTC were treated with 0.3, 1, or 10 nM trametinib, and at 10 nM trametinib, ERK1/2 phosphorylation was completely inhibited after 4 h (Fig. 1A). Trametinib (10 nM) completely inhibited ERK1/2 phosphorylation within 10 min and continued to inhibit it for 24 h (data not shown) without altering total ERK1/2 (Fig. 1A). PGC-1␣ mRNA expression increased 1.8fold at 1 h and 2.8-fold at 4 h before decreasing to control levels at 24 h after trametinib exposure (Fig. 1B). The increase in PGC-1␣ mRNA was linked to increased nuclear encoded mediators of MB and gene targets of PGC-1␣, including mitochondrial transcription factor A (TFAM), nuclear respiratory factor-1 (NRF1), and NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) at 1, 4 and 24 h after trametinib treatment ( Fig. 1, C-E). These results reveal that ERK1/2 represses PGC-1␣ mRNA expression and its downstream target genes under physiological conditions and that inhibition of ERK1/2 activation results in a rapid induction of PGC-1␣ mRNA and other downstream MB genes.
Other kinases, such as p38 and AKT, are known to target FOXO3a for phosphorylation (20,21); however, p38 and AKT phosphorylation did not change in the presence of trametinib (Fig. 2C).
To exert its transcriptional effects, FOXO3a shuttles between the cytosol and the nucleus (22). was responsible for the funding acquisition.
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. High levels of site-specific RNA editing are found in transcripts that are expressed in the CNS. Since inosines prefer to base-pair with cytosine (C), a guanosine residue is inserted when cDNA is generated. Inosine is read as if it were guanosine by the translational machinery thus recoding the mRNA.
The second type of editing occurs within long dsRNA structures (>100 bp) that are deaminated at multiple sites. It has been demonstrated that up to 50% of the adenosine residues within these long dsRNAs can be deaminated so therefore this type of editing is referred to as hyper or promiscuous editing [48,59]. The deamination of an adenosine within an A-U base pair duplex produces the less stable I-U wobble pair, while deamination within A-C mismatches produces the more stable I-C pairs. These editing events are found within RNA duplex structures formed by inverted Alus and other repetitive elements within the non-coding regions of transcripts such as introns and 3ʹUTR regions as well as within transcribed intragenic regions [60][61][62][63][64][65]. The presence of A-to-I modifications in transcripts encoding Alu elements and other hyperedited transcripts regulate the structure and stability of the dsRNAs and inosinecontaining dsRNAs have been shown to play an essential role in regulating the innate immune response [66,67]. This type of editing will be discussed later.
RNA Editing Resulting in Recoding
When compared to mammals invertebrates have a significantly higher level of editing that results in recoding (for review, see [68]). In octopus, editing of transcripts encoding a potassium channel has been shown to vary depending on the location of the species, being highly edited in cold waters, whereas tropical water species are mostly unedited [69]. This elegant experiment suggests that RNA editing can respond to environmental queues.
The transcript encoding one of the α-amino-3-hydroxy-5-methylisoxasole-4propionate (AMPA) receptor subunit is the best studied mammalian site-specific editing event [70,71]. The AMPA receptor is a glutamate-gated ion channel that mediate fast synaptic transmission at excitatory synapses which are important for neuronal development, long term potentiation and synaptic plasticity. AMPA receptors are activated by L-glutamine which triggers an influx of calcium (Ca 2+ ) into the neuron. The Ca 2+ permeability of the AMAP receptors is dependent on the tetrameric composition of the subunit of the glutamate receptor [72]. AMPA receptors containing the glutamate receptor 2 (GRIA2) subunit exhibit lower Ca 2+ permeability compared to those without it and this is due to the presence of single amino acid residue, arginine (R) in the pore of the channel [73]. When comparing cDNA and genomic sequences encoding the GRIA2 subunit, it was found that the R codon (CGG) in the cDNA was a glutamine (Q) codon (CAG) in the genomic sequence [70]. ADAR2 is the enzyme that catalyzes this critical site specific editing event at this Q/R site [74]. This editing event also regulates the heterotetrameric assembly of the AMPA receptors by retaining the GRIA2 subunits within the endoplasmic reticulum therefore increasing the time required for it to be transported to the synaptic surface [75,76] Editing at this Q/R site is highly efficient >99% in the CNS and it dictates the Ca 2+ permeability of the AMPA receptor. It is therefore is a key determinant in neuronal Ca 2+ homeostasis, as the edited isoform, GRIA2 (R), is impermeable to Ca 2+ .
The early onset epileptic seizures and premature death of both Adar2 null mice and mice heterozygous for an uneditable GRIA2 allele demonstrated the physiological importance of this editing event [77,78]. The Adar2 null phenotype is rescued by generating a transgenic mouse containing the edited GRIA2 allele [71]. In addition to the Q/R site, the GRIA2 transcript is also edited at the R/G site [79]. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine
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Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. Stimulation of cells with 5-HT induced significant co-sequestration of both receptors and CD59, a complement regulatory protein, as well as fluorescent transferrin into the perinuclear region (Fig. 6, A and B). Thus, sustained stimulation of receptors leads to sequestration of recycling molecules. Treatment with cPKC and PLD inhibitors efficiently blocked sequestration of both CD59 and transferrin in response to 5HT, suggesting that agonist-induced cPKC/PLD-dependent 5-HT 2A R sequestration globally affects the intracellular trafficking of recycling membrane components.
5-HT Induces Global Sequestration of Molecules to Perinuclear Recycling Compartment and Can Affect Cellular Fate of
The significance of 5-HT-induced formation of the pericentrion in regulation of trafficking and signaling of other membrane receptors was evaluated next, and for these studies, the effects of 5-HT on the endogenous EGFR were examined because PMA has been shown to regulate the trafficking of EGFR. Treatment with 5-HT led to the simultaneous sequestration of both 5-HT 2A R and the EGFR in the perinuclear region (Fig. 7A). Moreover this translocation occurred in a cPKC-and PLD-dependent manner because it was inhibited by both Gö6976 and 1-butanol (Fig. 7A).
Interestingly it has been shown previously that activation of PKC with PMA diverts internalized EGFR from a degradative to a recycling pathway(s) (21). Thus, we wondered whether the 5-HT 2A R displayed the capacity to regulate the fate of the EGFR. When cells were treated with epidermal growth factor, there was significant loss of the EGFR protein; however, pretreatment with either PMA or 5-HT prevented/ameliorated the loss of the EGFR (Fig. 7B). Importantly the protective effects of PMA and 5-HT were inhibited by 1-butanol (Fig. 7B), whereas the secondary alcohol (2-butanol) did not have any effect (data not shown).
PAR-1 provides an example of a GPCR that is rapidly internalized and degraded following its activation (17,18), and thus, it was chosen for additional study. First we confirmed that treatment with the PAR-1 agonist peptide (SFLLRN) led to significant degradation of PAR-1, whereas PMA caused sequestration of PAR-1 as analyzed by confocal microscopy (Fig. 8A). Most interestingly, however, treatment of cells with 5-HT led to co-sequestration of both receptors in a cPKCand PLD-dependent manner, and when cells were pretreated with 5-HT, SFLLRN did not induce degradation (Fig. 8B). Taken together, these results indicate that sustained stimulation with 5-HT changes the cellular fate of membrane receptors that are normally targeted for degradation.
DISCUSSION
The results from this study reveal dramatic effects of sustained stimulation of cPKC by GPCRs on the trafficking of several proteins leading to their sequestration in the perinuclear region. It is shown that sustained stimulation of G q -coupled GPCRs leads to their sequestration into a subset of the Rab11-positive recycling compartment. This sequestration was cPKC-dependent and required continuous cPKC activation and PLD activity. Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice
Background Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. Measurement of damaged infected cells was an important tool to measure CTL function [74,79,80]. Application of such methods is required in further investigations regarding TgDPA antigen to highlight its role in CTL response stimulation and resistance development against T. gondii infection.
Conclusion
Our study demonstrated that the pTgDPA delivered as a single protein is an antigen with the potential of inducing and regulating significant levels of humoral as well as cellular (T H 1, T H 2 and T H 17) immune responses against acute T. This finding may encourage more investigations in evaluating the immunogenicity of DPA based vaccines against Toxoplasmosis.
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Domain: Biology Medicine<|endoftext|>Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions*
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. S. reviewed and edited the manuscript; J. B. C. and R. G. S. performed data visualization; and R. G. was responsible for the funding acquisition.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. In addition SEPT9 is recruited to ARP2/3-(for Listeria) and ARP2/3-N-WASP-based (for Shigella) actin-tails that cytosolic Listeria and Shigella use for intracellular motility. Moreover SEPT9 is essential for the entrapment of cytosolic Shigella in septin cages, which restrict the actin-based bacterial motility (92). We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. 6D). The abundance of SEPT9 v2 transcripts was considerably lower than v1 and v3 in HeLa cells, as SEPT9 variants v2 remained undetected using a different variant-specific set of primers (data not shown). This suggests that SEPT9(a) and SEPT9(c) represent the predominant SEPT9 isoforms in HeLa cells.
Next we assessed the impact of SEPT9a/c on EPEC interactions with HeLa cells. SEPT9 knockdown by siRNA in HeLa cells successfully repressed the expression of isoforms a-c (Fig. 6E, left panel). Using this approach, we analyzed EPECmediated cytotoxicity as a measure of EPEC virulence. SEPT9 knockdown resulted in significantly reduced LDH-release and cell death of EPEC WT-infected HeLa cells compared with cells transfected with nontargeting control siRNA (Fig. 6E Forty-eight hour post-transfection, cells were lysed and 15 g of HeLa cell lysate was subjected to 12% SDS-PAGE and subsequent Western right panel). No significant difference was observed for EPEC ⌬escN-infected cells, thus confirming our finding that EPECmediated targeting of SEPT9 was T3SS-regulated (Fig. 6E, right panel). We then tested whether the observed impact on host cell death is because of SEPT9 regulation of EPEC adherence to the host cell surface. Knockdown of SEPT9 resulted in a significant decrease of EPEC WT adherence to HeLa cells (Fig. 6F). Consistent with the cytotoxicity results, no significant changes in the number of adherent bacteria were observed for EPEC ⌬escN-infected cells during SEPT9 knockdown. These data strongly indicate that SEPT9 plays an important role in EPEC-host cell interactions and that SEPT9 is involved in T3SS-mediated EPEC adherence to the host cell surface.
We then assessed whether the T3SS-mediated phosphorylation event in the N-terminal SEPT9 serine residue is responsible for SEPT9 involvement in EPEC virulence. We used site-directed mutagenesis to generate FLAG-tagged SEPT9(a) constructs that carried a replacement of Ser-30 by either alanine to prevent phosphorylation (FLAG-SEPT9-S30A) or glutamate to mimic constitutive phosphorylation (FLAG-SEPT9-S30E). The transfection conditions used for subsequent experiments were chosen based on an earlier study to ensure low expression and thus allow for functional integrity and biological relevance of the transiently expressed FLAGtagged SEPT9 constructs ((38), Experimental Procedures). Western blot analysis of HeLa cells transfected with respective constructs confirmed that both mutants and wild-type (FLAG-SEPT9-WT) expressed similar levels of FLAG-tagged SEPT9 (FLAG-SEPT9), indicating that neither Ser-30 substitutions had a negative impact on protein stability (Fig. 7A). To assess whether SEPT9 phosphorylation affects EPEC adherence, we transfected HeLa cells with wild-type or mutant SEPT9 constructs. We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine
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14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
To further explore the potential signaling pathways associated with mammalian Eag, we set forth to identify novel binding partners of rEag1 channels in the brain. Since the rEag1 protein contains a long cytoplasmic C-terminus spanning over 470 amino acids, we generated three more GST fusion proteins that divided the Cterminus into three regions: GST-C1 (amino acids 492-724), GST-C2 (amino acids 723-848), and GST-C3 (amino acids 835-962). GST pull-down assay indicated that GST-C1 displayed the highest 14-3-3h-binding efficiency, virtually identical with that of GST-C0 (Fig. 3B). This observation suggests that the proximal region of the rEag1 C-terminus may harbor a major 14-3-3hinteracting domain. Two structural domains have been defined for this region of the rEag1 protein: the C-linker and the cyclic nucleotide-binding homology domain (CNBHD) [1,26]. We therefore made two additional GST fusion proteins that encoded either the C-linker (GST-C1-1: amino acid 493-560) or the CNBHD (GST-C1-2: amino acids 561-672) region. As depicted in Figure 3C, 14-3-3h preferentially bound to the GST-C1-2 fusion protein, implying that the CNBHD may contribute to the Cterminal interaction of rEag1 with 14-3-3h.
Interaction of 14-3-3h with rEag1 in heterolgous and native cells
To further confirm the foregoing GST pull-down results, we tested the association of 14-3-3h with full-length rEag1 by transiently co-expressing myc-14-3-3h and rEag1 proteins in HEK293T cells. As shown in the left panel of Figure 4A, upon immunoprecipitating myc-14-3-3h with the anti-myc antibody, a significant rEag1 protein signal was detected on the immunoblot. Conversely, myc-14-3-3h was also immunoprecipitated with the anti-rEag1 antibody (Fig. 4A, right panel), indicating that rEag1 indeed co-existed in the same protein complex with 14-3-3h. By contrast, no significant co-immunoprecipitation pattern was found for 14-3-3h and rEag2, an isoform of rEag1 (Fig. 4A, left panel).
In most cases, 14-3-3 proteins bind to targets containing specific phosphoserine or phosphothreonine motifs [27,28], although some non-consensus 14-3-3-binding phosphorylation motifs have been reported as well [29]. Moreover, 14-3-3 may also bind to unphosphorylated ligands [30,31]. Upon analyzing the amino acid sequence of rEag1, we did not find any consensus or putative 14-3-3-binding phosphorylation motif. To further address this issue, we compared the effects between a phosphatase inhibitor (okadaic acid) and a protein kinase inhibitor (staurosporine). The modulatory effects of the two agents on the status of cellular phosphorylation were confirmed by the dramatic change in the phosphorylation state of the protein kinase Akt (Fig. 4B and 4C, upper panels). As illustrated in the lower panels of Figures 4B and 4C, neither okadaic acid nor staurosporine treatments led to noticeable change in the 14-3-3h-binding efficiency of rEag1, consistent with idea that 14-3-3h may interact with rEag1 in a phosphorylation-independent manner.
Given that both 14-3-3h and rEag1 proteins are abundantly expressed in the brain, it is imperative to determine whether the potential interaction of these two proteins can also be verified in neurons. Crude membrane (P2) fractions prepared from rat forebrain homogenates were subject to immunoprecipitation with the anti-14-3-3h antibody, followed by immunoblotting with the anti-rEag1 antibody. As demonstrated in Figure 5A, rEag1 was effectively co-immunoprecipitated with 14-3-3h, suggesting that in the rat brain, endogenous 14-3-3h and rEag1 co-existed in the same protein complex.
Next we asked if 14-3-3h and rEag1 shared overlapping subcellular localization in neurons. (TIF)
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Domain: Biology Medicine<|endoftext|>Sources of heterogeneity in human monocyte subsets
Highlights • Variation in monocyte phenotype is explored using the markers CD14, CD16, HLA-DR, CX3CR1 and CCR2.• The CD14++CD16+ monocytes exhibit a spectrum of markers dependent on location within the gate.• Monocyte phenotype varies dependent on genetic background and history of exposure to infection.• Processing technique for PBMC purification does not lead to changes in monocyte subsets.• Processing technique for purification can alter intensity but not pattern of marker expression.
Introduction
Peripheral blood monocytes, which represent around 10% of circulating leukocytes in humans, are recognized as the largest pool of circulating progenitor cells and form a vital part of the immune system [1,2]. In a second blood draw, fresh PBMCs were purified, and these were stained on the same day as their cryopreserved cells. Cryopreserved cells show a greater proportion of monocytes as a percentage of live gated cells, as shown in Fig. 4A (z = −2.67, p = 0.004). However there were no significant differences in the proportion of subsets as tested by one-way ANOVA and repeated measures as shown in Fig. 4B. Differences in cell surface expression between fresh and cryopreserved monocytes are indicated in Fig. 5. No differences are seen in CD14, HLA-DR or CCR2 expression (Fig. 5A, C and D), but significant differences are seen in almost all subsets for CD16 and CX3CR1 surface expression ( Fig. 5B and E, respectively), with fresh PBMCs showing a higher MFI for all subsets in both markers.
African and Caucasian donors show differences in proportions of monocyte subsets
As the majority of studies investigating monocytes have taken place in high income areas, we were interested in whether the phenotypic patterns we have characterized were a feature of the study population or if they can be transferred across populations. We therefore undertook an investigation to phenotype monocytes from donors of different ethnicities. Fig. 6A-C illustrates the differences seen in flow analysis between (A) rural African monocytes (B) urban African monocytes and (C) Caucasian monocytes. There is a stark difference in the proportion of CD16 and CD14 expressing subsets between the ethnicities. Fig. 6D shows the proportions of the five subsets in the whole African and Caucasian populations that we sampled. Caucasians are exhibiting a significantly greater proportion of regCD14 cells compared to rural Africans (Fig. 6D, 1), while monocytes from Africans have a significantly greater proportion of all other subsets except dpCD14 (Fig. 6D, 2-5). Fig. Importantly our study indicates that conformity across research groups in gating of these subsets is necessary in order to compare studies.
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Domain: Biology Medicine<|endoftext|>
Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. On the other hand, we identified only CCS K in the urban vicinities of Ubatuba. We should expect that, if these lineages represent indeed, distinct species, they should display any reproductive isolation mechanism in this 'contact zone'. It is important to notice that Fouquet et al. [7] found spatial overlap among CCS Ma, J and K but no allele sharing between them. Nevertheless, we found that populations of CCSs Ma, J and K from Ubatuba and its vicinities share a haplotype (h16). Additionally, all morphological and acoustic parameters overlapped among these populations and no evidence of such distinctiveness could be found.
Campos et al. [83] studied the karyotype, among other leptodactylid species, from the three clades of A. marmorata, and their results corroborate the hypothesis that they are the same species. We analyzed two specimens from that study: CFBH 11512, from the municipality of Santa Branca, state of São Paulo (corresponding to CCS Ma) and CFBH 17137, from Alcatrazes Island, state of São Paulo (corresponding to CCS J). No cytogenetic difference was found between the two samples [83]. However, their samples from the municipalities of Salesópolis and São Luís do Paraitinga, state of São Paulo (which correspond J and CCS Ks in our study, respectively) showed the chromosome pair 12 metacentric, differing from the specimens from Santa Branca and Alcatrazes Island, which had the same chromosome pair telocentric. Campos et al. [83] suggested that this difference was due to intraspecific geographic variation and the data presented herein corroborate that interpretation.
It is important to point out that we assessed the taxonomic status of A. marmorata using molecular, acoustic, and morphological data, but there are still other character sets to be assessed (e.g. ecology, behavior, and tadpole morphology). As noted by de Queiroz [27], closely related species might be in "gray zones" (i.e., time in species evolution where alternative species concepts and character sets come into conflict, making it difficult for us to recognize species) and additional characters put into analysis may shed new light on the taxonomy of A. marmorata and lead to a more precise interpretation.
Adenomera marmorata is a widespread species showing rich phylogenetic structure and remarkable acoustical and morphological variation. These aspects make this species an interesting model to studies on diversification processes in the Atlantic Forest. Table. Acoustic terminology and definitions for the automated analysis of acoustic traits. (DOCX)
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Domain: Biology Medicine
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Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. K) considered as confirmed on the basis of the concordant mtDNA and nuDNA data but meager acoustic and morphological data suggesting phenotypic divergence. Despite the important contribution of Fouquet et al. [7] to the diversity patterns in Adenomera, it was beyond the scope of that study to formally describe candidate species. Therefore, a formal taxonomic review based on an integrated approach is still needed to validate the species hypothesis formerly proposed by Fouquet et al. [7] as confirmed candidate species (hereafter CCS). To avoid confusion between the nominal species Adenomera marmorata and the CCS Adenomera marmorata as defined by Fouquet et al. [7], hereafter we will refer to the CCSs Adenomera marmorata, Adenomera sp. J, Adenomera sp. K as CCS Ma, CCS J and CCS K, respectively.
Assigning populations to Adenomera marmorata is a well-known problem due to the high level of inter and intrapopulation polymorphism, the lack of an exact type locality and a brief original description. Additionally, there are few available synonyms under the name Adenomera marmorata [11]. Therefore, it is often difficult for herpetologists to confidently associate populations with species names. Given that A. marmorata is the type species of the genus, clarifying its taxonomical boundaries should be relevant to understand species' identities in the genus, especially those distributed across the Atlantic Forest domain. Notwithstanding, taxonomical contributions for the genus Adenomera within this domain are seldom published. Before the recent description of A. kweti [15], the most recent taxonomical contribution for the genus in the Atlantic Forest domain was published a decade ago [16].
Herein we aim to delimit the species Adenomera marmorata by evaluating the taxonomic status of the confirmed candidate species (CCS) defined by Fouquet et al. [7] (herein referred as CCS J and CCS K), using an integrative taxonomy protocol based on a comprehensive dataset of phenotypic (morphology and calls) and molecular data. based on the following criteria: 1) cladogram topology and 2) identification of at least one diagnostic phenotypic evidence that supports the independency of evolutionary lineages from its sister clade.
Molecular analyses
Taxon sampling. We obtained new DNA sequences from 149 tissue samples from CCS Ma (n = 73), CCS J (n = 51), and CCS K (n = 25). (DOCX)
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Domain: Biology Medicine<|endoftext|>Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. Next cells were incubated with 0.5 mg/ml (in PBS) sulfo-NHS-SS-biotin (Pierce) for 30 min on ice. After incubation, excess biotin was quenched by washing with icecold 15 mM glycine. Next cells were washed with prewarmed minimal essential medium, 0.1% bovine serum albumin containing 100 g/ml cycloheximide and incubated for 1 h at 37°C before treatment. All treatment conditions were done in duplicate. To measure total biotin associated with the cells, one set of samples was harvested immediately after treatment. To measure the internalized biotin, the second set of samples was stripped from cell surface biotin by washing three times for 5min with ice-cold GSH cleavage buffer (50 mM GSH, 75 mM NaCl, 1 mM EDTA, 1% bovine serum albumin, 0.75% 10 N NaOH). Cell lysates were subjected to immunoprecipitation with streptavidin-agarose (Novagen, San Diego, CA) and analyzed by Western blot to detect 5-HT 2A R-YFP. The amount of internalized receptor was determined as a percentage of total cell-associated biotin.
Statistics-Statistical significance was determined using oneway analysis of variance with Bonferroni post-test (GraphPad Prism, version 4). p values lower than 0.05 were considered to be statistically significant.
Continuous Exposure to Agonist Induces Sequestration of 5-HT 2A Receptors into the Rab11-positive Compartment-
The agonist-induced internalization of the 5-HT 2A R has been reported recently (20); however, the detailed trafficking of 5-HT 2A R following continuous exposure to its natural ligand, 5-HT, has not yet been elucidated. To study the effects of 5-HT more closely, confocal microscopy studies were performed in HEK293 cells transiently transfected with 5-HT 2A R-YFP. Immediately after agonist treatment (1 min), 5-HT 2A R underwent endocytosis; however, continuous treatment with agonist (30 min to 2 h) induced redistribution of internalized 5-HT 2A R and its sequestration in a perinuclear compartment ( Fig. 1A and supplemental Fig. S1). Similar sequestration was observed for the AT1AR upon sustained ligand action (Fig. 1B). To study cellular localization of sequestered receptors, colocalization with markers of different compartments was performed. The sequestered 5-HT 2A R colocalized with, but extended beyond, Rab11 (Fig. 1C), a marker of the perinuclear recycling compartment. There was no colocalization with Lamp1 (lysosomal marker) or giantin (Golgi marker). Brefeldin A, which disrupts the Golgi, did not inhibit 5-HT 2A R sequestration (Fig. Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. Acad. Sci. U. S. A. 104, 18718–18723; Avitabile, E., Forghieri, C., and Campadelli-Fiume, G. (2007) J. Virol. 81, 11532–11531); it was not determined whether they led to biochemical complexes. Infected cells harbor a gD-gH complex (Perez-Romero, P., Perez, A., Capul, A., Montgomery, R., and Fuller, A. O. (2005) J. Virol. 79, 4540–4548). We report that gD formed complexes with gB in the absence of gH/gL and with gH/gL in the absence of gB. Complexes with similar composition were formed in infected and transfected cells. They were also present in virions prior to entry and did not increase at virus entry into the cell. A panel of gD mutants enabled the preliminary location of part of the binding site in gD to gB to the amino acids 240–260 portion and downstream with Thr304-Pro305 as critical residues and of the binding site to gH/gL at the amino acids 260–310 portion with Pro291-Pro292 as critical residues. The results indicate that gD carries composite-independent binding sites for gB and gH/gL, both of which are partly located in the profusion domain.
Herpes simplex virus (HSV) 2 entry into the cell occurs by fusion, and requires a multipartite apparatus made of a glyco-protein quartet: gD, gB, and the heterodimer gH/gL; for reviews, see Refs. [1][2][3]. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine
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Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro Objective. Negative pressure was found to significantly inhibit the transcription of ToxA, RhlA, LasB, LasI, and RhlI and the expression of these genes in the negative pressure group was 0.3-, 0.7-, 0.68-, 0.21-, 0.11-fold that of the control group, respectively (Figure 4). The repression of these genes under negative pressure supports the observed reduction in virulence factors and biofilm formation of P. aeruginosa.
Discussion
In recent years, physical therapies have been increasingly popular in the management of contaminated wounds owing to their satisfying wound closure and low risk of microbial resistance [25]. In particular, NPWT has been shown to promote the healing rates and prevent wound infections by multiple mechanisms, including decreasing edema, removal of wound exudates, and translating physical stimulation to signal transduction in cells [26,27]. Previous studies indicated that negative pressure conditions caused by NPWT could alter the gene expression and the function of host cells in vitro, such as bone marrow MSCs and keratinocytes [19,20,28]. However, its potential effects on P. aeruginosa and virulence factors have not been studied yet. In this study, we investigated the effect of negative pressure on the proliferation, virulence factor secretion, and virulenceregulated gene expression of P. aeruginosa, which is one of the most frequently isolated pathogens during wound infections [3].
In this study, the negative pressure value (−125 mmHg) was consistent with the clinical use of negative pressure in NPWT, and the O 2 tension was kept at 20% during bacterial culture in order to reduce interference from low oxygenation [29]. Colony diameter and growth curve indicated that negative pressure conditions could significantly inhibit the proliferation and growth rate of P. aeruginosa. Physical stimulations caused by pressure variation may contribute to this inhibition. Similarly, Liu et al. found that NPWT could decrease proliferation of P. aeruginosa within the burn wound and reduce mortality in a murine model [6]. Previous studies have found that physical stimulations, such as shear stress and hydrostatic pressure, could decrease the growth rate of S. aureus, attenuate bacterial virulence, and increase susceptibility to antimicrobial treatment [30,31]. Furthermore, significant decrease in metabolic functions, such as carbohydrate metabolism and protein synthesis, was also observed under shear stress conditions [32]. Thus, it is hypothesized that negative pressure might inhibit the growth of P. aeruginosa.
Authors' Contributions
Guo-Qi Wang, Tong-Tong Li, and Zhi-Rui Li contributed equally to this work.
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Domain: Biology Medicine<|endoftext|>
Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. Data are shown as min-max when applicable. Specimens from clade J1 are geographically circumscribed within the range of calde J2; i.e. specimens from clade J1 are restricted to the municipalities of Cubatão and Santo André, other individuals from these localities were also recovered in clade J2. The CCS Ma includes two main clades, Ma1 and Ma2, both well-supported. The clade Ma1 lies primarily in the inland eastern São Paulo state, reaching its northern coast (including Picinguaba, a locality within the municipal limits of Ubatuba); the clade Ma2 lies in the southern coast of Rio de Janeiro state and adjacent Serra do Mar, including the presumed type locality of the species Adenomera marmorata.
We recovered 47 haplotypes for POMC and 77 haplotypes for RAG fragments. The TCS network for POMC gene revealed that the most abundant haplotypes were shared among all CCSs (J, K, and Ma) and the second most abundant haplotype (h17) was shared between CCS K (clade K2) and CCS Ma (clade Ma2). Addittionally, CCS K (clade K1) and CCS Ma (clade Ma1) lineages share two haplotypes (h20 and h23; Fig 2). In the corresponding network for the RAG fragment, there is essentially no haplotype sharing among lineages, except for three haplotypes (h4, h16, and h18). All CCSs share the haplotype h16 (clades Ma1, J2, and K2); CCS K and CCS Ma share the haplotype h4 (clades Ma1, Ma2, and J2); and CCS J and CCS Ma share haplotype h18 (clades Ma1 and J2; Fig 2).
Phenotypic analyses: Morphology and advertisement call
Morphological variation among the CCSs J, K and Ma did not allow us to distinguish them from each other. The SVL completely overlapped among the three groups of specimens ( Fig 3). Dorsolateral stripe was present in specimens in populations of all three CCSs. Tubercles on the dorsal surface of the tibia were present in CCS Ma and CCS K. Specimens from continental populations of CCS J did not have tubercles on the tibia. Nevertheless, specimens of CCS J from the Alcatrazes Island had tubercles on dorsal surface of the tibia similar to CCS Ma and CCS K (Fig 4). Specimens of CCS J from Alcatrazes Island are also larger (male SVL: 25.6 mm, n = 1; maximum female SVL: 27.3, n = 3) than those of continental populations of CCS J (maximum male SVL: 20.5 mm, n = 5; maximum female SVL: 19.4 mm, n = 9).
We analyzed the advertisement calls of 51 individuals of CCS Ma, J, and K from 10 localities (municipalities of Guapimirim, Itatiaia, Maricá, Petrópolis, and Rio de Janeiro, state of Rio de Janeiro, and Mogi das Cruzes, Nazaré Paulista, Santo André, São Luís do Paraitinga, and Ubatuba, state of São Paulo), totaling 1951 calls. The advertisement calls of the three lineages showed similar structure: a single, nonpulsed note emitted at regular intervals, having or not a frequency upsweep along the note (Fig 5). The dominant frequency coincides with the fundamental harmonic. Although CCS J tended to have calls with shorter durations and higher in pitch than those CCS Ma and CCS K, the acoustic traits analyzed usually overlapped among the them ( Table 4).
Taxonomic decision
Redescription of the holotype. The specimen is completely faded and with both forearms detached from the body, although some traits are still present. (Fig 8). (DOCX)
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Domain: Biology Medicine
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Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells
Background EDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. Moreover, transport of modified RTA to the cytosol, in contrast to wild-type RTA, appeared to be EDEM1-and EDEM2-independent [23,24]. The introduced P250A mutation alters the secondary structure of RTA into a more helical structure suggesting that EDEMs recognition might be determined by the structure of the ERAD substrate. Recognition of protein substrates by ER chaperones remains poorly defined. In the case of EDEM proteins it has been suggested that besides glycans, EDEM1 can recognize misfolded regions of aberrant proteins [19] such as exposed hydrophobic domains [10,37]. In this study, we have verified that changed hydrophobicity of protein determinants may influence protein substrate interaction with EDEM1 and EDEM2. We have demonstrated that ricin A-chain and a model misfolded protein, BACE457 carrying mutations that significantly decreased their hydrophobicity, are poorly recognized by EDEM1 and EDEM2 in contrast to their wild-type counterparts. Moreover, degradation of mutated BACE457 with decreased hydrophobicity is significantly reduced. Thus, we conclude that the chaperone proteins EDEM1 and EDEM2, similarly to the so called 'classical chaperones', recognize hydrophobic regions of the protein substrates and interact with determinants that posses relatively high degree of hydrophobicity.
Biophysical characteristics of RTA mutants
To characterize the correct folding and overall stability of RTA DHF and RTA IHF , their sensitivity to trypsin ( Figure 2A) and pronase (see Additional file 2) was compared to that of wild-type RTA. Digestion patterns for the ricin A-chain mutants and wild-type counterpart were the same, confirming equal in vitro stability of mutant RTA DHF, RTA IHF and the wild-type protein ( The obtained modified ricin A-chain proteins were reassociated with ricin B-chain to form a holotoxin (RTA DHF :RTB, RTA IHF :RTB; see Methods). To ensure that the introduced mutations were not affecting the reduction of the disulfide bond, either by increasing or decreasing the possibility of reductive cleavage, mutant RTA DHF, RTA IHF and wild-type holotoxin were incubated with increasing amounts of dithiothreitol (DTT). The results showed no significant difference in the RTA mutants reducibility in comparison to wild-type holotoxin ( Figure 2B). The denatured forms of ricin were reduced by much lower concentrations of reducing agent ( Figure 2B), what also indicate that native forms of ricin holotoxin, wild-type and mutant forms DHF and IHF are properly folded.
Mutations that change hydrophobicity of RTA influence the binding of modified proteins to the cell surface as well as affect their endosomal-lysosomal degradation Abolished cytotoxicity of ricin DHF and ricin IHF is at least partially connected with a decreased intracellular level of both proteins. To further investigate the reason for the reduced amounts of both mutated forms of ricin, measurement of binding of ricin holotoxin DHF and IHF to the cell surface was performed. Cells were incubated with ricin at 0°C. This temperature stabilizes ricin bound the cell surface [40]. In this experiment Western blot analysis with anti-RTA antibodies was applied ( Figure 4B). In the case of both mutated forms of ricin (DHF and IHF), there was a significant decrease in the amount of cell surface-bound holotoxin proteins with changed hydrophobicity in comparison to the wildtype protein ( Figure 4B). Observed differences were not due to the decreased stability of ricin DHF and ricin IHF after incubation on ice, as presented in Figure 4C. These data suggest surprisingly that introduced mutations in the hydrophobic region of RTA affect the binding of ricin IHF and ricin DHF to the cell surface. This may explain decreased intracellular levels of both forms of ricin ( Figure 4A). Proteins were detected by chemiluminescence reagent SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific).
Immunofluorescence microscopy
HEK293 cells transfected with cDNA encoding BACE457 , BACE457 DHF or BACE457Δ were grown on coverslips.
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. The AMPA receptor is a glutamate-gated ion channel that mediate fast synaptic transmission at excitatory synapses which are important for neuronal development, long term potentiation and synaptic plasticity. AMPA receptors are activated by L-glutamine which triggers an influx of calcium (Ca 2+ ) into the neuron. The Ca 2+ permeability of the AMAP receptors is dependent on the tetrameric composition of the subunit of the glutamate receptor [72]. AMPA receptors containing the glutamate receptor 2 (GRIA2) subunit exhibit lower Ca 2+ permeability compared to those without it and this is due to the presence of single amino acid residue, arginine (R) in the pore of the channel [73]. When comparing cDNA and genomic sequences encoding the GRIA2 subunit, it was found that the R codon (CGG) in the cDNA was a glutamine (Q) codon (CAG) in the genomic sequence [70]. ADAR2 is the enzyme that catalyzes this critical site specific editing event at this Q/R site [74]. This editing event also regulates the heterotetrameric assembly of the AMPA receptors by retaining the GRIA2 subunits within the endoplasmic reticulum therefore increasing the time required for it to be transported to the synaptic surface [75,76] Editing at this Q/R site is highly efficient >99% in the CNS and it dictates the Ca 2+ permeability of the AMPA receptor. It is therefore is a key determinant in neuronal Ca 2+ homeostasis, as the edited isoform, GRIA2 (R), is impermeable to Ca 2+ .
The early onset epileptic seizures and premature death of both Adar2 null mice and mice heterozygous for an uneditable GRIA2 allele demonstrated the physiological importance of this editing event [77,78]. The Adar2 null phenotype is rescued by generating a transgenic mouse containing the edited GRIA2 allele [71]. In addition to the Q/R site, the GRIA2 transcript is also edited at the R/G site [79]. The transcript encoding the GRIK2 subunit of the high affinity kainite (KA) receptor is edited at the Q/R site in transmembrane segment 1 (TM1) and also at the I/V and Y/C sites in TM2 [80]. Editing at these positions affects the Ca 2+ permeability of the KA receptor channel. In contrast to the AMPA receptors, editing at the Q/R site of the GRIK2 transcript increases the Ca 2+ permeability of the KA receptor [80]. Burns and colleagues demonstrated that transcripts encoding the G-protein-coupled 5-HT2C undergo editing at sites within the second intracellular loop which results in 3 amino acid changes of isoleucine to valine (I/V), asparagine to serine (N/S) and isoleucine to valine (I/V) [81]. RNA editing at these sites generates several 5-HT2C isoforms which are expressed at various levels in different brain regions [81]. The fully edited 5-HT2C isoform has a reduced affinity for the G-protein PLC which inhibits the downstream phosphatidylinositol signal pathway [81]. Therefore, RNA editing regulates the efficacy of receptor-G-protein coupling. The Kv1.1 transcript encoded by the voltage gated potassium channel gene is edited at a single site and results in an isoleucine-to-valine (I/V) recoding event [82]. The I/V site is located in a highly conserved region of the channel pore that is involved in modulating potassium ion flow and RNA editing at this site results in the fast inactivation of the Kv1.1 channel [82]. The Gabra-3 transcript encoding the α3 subunit of the GABAA receptor undergoes site-specific editing, resulting in an isoleucine to methionine (I/M) amino acid change [83]. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine
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Protective effects of various ratios of DHA/EPA supplementation on high-fat diet-induced liver damage in mice
Background A sedentary lifestyle and poor diet are risk factors for the progression of non-alcoholic fatty liver disease. In the present study, levels of inflammatory factors as well as those of their relative gene expression decreased obviously; the biggest reduction in TNF-α levels was found in the DHA/EPA 2:1 group, while the lowest levels of IL-1β and IL-6 were observed in the DHA/EPA 1:2 group with an n-6/n-3 ratio of 4:1. This result is consistent with the observation of inflammation-dampening effects of n-3 PUFAs in the liver and a decreased inflammatory response in fat-1 mice that is associated with significantly reduced hepatic gene expression of TNF-α, IL-1β, and IL-6 [40].
Cytokines involved in the complex inflammatory response network can be modulated by various activators and inhibitors, thus, activating various reactions in inflammatory pathways [41], which may amplify the inflammatory reaction and result in tissue injury if uncontrolled [42]. Among the typical inflammatory responses of metaflammation, activating protein-1 (AP-1, including c-Jun and c-Fos) [43] is a relevant type of inflammatory transcription factor [44] and an important signal transduction pathway component of proinflammatory mediator expression that is independent of NF-κB [31]; its activity might be regulated by gene transcription levels and protein concentration [45]. The results of our present study suggest that consumption of DHA/EPA significantly suppressed the expression of c-Jun and c-Fos proteins and their respective genes, with ratios of 1:1 and 1:2 showing better effects than those by DHA, in HFD-induced mice. Consequently, DHA/EPA reduced the expression of c-Jun and c-Fos proteins and weakened the activity of AP-1, which may decrease the expression of inflammatory factors; thus, this may have attenuated the activation of inflammatory pathways as a lower DHA/EPA ratio was found to be more effective at alleviating an inflammatory response.
Conclusions
In summary, supplementation with various DHA/EPA ratios composed of fish, algae, and sunflower seed oils with an n-6/n-3 ratio of 4:1 was beneficial by means of promoting lipid metabolic processes, attenuating steatosis, and hepatic lipid accumulation, and by relieving hepatocyte oxidative damage by regulating the serum lipid profiles, controlling inflammatory reactions, and balancing lipid peroxidation. These effects of n-3 PUFA on lipid metabolism may be linked to the improvement of Fra1 expression and the attenuated activity of c-Jun and c-Fos, ultimately reducing the severity of a lipid metabolism disorder and liver damage to some extent. DHA/EPA did not yield better results on modulation of the serum lipid profile, oxidative damage, and expression of lipid metabolismrelated genes, but were more effective at controlling inflammatory factors as compared to that by DHA. Further research is required to explain this discrepancy.
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Domain: Biology Medicine<|endoftext|>Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. In gD ⌺218 -240 , the 218 -240-aa segment, carrying ␣-helix3, was substituted with a 18-aa-long Ser-Gly linker. In gD ⌬ 61-218 , the deleted sequences were replaced with a 18-mer Ser-Gly linker. gD ⌬6 -60 carries the indicated deletion. B, gD cell surface expression measured in transfected 293T cells by CELISA with mAb H170 (or mAb BD80 for gD ⌬6 -60 ). The extent of expression is relative to cells expressing wt gD (100%). C, cell-cell fusion in 293T cells transfected with wt or mutant gD, plus gB, gH, and gL. All other details as in legend to Fig. 1C. D, infectivity complementation of gD mutants. 293T cells in T 75 flasks were transfected with one of the gD mutants and 4 h later were infected with ⌬gD-HSV (FgD). In this assay, progeny virus harvested at 18 h after infection is complemented by the transiently expressed transgenic gD; it was titrated in R6 cells (which express wt gD and allow plaque formation); titer was expressed as PFU/ml. It can be seen that all of the gD mutants were dramatically hampered in infection, relative to wt gD. In all histograms, each column represents the average of triplicate samples. The bars denote Ϯ S. E. E, extracellular virions from the experiment shown in D ultracentrifuged and analyzed by WB for the presence of gD and for comparison of gB. It can be seen that, except gD ⌬ 61-218 , all mutant forms of gD were incorporated in virions.
N-terminal degradation product. Its reactivity to mAb H170, used for WB, which recognizes an epitope located at aa 1-23, indicates that this is an N-terminal peptide. Interestingly, with respect to gD ⌺240 -310 and gD ⌺260 -310 , gH st pulled down the 38-kDa N-terminal species and only small amounts of the higher molecular mass species (Fig. 7A). The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine<|endoftext|>
Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. marmoratafrom Alcatrazes Island (CCS J) are much larger than specimens from other populations (male minimum SVL 25.6 mm, n = 1, female minimum SVL 25.7 mm, n = 4; other mainland populations male maximum SVL 22.4, n = 37, female maximum SVL = 23.4 mm, n = 24). Vertebrate gigantism on islands has massively been reported in the literature ( [78][79][80]). Nevertheless, Rebouças et al. [80] found opposite results, (i.e. dwarfism) analyzing populations of A. marmorata from Ilha Grande Island. Variation in size of isolated insular communities may be related to genetic drift or to their local ecological processes, such as predation pressure, competition, and availability of resources [80][81][82].
Populations from CCSs J, K, and Ma were never found in syntopy. Nevertheless, the north coast of São Paulo deserves special attention. The northernmost distribution of CCSs J and Ma (clade Ma1) is on Ilha das Couves Island and Picinguaba, respectively-both localities within the municipal limits of Ubatuba, state of São Paulo. On the other hand, we identified only CCS K in the urban vicinities of Ubatuba. We should expect that, if these lineages represent indeed, distinct species, they should display any reproductive isolation mechanism in this 'contact zone'. It is important to notice that Fouquet et al. [7] found spatial overlap among CCS Ma, J and K but no allele sharing between them. Nevertheless, we found that populations of CCSs Ma, J and K from Ubatuba and its vicinities share a haplotype (h16). Additionally, all morphological and acoustic parameters overlapped among these populations and no evidence of such distinctiveness could be found.
Campos et al. [83] studied the karyotype, among other leptodactylid species, from the three clades of A. marmorata, and their results corroborate the hypothesis that they are the same species. We analyzed two specimens from that study: CFBH 11512, from the municipality of Santa Branca, state of São Paulo (corresponding to CCS Ma) and CFBH 17137, from Alcatrazes Island, state of São Paulo (corresponding to CCS J). No cytogenetic difference was found between the two samples [83]. (DOCX)
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Domain: Biology Medicine
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This below document has 2 sentences that end with 'for 3 hours',
2 sentences that end with '3 hours at 37°C'. It has approximately 606 words, 26 sentences, and 8 paragraph(s).
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Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells
Background EDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. The concentration of the protein solutions were 30 μg/ml. Spectra recorded from 190 to 240 nm with 1-nm step size were averaged from three accumulations and were corrected against the buffer.
Measurements of protein synthesis
HEK293 cells were washed in leucine free Hepes-buffered medium and incubated with the same type of medium with different concentrations of wild-type or mutant ricin DHF or IHF for 3 hours. The cells were then incubated in leucine free medium supplemented with 1 μCi/ml [ 3 H] leucine for 20 minutes at 37°C. Cells were extracted with 5% trichloroacetic acid (TCA) for 20 minutes, followed by a wash (5 minutes) in 5% TCA and subsequently dissolved in 0.1 M KOH. The cell-associated radioactivity was measured. The results are expressed in percent of [ 3 H]leucine incorporated in cells incubated without toxin. Deviations between duplicates did not vary by more than 10%.
Cell surface binding of wild-type ricin, IHF ricin and DHF ricin was measured after 30 min incubation with ricin (~100 ng/ml) at 0°C in Hepes medium. After incubation, cells were washed four times with ice-cold Hepesbuffered medium. For the wild-type ricin, IHF ricin and DHF ricin visualization, Western blot with anti-RTA antibodies was employed. Stability of all types of ricin was verified in a separate control experiment after 30 min incubation on ice. Proteins were visualized by SDS/PAGE and Coomassie Blue staining. For degradation measurement, cells were treated either with bafilomycin A1, pepstatin A, CA074 methyl ester, or a combination of pepstatin A and CA074 methyl ester, for 30 minutes, and then incubated with wild-type ricin, IHF ricin or DHF ricin for 3 hours. To determine the total amount of ricin remaining in the cells after degradation, Western blot with anti-RTA antibodies was performed. Concentrations of inhibitors are indicated in the figure legends ( Figure 4 and see Additional file 3).
Sulfation of wild-type ricin sulf-1 and mutants with increased or decreased hydrophobicity HEK293 cells were incubated with approximately 500 ng/ml of wild-type, DHF ricin or IHF ricin sulf-1 in Hepes medium for 3 hours at 37°C. For the sulfated ricin analysis, cells were incubated with 0.2 mCi/ml Na 2 35 SO 4 in DMEM without sulfate for 3 hours before toxin was added, and the incubation was continued for the next 3 hours at 37°C. Cells were then incubated twice (5 min) in a 0.1 M lactose solution at 37°C, to remove surface bound toxin, then washed once with cold PBS, and lysed (lysis buffer: 0.1 M NaCl, 10 mM Na 2 HPO 4 , 1 mM EDTA, 1% Triton X-100, pH 7.4) in the presence of a protease inhibitor mixture (Roche Diagnostics). Wild-type ricin, IHF ricin or DHF ricin were immunoprecipitated from the fractions with rabbit anti-ricin antibodies and immobilized on protein A-Sepharose CL-4B (GE Healthcare). Finally, the beads were washed with cold PBS supplemented with 0.35% Triton X-100, and the adsorbed material was analyzed by SDS-PAGE (12%) under non reducing conditions. For the detection of 35 SO 4 2− -labeled ricin, dried membranes were exposed to Kodak BioMax MR film (Kodak, Rochester, NY) at room temperature. The radioactivity in the cell lysates was measured to detect possible differences in the total amount of isotope incorporated under different conditions. Proteins were detected by chemiluminescence reagent SuperSignal WestPico Chemiluminescent Substrate (Thermo Scientific).
Immunofluorescence microscopy
HEK293 cells transfected with cDNA encoding BACE457 , BACE457 DHF or BACE457Δ were grown on coverslips.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. Cell lysis was performed similarly to what has been previously described (21). Briefly, cells were resuspended in 50 mM ammonium bicarbonate and 1% (w/v) sodium deoxycholate and boiled for 5 min. MgCl 2 was added to a final concentration of 1.5 mM and DNA was digested by Benzonase endonuclease (Santa Cruz Biotechnology, Dallas, TX) at room temperature for 30 min. After combining the lysates of light, medium and heavy labeled cells (1:1:1), proteins were reduced with 10 mM DTT at room temperature for 30 min and alkylated with 55 mM iodoacetamide at room temperature for 20 min in the dark. Trypsin (Promega, Fitchburg, WI) digestion was performed at a trypsin-toprotein ratio of 1:100 (w/w) under constant agitation at 37°C for 16 h. Peptides were desalted with C18 StAGE Tips (25). Phosphopeptides were enriched using lactic acid-modified titanium dioxide (GL Sciences, Tokyo, Japan) and consecutively eluted by 5% (v/v) ammonium hydroxide and 0.5% (v/v) pyrrolidine as previously reported (26,27). Eluted phosphopeptides were desalted with C18 StAGE Tips (25) prior to analysis by LC-MS/MS. NanoLC-MS/MS Analysis-Phosphopeptides were separated using an 1100 series nanoflow high-performance liquid chromatography (HPLC) instrument (Agilent) equipped with a two column set up. Samples were loaded onto a 2 cm long, 100 m inner diameter fused silica trap column containing 5.0 m Aqua C18 beads (Phenomenex, Torrance, CA) for 10 min in buffer A (0.5% acetic acid) at a flow rate of 5 l/min prior to analytical separation. Analytical separation was accomplished using a 200 mm in length and 75 m of inner diameter column containing 3 m ReproSil-Pur C18-AQ material (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) at a flow rate of 250 nL/min. Both trap and analytical columns were prepared in-house. Peptides were eluted from the analytical column by altering the gradient from 100% buffer A (0.5% acetic acid) to 40% buffer B (0.5% acetic acid, 80% acetonitrile) over 90 min. The eluting peptides were directly infused into an LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific) via ESI. MS and MS/MS information was collected in a data-dependent manner using the following settings: one full scan (resolution 60,000; m/z 300 -1600) followed by top 15 MS/MS scans using collision-induced dissociation in the linear ion trap mass spectrometer (ITMS; min. signal required: 200, isolation width: 3 Th, normalized collision energy: 35, activation Q: 0.25, activation time: 10 ms) using dynamic exclusion (repeat count: 1, repeat duration: 30 s, exclusion list size: 500, exclusion duration: 60 s). AGC targets of 1 ϫ 10 6 and 3 ϫ 10 4 with maximum fill times of 250 and 100 ms were used for MS and MS-MS scans, respectively.
Because of higher sample complexity, peptides for whole proteome analysis were analyzed on the Q Exactive mass spectrometer (ThermoFisher Scientific) coupled to a two-column EASY-nLC1000 system. Trap and analytical column set up were similar as described above, with the analytical column being 400 mm in length and containing 1.9 m ReproSil-Pur C18-AQ material (Dr. Maisch GmbH). We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine
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Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro Objective. The data were recorded as ng/mL. Rhamnolipid was quantified by orcinol method, as previously described with a few modifications [23]. Briefly, 400 L supernatant from the bacterial culture was extracted twice using 600 L diethyl ether. The ether layer was transferred to a fresh tube for evaporation. Residues were dissolved in 150 L H 2 O, 100 L 1.6% orcinol (Sigma), and 750 L 60% sulphuric acid (H 2 SO 4 ). After heating for 30 min at 80 ∘ C, all the tubes were cooled at room temperature for 30 min and absorbance was recorded at 421 nm. The concentrations of rhamnolipid were calculated by multiplying rhamnose values by a coefficient of 2.5, as previously described [24].
The elastase activity was measured by the elastin-Congo red assay, as previously described [23]. Briefly, 100 L supernatant from 24 h LB cultures was added to tubes containing 10 mg of elastin-Congo red (Sigma) and 900 L Na 2 HPO 4 (pH 7.0). Tubes were incubated for 4 h at 37 ∘ C under shaking conditions and the absorbance was recorded at 495 nm after removing the precipitate by centrifugation.
Static Biofilm Assays.
To observe the influence of negative pressure on biofilm formation, 18 × 18 mm cover glass was put into a 35 mm culture dish, and each dish was incubated with 2 mL LB broth containing 10 6 P. aeruginosa for 24 h in a constant temperature incubator at 37 ∘ C. After 24 h, each cover glass was washed three times with phosphate buffered saline (PBS) to remove planktonic bacteria. The P. aeruginosa glycocalyx was visualized by staining with 50 g/mL of Alexa Fluor 647 conjugate of Con A (Life Technologies, USA) for 15 min at room temperature in the dark as previously described with a few modifications [4]. Biofilm formation was observed through fluorescence microscopy (Olympus BX51).
Quantitative RT-PCR.
Bacteria were isolated from the LB medium for quantitative RT-PCR analysis as previously described [21]. Primers used to amplify ToxA, RhlA, LasB, LasI, and RhlI, as well as the reference gene, RpoD, are shown in Table 1. Briefly, total RNA was extracted using an RNAprep Pure Cell/Bacteria Kit (TIANGEN, China) according to the manufacturer's instructions. Total RNA was
Statistical
Analysis. SPSS 17.0 was used for the statistical analysis. The measurement data were expressed as mean ± SD and compared between the two groups using Student'stest. aeruginosa.
Authors' Contributions
Guo-Qi Wang, Tong-Tong Li, and Zhi-Rui Li contributed equally to this work.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. When HeLa cells were transfected with FLAG-SEPT9-S30A, EPEC WT adherence was significantly decreased when compared with FLAG-SEPT9-WT transfected HeLa cells (Fig. 7B). However, when cells were transfected with FLAG-SEPT9-S30E, EPEC WT adherence was similar to cells transfected with FLAG-SEPT9-WT. EPEC ⌬escN-infected HeLa cells showed no significant difference in bacterial adherence. These data not only revealed that blocking Ser-30 phosphorylation impairs EPEC WT adherence but also that phosphorylation mimicking by glutamate leads to a functional rescue and promotes EPEC WT adherence to the host cell.
Adherence to host cells is a key strategy of EPEC virulence and is functionally connected with EPEC-induced host cell death in cell culture models. Thus, assessment of cell death by LDH-release analysis was used as an independent approach to confirm the previous findings. When compared with FLAG-SEPT9-WT transfected cells, HeLa cells transfected with FLAG-SEPT9-S30A and infected with EPEC WT displayed less cytotoxicity (Fig. 7C, p ϭ 0.067). In contrast, cells transfected with FLAG-SEPT9-S30E and infected with EPEC WT showed a significant increase in LDH-release when compared with FLAG-SEPT9-S30A transfected and EPEC WTinfected cells. This again confirms the functional rescue by the phospho-mimicking mutant and underlines the critical role of SEPT9 phosphorylation in EPEC interactions with host cells. As before, no significant differences were noted in EPEC ⌬escN-infected cells.
Collectively, these SEPT9 studies validate and functionally characterize the phosphoproteomic data. These results suggest that SEPT9 is a novel key host player in EPEC pathogenesis controlling the pathogen's attachment to the host cell surface. Furthermore, T3SS-mediated phosphorylation of Ser-30 in SEPT9 is critical for efficient bacterial adherence. DISCUSSION Alterations in host protein phosphorylation following infection can provide substantial insight into the molecular mechanisms by which human pathogens exploit host cell signaling to cause disease. Here we present the first study that examines the global impact of an extracellular diarrheagenic bacterial pathogen on the host phosphoproteome.
Consistent with this hypothesis we observed a less profound impact on the host phosphoproteome by EPEC compared with earlier phosphoproteomic profiling studies of Shigella or Salmonella (19,20). Nevertheless, our unbiased and comprehensive phosphoproteome analysis revealed that EPEC still triggers extensive changes in the host phosphoproteome far beyond what was established from targeted protein studies.
We observed a time-dependent increase of regulated phosphosites, which correlated to increased pathogen exposure. As expected, the impact on host phosphorylation was more profound in EPEC WT-infected cells when compared with T3SS-deficient mutant-infected cells. The observed T3SS-dependent alterations mainly affected cellular components that are known targets of pathogens: e.g. MAPKs have been implicated as an important signaling hub during host infection with EPEC and various other bacterial pathogens, and are often targeted by translocated bacterial virulence factors (16,48,(77)(78)(79)(80). Based on kinase enrichment, motif and T3SSregulated phosphosite analysis as well as the identification of a T3SS-regulated MAPK activation loop phosphopeptide, our results emphasize the central role of MAPK during EPEC infection. We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine
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Sources of heterogeneity in human monocyte subsets
Highlights • Variation in monocyte phenotype is explored using the markers CD14, CD16, HLA-DR, CX3CR1 and CCR2.• The CD14++CD16+ monocytes exhibit a spectrum of markers dependent on location within the gate.• Monocyte phenotype varies dependent on genetic background and history of exposure to infection.• Processing technique for PBMC purification does not lead to changes in monocyte subsets.• Processing technique for purification can alter intensity but not pattern of marker expression.
Introduction
Peripheral blood monocytes, which represent around 10% of circulating leukocytes in humans, are recognized as the largest pool of circulating progenitor cells and form a vital part of the immune system [1,2]. 3C). Gating in such a manner would identify any differences occurring both within this group, as well as between this group and the adjacent subsets. For the purposes of this manuscript we wished to maintain a distinction between what has been previously published and agreed to (three subsets defined as CD14++CD16−, CD14++CD16+ and CD14+CD16++) and the monocyte groupings as we defined them here. We thus decided to designate the CD14++CD16− as regCD14 monocytes and the CD14+CD16++ as regCD16 monocytes. The gating of all five subsets is shown in Fig. 3A with the proportions of each of the subsets illustrated in Fig. 3B.
Rather than distinct subsets, the monocyte gate consists a spectrum of progressively changing phenotypic markers
From the larger cohort of 62 Africans we analyzed the five monocyte groupings with respect to changes in their surface expression of the phenotypic markers CCR2 and CX3CR1, and the MHC receptor HLA-DR. Fig. 3C-E shows the mean expression levels of these markers in each of the monocyte groupings. The significantly elevated level of HLA-DR in the HLADRhi subset compared to dpCD16 (p < 0.001) and the regCD14 cells (p < 0.001) may indicate an increased activation status in these cells (Fig. 3C). CCR2 shows a spectrum of expression levels with the highest on the regCD14 monocytes, decreasing with increasing CD16 expression (Fig. 3D). The CD14high expressing monocytes (regCD14, dpCD14 and HLADRhi) do not show significant differences in CCR2 expression, however with increasing CD16 expression (transitioning the subset from regCD14 towards regCD16) there is a concurrent decrease in CCR2 expression (from HLADRhi to dpCD16: MFI difference = −55.48, p = 0.001, and from dpCD16 to regCD16 MFI difference = −63.68, p < 0.001). In contrast, CX3CR1 shows significant differences in expression level across all subsets (Fig. 3E), with the lowest expression of CX3CR1 on CD14++ monocytes (regCD14 and dpCD14) as previously reported [35]. Interestingly, the highest expression is in the dpCD16 monocytes with a significant decrease in expression between dpCD16 and regCD16 monocytes (dpCD16 to regCD16 MFI difference = −14,414, p < 0.001) (Fig. 3E). The regCD16 monocytes show a significant decrease in marker expression compared to dpCD16 for all analyzed markers (Fig. 3C-E).
Total monocyte number but not subset proportion differs between fresh and cryopreserved PBMCs
To investigate whether monocytes change their expression levels and phenotype dependent on cryopreservation, nine donors of Caucasian or African descent, with predominantly urban backgrounds, had a collection of peripheral blood for PBMC purification and cryopreservation. Importantly our study indicates that conformity across research groups in gating of these subsets is necessary in order to compare studies.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis*
Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation-mimicking SEPT9 mutant rescued these effects. Collectively, this study provides the first global analysis of phosphorylation-mediated processes during infection with an extracellular, diarrheagenic bacterial pathogen.
Diarrheagenic E. coli are a major global health burden and cause much morbidity and mortality worldwide. Enteropathogenic E. coli (EPEC) 1 is the causative agent of potentially fatal infantile diarrhea and remains an endemic health threat for children in developing countries. EPEC and the closely related enterohemorrhagic E. coli (EHEC) belong to the group of at-taching and effacing (A/E) pathogens that form distinct A/E lesions on the surface of intestinal epithelial cells causing the loss of the characteristic intestinal brush border architecture (1).
Upon attachment to intestinal cells, EPEC uses a syringelike molecular apparatus, the type III secretion system (T3SS), to inject at least 25 unique bacterial effector proteins into the host cell (2)(3)(4). Once translocated into mammalian cells, these effectors manipulate a wide range of host signaling pathways, thereby subverting host cell function and promoting virulence (5). We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine
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Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. 3C). Thus, activation of PKC is necessary and sufficient to induce the sequestration of GPCRs.
Next the effects of persistent ligand action on cPKC activity were determined by evaluating the phosphorylation of PKC␣/ II on Thr-638/641. The results demonstrated that treatment of C6 and HEK293 cells with agonist caused an increase in the phosphorylation of Thr-638/641 that persisted for up to 2 h of treatment (Fig. 3D), thus demonstrating sustained activation of cPKC.
Importantly treatment of cells with the cPKC inhibitor following ligand-induced sequestration (Fig. 3A, POST) also caused dispersal of the sequestered 5-HT 2A receptors. Thus, sustained activity of cPKC is required for maintaining sequestration of receptors.
To further elucidate the role of cPKC phosphorylation in 5-HT 2A R sequestration, PKCII autophosphorylation sites (Thr-641 and Ser-660) were mutated to alanine, thus generating a double alanine (DA) mutant of PKCII. 5-HT stimulation of cells co-transfected with 5-HT 2A R and WT PKCII caused perinuclear sequestration of both 5-HT 2A R and PKC (Fig. 3E). However, when cells were co-transfected with the 5-HT 2A receptor and the DA mutant of PKCII, neither PKC nor the receptor was sequestered to the perinuclear region, although the DA mutant did translocate to the plasma membrane. When DA-PKCII was expressed alone it showed a cytoplasmic distribution, typically observed for PKC in unstimulated cells (Fig. 3E, bottom panel). However, when coexpressed together with 5-HT 2A R, there was a considerable amount of the DA-PKCII localized to the membrane without agonist stimulation. It was previously shown that PKCII mutated at S660A,T641A and mutated at both autophosphorylation sites (DA) does not dissociate efficiently from the membrane after activation (19). Because 5-HT 2A R-YFP has a small amount of constitutive activity even in the absence of agonist, the results suggest that DA-PKCII is activated in cells coexpressing 5-HT 2A R. In accordance with that, the 5-HT 2A R antagonist ketanserin completely prevented this basal localization of DA-PKCII to the plasma membrane (Fig. 3E, lower panel) It should be noted that although the DA mutant completely blocked 5-HT-induced 5-HT 2A R sequestration, a fraction of receptors was internalized and localized to the early endosomes (data not shown). Taken together, these results imply an important role for cPKC in the regulation of 5-HT 2A R sequestration into the pericentrion.
PLD Activity Is Required for Receptor Sequestration-It was
shown previously that PLD activation was required for translocation of PKC␣/II and formation of the pericentrion (8). To further define the mechanism of receptor sequestration, it became important to determine whether the PLD pathway was involved in agonist-induced 5-HT 2A R sequestration. Inhibition of PLD with 1-butanol abolished the perinuclear sequestration of receptors; however, treatment with 2-butanol, a positional isomer of 1-butanol that does not inhibit PLD, was without effect (Fig. 4A). Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro Objective. aeruginosa levels could not be confirmed under NPWT in vivo. Besides, few studies have reported the bacteria in wounds, secondary to negative pressure treatment, particularly with regard to P. aeruginosa proliferation, virulence, and gene expression. Previous studies have indicated that negative pressure induced by NPWT could alter the gene expression and proliferation of bone marrow mesenchymal stem cells [19,20]. Our previous work had shown that negative pressure had an effect on the growth, secretion, and biofilm formation of Staphylococcus aureus [21].
The aim of this study was to evaluate the influence of negative pressure on the proliferation, virulence factor secretion, biofilm formation, and the virulence-regulated gene expression of P. aeruginosa in vitro.
Bacterial Strain and Preparation
. P. aeruginosa laboratory strain PAO1 carrying the gene encoding the green fluorescent protein (GFP) was obtained from the laboratory of the Chinese PLA Institute for Disease Control and Prevention (Beijing, China). P. aeruginosa was grown overnight and cultured in Luria broth at 37 ∘ C until log-phase was achieved. Optical density at 600 nm wavelength was measured. An optical density of 1.0 was equivalent to 10 5 colony-forming units per microliter, as determined by a standard curve.
Growth
Conditions. The bacterial culture protocol was based on our previously published model of in vitro negative pressure condition [21]. In brief, negative pressure condition was created for bacterial growth and an airtight chamber was used as the incubator. The air was sucked from the chamber by a vacuum pump device (provided by Professor Hu Lei, Beihang University, Beijing, China), which could automatically produce and maintain the negative pressure at −125 mmHg. The O 2 concentration was constantly maintained at 20%, as adequate amount of room air was introduced into the incubator every 15 min. Bacterial culture was performed in culture dishes (Corning Life Sciences, USA) with a diameter of 35 mm at 37 ∘ C. Each of the dishes contained 2 mL LB medium and 10 6 P. aeruginosa (in a volume of 10 L) was added. aeruginosa.
Authors' Contributions
Guo-Qi Wang, Tong-Tong Li, and Zhi-Rui Li contributed equally to this work.
==
Domain: Biology Medicine<|endoftext|>Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. Recently, a receptor for gB was described (23). Of note, although for virus-to-cell entry and for cell-cell fusion, fusion requires the simultaneous presence of gB and gH/gL, fusion of perinuclear virions with the outer nuclear membranes appears to necessitate either gH/gL or gB (24). Furthermore, it has been reported that HSV fusion may be preceded by a hemifusion intermediate mediated by gD and gH/gL (25).
A major focus of current research is definition of proteinprotein interactions. With respect to HSV entry/fusion, to understand how the four glycoproteins cross-talk to each other, it is pivotal to detect which complexes are formed by the glycoproteins in their prefusion and fusion-active conformations, and, ultimately, the chain of interactions that signal the gD encounter with its cellular receptor and culminate in activation of gB and gH/gL. The proposed model of gD-activated entry/ fusion (2, 9 -12, 26, 27) envisions that (i) gD ectodomain is organized in two functionally and topologically distinct regions, the N-terminal one, spanning amino acids (aa) 1 to ϳ240/260, carrying the receptor binding sites, and the C-terminal one (aa 240/260 -310), carrying the profusion domain required for fusion but not for receptor binding; (ii) the unliganded gD adopts an auto-inhibited conformation, whereby the C-terminal domain folds around the N-terminal one and occupies or hinders the receptor-binding sites; (iii) gD undergoes a closed-to-open switch in conformation, whereby the C-terminal domain is dislodged from its binding site and exposes the profusion domain; and (iv) the active form of gD ultimately leads to the activation of gH/gL and gB. It was hypothesized that gB and gH/gL activation occurs through their recruitment to activated gD and that the C-terminal profusion domain carries the actual binding sites for gB and gH/gL (9,12). An alternative possibility is that the C-terminal profusion domain simply enables the conformational changes in gD but does not carry the actual binding sites for gB and gH/gL.
The objective of this work was to provide biochemical evidence for complex formation among the glycoprotein quartet, to verify whether complexes are present in infected cells and in virions and whether they are formed at virus entry into the cell. We analyzed the composition of the complexes by two approaches, co-immunoprecipitation and a pulldown assay that exploits the ability of One-strep-tagged proteins to be specifically retained by the Strep-Tactin resin. Complexes with undistinguishable composition were detected in infected and transfected cells and in virions prior to entry into the cell. A panel of mutants enabled the preliminary location of part of the gD regions critical to gB-and gH/gL-binding sites at the profusion domain.
Antibodies-R8 polyclonal antibody (pAb) to gD and BD80 monoclonal antibody (mAb) to aa 264 -275 epitope of mature gD were generously provided by Dr. G. H. Cohen and Dr. R. Eisenberg; mAbs HD1, HC1, and H233 were a gift of Dr. Reema Mendez. pAbs to gH and gL were a gift from Dr. Isabel Neal (Cambridge, UK) and D. Sydney (Portland). mAb H170 (reactive to aa 1-23 epitope), H1817, and H633 were purchased from Goodwin Institute. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine
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New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. They found that the adenosine base at the editing site is more flexible and is inclined to move out into the minor groove. Beal and colleagues who were investigating the protein-RNA conformational changes that occur during deamination, found that nucleotides near the editing site on the opposite strand of the RNA become hypersensitive to hydrolytic cleavage after binding by the dsRBD of ADAR2 [53], indicating that RNA binding by ADAR changes the conformation of the RNA. In the same study, a tryptophan fluorescent probe was used to demonstrate conformational changes in the deaminase domain of ADAR2 in the presence of an RNA substrate.
Despite being isolated and characterized over 20 years ago, there is currently no structural information for a full-length ADAR containing both the dsRBDs and the deaminase domains. Nor is there any co-crystal of an ADAR protein with its dsRNA substrate. This is due to the modular nature of these enzymes which is a source of flexibility that makes in vitro studies and crystallization experiments more challenging. Another open question in the field is the role of dimerization of ADARs in the function of this family of enzymes. The ADAR proteins were shown to form dimers in vitro and in vivo [54][55][56]. However, it is still controversial whether the dimerization is essential for the activity [57] and whether it occurs in a RNA-dependent [54,55] or independent manner [58].
The Two Types of A-to-I RNA Editing
A-to-I editing by ADARs can be classified into two types: site-specific and promiscuous editing. Site-specific editing occurs within short, imperfectly-paired regions of dsRNA that can result in recoding of open reading frames to generate proteins with altered functions. High levels of site-specific RNA editing are found in transcripts that are expressed in the CNS. Since inosines prefer to base-pair with cytosine (C), a guanosine residue is inserted when cDNA is generated. Inosine is read as if it were guanosine by the translational machinery thus recoding the mRNA.
The second type of editing occurs within long dsRNA structures (>100 bp) that are deaminated at multiple sites. It has been demonstrated that up to 50% of the adenosine residues within these long dsRNAs can be deaminated so therefore this type of editing is referred to as hyper or promiscuous editing [48,59]. The deamination of an adenosine within an A-U base pair duplex produces the less stable I-U wobble pair, while deamination within A-C mismatches produces the more stable I-C pairs. These editing events are found within RNA duplex structures formed by inverted Alus and other repetitive elements within the non-coding regions of transcripts such as introns and 3ʹUTR regions as well as within transcribed intragenic regions [60][61][62][63][64][65]. The presence of A-to-I modifications in transcripts encoding Alu elements and other hyperedited transcripts regulate the structure and stability of the dsRNAs and inosinecontaining dsRNAs have been shown to play an essential role in regulating the innate immune response [66,67]. This type of editing will be discussed later.
RNA Editing Resulting in Recoding
When compared to mammals invertebrates have a significantly higher level of editing that results in recoding (for review, see [68]). In octopus, editing of transcripts encoding a potassium channel has been shown to vary depending on the location of the species, being highly edited in cold waters, whereas tropical water species are mostly unedited [69]. This elegant experiment suggests that RNA editing can respond to environmental queues.
The transcript encoding one of the α-amino-3-hydroxy-5-methylisoxasole-4propionate (AMPA) receptor subunit is the best studied mammalian site-specific editing event [70,71]. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine<|endoftext|>DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice
Background Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. In this investigation, our data demonstrated that both cell subtypes were significantly accumulated in response to immunization with TgDPA. This result corresponds with reports regarding immunological responses to T. gondii antigens [16,[68][69][70][71][72].
As a result of these significant immunological changes, pTgDPA vaccinated mice survived for a longer time compared to the control groups in this research. However, due to uncontrolled parasite replication, pTgDPA mice ultimately succumb during late acute infection. It indicated that the DNA vaccine of pTgDPA did not provide complete protection. However, investigations concerning this protein should further be conducted.
One of the prominent advantages of DNA vaccine application is their induction of CTL cells. CTL cells kill the pathogen infected cells mainly by inducing apoptosis [74]. T. gondii maintains its survival and replication by interfering with infected cells apoptosis, blocking an important pathway known as caspase cascade [75][76][77][78]. Measurement of damaged infected cells was an important tool to measure CTL function [74,79,80]. Application of such methods is required in further investigations regarding TgDPA antigen to highlight its role in CTL response stimulation and resistance development against T. gondii infection.
Conclusion
Our study demonstrated that the pTgDPA delivered as a single protein is an antigen with the potential of inducing and regulating significant levels of humoral as well as cellular (T H 1, T H 2 and T H 17) immune responses against acute T. This finding may encourage more investigations in evaluating the immunogenicity of DPA based vaccines against Toxoplasmosis.
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Domain: Biology Medicine
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Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. Transfected 293T cells in T 25 flasks received the appropriate plasmid mixture together with Arrest-in (Celbio, Milano), in the amount of 1.5 g of DNA for each plasmid (1ϫ amount). When indicated, pBEC was co-transfected at 3 g/T 25 flask ( 2X amount). Transfection mixtures in which one or more plasmids were omitted contained the HER2-encoding plasmid in place of the omitted plasmid, such that the total amount of DNA transfected in each flask did not vary. The cells were harvested 18 h after infection or transfection, prior to the appearance of syncytia, without freezing. For co-immunoprecipitation, in a typical experiment cells from two replicate T 25 flasks (or one T 75 flask) were solubilized in 200 (or 300) l of EA1 buffer ( 50 mM HEPES, 250 mM NaCl, 0.1% Igepal) containing the protease inhibitors N ␣ -p-tosyl-L-lysine chloromethyl ketone hydrochloride and N ␣ -p-tosyl-L-phenylalanine chloromethyl ketone (final concentration, 0.3 mM each), as described (28), at 4°C for 20 min. The lysates were centrifuged at 14,000 rpm for 30 min. The supernatants were cleared by incubation for 1 h at 4°C with a preimmune rabbit serum (2 l of serum/200 l of supernatant) followed by absorption to protein A-coupled Sepharose (10 mg) (Sigma-Fluka, Milano). The cleared unbound fraction was then incubated with pAb R8 to gD (2 l/200 l) overnight at 4°C, and thereafter with 10 mg of protein A-coupled Sepharose for 1 h at 4°C. The beads were washed three times with EA1 buffer containing the protease inhibitors and once with 50 mM Tris-HCl, pH 7.5, and 15 mM NaCl. gH st pulldown experiments were carried out from transfected 293T cells (2 T 25 For both co-immunoprecipitation and pulldown experiments, the material deriving from two flasks was solubilized with 120 l of sample buffer ( 2% sodium dodecyl sulfate, 5% -mercaptoethanol, 50 mM Tris-HCl, pH 7, and 2.5% sucrose), boiled, and loaded in two different gels: one for Western blot (WB) detection of gD and gB and one for detection of gH, gL, and gC.
Western Blot-Proteins separated by 8.5% PAGE were transferred to Hybond ECL nitocellulose membrane from GE Healthcare (Milano) and developed by Amersham Biosciences ECL Advance Western blotting detection kit (GE Healthcare).
Cell-Cell Fusion Assay-The luciferase-based cell-cell fusion assay was performed as detailed (49,50) by means of a luciferase assay system from Promega (Florence, Macao) in 293T cells. The total amount of transfected plasmid DNA was made equal by the addition of human epidermal growth factor receptor 2 plasmid DNA. All of the samples were run in triplicate.
Infectivity Complementation-The assay was performed as detailed elsewhere (46). Briefly, 293T cells in T 75 flasks were transfected with the appropriate gD plasmid. Four h later, the cells were infected with a gD Ϫ/ϩ stock of FgD (3 PFU/cell). Unpenetrated virions were removed by two phosphate-buffered saline rinses and inactivated by means of 40 mM citric acid, 10 mM KCl, 135 mM NaCl, pH 3, for 1 min. The monolayers were then rinsed twice with phosphate-buffered saline and overlaid with medium containing 1% fetal calf serum. The cells were incubated overnight at 37°C. The extracellular progeny virions were ultracentrifuged, analyzed by WB, and titrated in the gD-complementing R6 cells.
RESULTS
Tagging of HSV-5 Glycoproteins-Biochemical evidence of complex formation among the HSV glycoprotein quartet by means of co-immunoprecipitation or pulldown experiments relied on WB-positive antibodies, able to detect the complexed glycoproteins. The most limiting factor throughout this study was the detection of gH and gL, which greatly affected the sensitivity of the assays. To improve their detection, gH and gL were tagged with the following epitopes: 5E1 (51) plus or minus polyhistidine, V5-polyhistidine, and One-strep-tag. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine<|endoftext|>Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. Anti-giantin antibody was from Covance Research Products, Inc. (Berkeley, CA). Anti-Rab11 and anti-green fluorescent protein (GFP) antibody were from Zymed Laboratories Inc./Invitrogen. Anti-EEA1 and anti-PKC␣ antibody were from BD Biosciences. Anti-HA antibody was from Roche Applied Science. Anti-EGFR antibody was from Upstate Biotechnology (Lake Placid, NY). Polyclonal rabbit antibody specific for phosphorylated PKC␣/II at Thr-638/641 was from Cell Signaling Technology (Danvers, MA). Anti-Lamp1 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-CD59 antibody was a generous gift from Dr. Stephen Tomlinson (Medical University of South Carolina, Charleston, SC). Serotonin, ketanserin, epidermal growth factor, and all other chemicals were from Sigma.
Cell Culture-HEK293 cells were maintained in minimal essential medium supplemented with 10% (v/v) fetal bovine serum in a 5% CO 2 incubator at 37°C. Cells were passaged every 3-4 days to maintain cells in logarithmic growth. C6 glioma cells were grown in high glucose Dulbecco's modified Eagle's medium and maintained as above.
Plasmids-All recombinant DNA procedures were carried out following standard protocols. The wild type pBK-CMV-GFP-PKCII was described previously (19). PKCII-mCherry was described previously (11). The 5-HT 2A R-yellow fluorescent protein (YFP) receptor construct was generated by cloning the cDNA sequence of human 5-HT 2A R into XhoI and BamHI sites of EYFP-N1 vector (Clontech, BD Biosciences). The HA-PAR1 was a gift from Dr. JoAnn Trejo (University of California, San Diego, CA). The cells with stable expression of AT1AR-GFP were a gift from Dr. Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>
Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. Furthermore, many species descriptions are based on few specimens from the same population (often because it is the only available material), which can lead to precipitated taxonomic conclusions. Our results show the robustness of delimiting species based on phenotypic and molecular datasets combined. We analyzed more phenotypic data from more localities than previously known for the A. marmorata clade (sensu Fouquet et al. [7]). The secondary lines of reproductive isolation evidences overlap among lineages, even those formerly used to distinguish one lineage from another (and thus, confirming the status of CCS of each lineage; Fouquet et al. [7]). Additionally, our PCA analysis was not able to distinguish any acoustic group.
The specimens of A. marmorata from the clade corresponding to CCS J, despite overlapping with the other two clades, showed a tendency to have a smaller SVL, shorter call length, and a higher dominant frequency. On the other hand, specimens of A. marmoratafrom Alcatrazes Island (CCS J) are much larger than specimens from other populations (male minimum SVL 25.6 mm, n = 1, female minimum SVL 25.7 mm, n = 4; other mainland populations male maximum SVL 22.4, n = 37, female maximum SVL = 23.4 mm, n = 24). Vertebrate gigantism on islands has massively been reported in the literature ( [78][79][80]). Nevertheless, Rebouças et al. [80] found opposite results, (i.e. dwarfism) analyzing populations of A. marmorata from Ilha Grande Island. Variation in size of isolated insular communities may be related to genetic drift or to their local ecological processes, such as predation pressure, competition, and availability of resources [80][81][82].
Populations from CCSs J, K, and Ma were never found in syntopy. Nevertheless, the north coast of São Paulo deserves special attention. The northernmost distribution of CCSs J and Ma (clade Ma1) is on Ilha das Couves Island and Picinguaba, respectively-both localities within the municipal limits of Ubatuba, state of São Paulo. (DOCX)
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Domain: Biology Medicine
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Taxonomists always have had intense discussions about how species should be delimited and recently many studies have used integrative approaches by combining molecular, morphological, and bioacoustic data. Head longer than wide; HW 34% SVL, HL 39% SVL. Snout rounded in dorsal and ventral views; canthus rostralis indistinct. Loreal region slightly concave. Nostril not protruding, dorsolaterally oriented, rounded. Eye protruding, eye diameter larger than eye to nostril distance. Tympanum distinct, rounded, less than half the eye diameter. Supratympanic fold no apparent. Jaw glands absent. Vocal sac single, internal. Vomerine teeth could not be observed. Choanae moderate, rounded. Tongue elongate; lateral and posterior margins free. Vocal slits elongate, parallel to jaw, extending from posterior portion to the first third of the mouth. Arms poorly preserved, detached from the body. Toes free; toe tips II, III, and IV expanded (toe tips I and V unexpanded). Inner and outer metatarsal tubercles distinct and slightly protruding, ovoid, approximately the same size. Due to the degraded condition of the feet, toe formula could not be observed. Subarticular tubercles rounded and well-developed. Posterior members small and robust; thigh length slightly smaller than shank length (THL 40% SVL and SHL 44% SVL). Metatarsal fold absent. (DOCX)
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Domain: Biology Medicine<|endoftext|>Reorientation of the first signal-anchor sequence during potassium channel biogenesis at the Sec61 complex
The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. [21]). The unusual behaviour of this very short TASK-1 chain underlines the flexibility of the environment provided by the ER translocon [6]. By analysing specific TASK-1 fragments in cultured mammalian cells, we identified a cohort of polypeptides that assume an inverted topology in vivo, and found that this phenotype is 'corrected' upon synthesis of the full-length protein.
These findings provide further evidence for the dynamic nature of Sec61-mediated TM integration and underline the complexity of the molecular events leading to the acquisition of a native membrane topology.
Materials
Where possible, amino acid changes were designed to preserve the biophysical properties of the resulting mutants by maintaining the G pred values for residues located in TMs [5] or provide a side chain of comparable size in hydrophilic loops. Cysnull (cysteine-null) versions of human TASK-1 (UniProt ID O14649) and Paramecium bursaria chlorella virus 1 Kcv (UniProt ID Q84568) genes were made to order (GenScript), and used valine to replace cysteine residues in TMs and serine to substitute for cysteine residues in hydrophilic regions. When synthesized in vitro, the wild-type and Cys-null versions of TASK-1 appear to be of identical size and N-glycosylation status upon SDS/PAGE, confirming that the alterations had not grossly perturbed membrane insertion (results not shown). The locations of novel cysteine probes were also chosen to minimize any perturbation of G pred values for membrane insertion [5]. Single cysteine residues were introduced into the constructs with QuikChange TM site-directed mutagenesis (Stratagene), and Nglycosylation sites were removed and inserted using the same approach. OPG (opsin N-glycosylation tag) derivatives of both proteins were produced from cDNAs containing an in-frame fusion of the coding region for residues 1-26 of bovine opsin (UniProt ID P02699) in place of the original start codon (TASK-1) or before it (Kcv), and all mutagenesis was confirmed by DNA sequencing. All in vitro studies used the pTNT vector (Promega), whereas for expression in HeLaM cells, OPG-TASK-1 derivatives were cloned into pCDNA3.1 + (Invitrogen). TorsinA Myc-His 6 [30] was used as a positive control for EndoH (endoglycosidase H) treatment of cell lysates (see Figure 5).
In vitro transcription and translation
Transcription templates were generated by PCR, in most cases incorporating a C-terminal V5 tag via the reverse primer, and RNA produced as described previously [11]. Ribosomebound integration intermediates were generated by translating the resulting transcripts in rabbit reticulocyte lysate at 30 • C for 40 min in the presence of canine pancreatic microsomes and [ 35 S]methionine/[ 35 S]cysteine followed by treatment with 0.1 mM aurintricarboxylic acid for 10 min at 30 • C, then 2.5 mM cycloheximide (cf. [10,11,13]). Alternatively, ribosome/nascent chain complexes were dissociated using 1 mM puromycin and 20 mM EDTA at 37 • C for 10 min. Membrane-associated components were isolated by centrifugation through a 750 mM sucrose cushion (120 000 g for 10 min at 4 • C) and membrane pellets were resuspended in 110 mM potassium acetate, 20 mM Hepes and 2 mM magnesium acetate (pH 7.2).
Cross-linking, immunoprecipitation and deglycoslation
Resuspended membrane fractions were treated with 1 mM BMH (bismaleimidohexane) cross-linker for 10 min at 30 • C, quenched with 5 mM 2-mercaptoethanol for 5 min and treated with 250 μg/ml RNase A for 5 min at 37 • C [13,31]. SDS was added to 1 % (v/v), samples were heated to 70 • C for 10 min and then processed further as described previously [31]. Antibodies used for immunoprecipitation were: mouse anti-V5 (Serotec), rabbit anti-Sec61α (Richard Zimmerman, Saarland University, Saarbrücken, Germany), rabbit anti-Sec61β and rabbit anti-TRAM (Bernhard Dobberstein, ZMBH, Heidelberg, Germany). Samples were deglycosylated using EndoH (New England Biolabs) according to the manufacturer's instructions or by adding recombinant enzyme to samples in SDS/PAGE sample buffer and incubated at 37 • C overnight.
Cell culture and DNA transfection
HeLaM cells were maintained in DMEM (Dulbecco's modified Eagle's medium) containing 10 % (v/v) FBS and 2 mM Lglutamine, 0.1 mM non-essential amino acids at 37 • C, 5 % CO 2 . Lipofectamine TM 2000 (Invitrogen) was used for transient transfection in accordance with the manufacturer's instructions, and cells were harvested after ∼18 h.
SDS/PAGE and sample analysis
Our aim was to investigate two aspects of TASK-1 biogenesis at the ER membrane: (i) the proximity of distinct TMs to the Sec61 translocon at different nascent chain lengths; and (ii) the orientation of the first TM, a type II signalanchor, during membrane integration. To this end, we employed in vitro translation and site-specific cross-linking, combined with non-native N-glycosylation sites as a topology reporter, to study integration intermediates. Notwithstanding technical limitations [33], both photo-and maleimide-mediated sitespecific cross-linking have provided useful insights into the components and mechanisms underlying polytopic membrane protein biogenesis at the ER (e.g. 1 kcal = 4.184 kJ.
Figure S4 Cysteine-dependent adducts with Kcv membrane integration intermediates
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Analysis of the recombinant PKR-ADAR1 chimera revealed that the editing activity was significantly impaired compared to wild-type ADAR1 [39]. Furthermore, small inhibitory RNA substrates that bind PKR were also able to inhibit the editing activity of the PKR-ADAR1 chimera. These results demonstrate that the dsRBDs play a role in substrate specificity which in turn influences the function of ADARs.
Differences also exist between the dsRBDs amongst the ADAR proteins. Mutagenic analysis of ADAR1 revealed that deletion of dsRBD2 has no effect on the editing activity of the enzyme whereas the deletion of dsRBD1 or dsRBD3 significantly reduces the its editing activity [40]. Interestingly, the two dsRBDs of ADAR2 align to dsRBD1 and dsRBD3 of ADAR1 [13]. Analysis of the two dsRBDs in ADAR2 demonstrated that dsRBD2 is required for editing of all substrates whereas dsRBD1 is only required for editing of a specific subset of substrates [38]. Furthermore, point mutations in dsRBD2 only affected editing of some of its substrates suggesting that it uses different amino acids to interact with different RNAs [36,37]. Together, these studies demonstrate that each of the dsRBDs recognize specific elements in the RNA substrate. [37]. The specific adenosine that is deaminated in the catalytic reaction is highlighted in red. In grey is shown where the deaminase domain is likely positioning during the reaction. PDB file: 2L3J. (B) Crystal structure of the catalytic domain of hADAR2 [41], secondary structures are colored from yellow (N-terminus) to red (C-terminus). The Zinc ion and inositol hexakisphosphate molecule (IP6) cofactor are highlighted and colored in blue. PDB file: 1ZY7.
The Catalytic Deaminase Domain
The structure of the deaminase domain of hADAR2 has been solved by X-ray crystallography [41]. This C-terminal 45 kDa domain comprises of 400 residues which form a globular structure similar to the deaminase domain found in other CDA family members [42][43][44][45]. This core deaminase motif consists of two α-helices (α2 and α5) and four β-strands (β1, β2, β5, and β8) [41]. Additionally, the active site of this deaminase domain contains one zinc ion and one molecule of inositol hexakisphosphate (IP6) (Figure 2b). Hydrolytic deamination catalyzed by ADARs uses a nucleophile comprised of a zinc atom (Zn 2+ ) and a water molecule (H20). The crystal structure of ADAR2 revealed that the Zn 2+ atom is coordinated by two cysteines (C451 and C516) and a histidine (H394) [41]. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine
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Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. Immediately after agonist treatment (1 min), 5-HT 2A R underwent endocytosis; however, continuous treatment with agonist (30 min to 2 h) induced redistribution of internalized 5-HT 2A R and its sequestration in a perinuclear compartment ( Fig. 1A and supplemental Fig. S1). Similar sequestration was observed for the AT1AR upon sustained ligand action (Fig. 1B). To study cellular localization of sequestered receptors, colocalization with markers of different compartments was performed. The sequestered 5-HT 2A R colocalized with, but extended beyond, Rab11 (Fig. 1C), a marker of the perinuclear recycling compartment. There was no colocalization with Lamp1 (lysosomal marker) or giantin (Golgi marker). Brefeldin A, which disrupts the Golgi, did not inhibit 5-HT 2A R sequestration (Fig. 1C). Taken together, sustained stimulation of receptors leads to their sequestration into a perinuclear recycling compartment.
Sequestration of 5-HT 2A Receptors Coincides with Sequestration of cPKC to the Pericentrion and Requires Sustained PKC
Activity-A previous study implicated PKC in agonist-induced 5-HT 2A R internalization (20), and our work has established a critical role for cPKC in the sequestration of recycling molecules into a subset of endosomes in the perinuclear region (6,7). In addition, it was demonstrated previously that sustained activation of cPKC by PMA not only caused sequestration of recycling components but also led to a concomitant translocation of cPKC␣ and -II but not -I into the pericentrion (8). These findings prompted us to investigate whether the observed 5-HT-induced sequestration of receptors involves cPKC. In control cells, the 5-HT 2A R was predominantly present on the cell surface, whereas cPKC localized in the cytosol. 5-HT induced rapid internalization of 5-HT 2A R and a translocation of cPKC to the plasma membrane. More prolonged agonist treatment brought about the sequestration of the 5-HT 2A R but also caused some co-sequestration of PKCII ( Fig. 2A).
Similar observations were made in C6 glioma cells that harbor endogenous 5-HT 2A R: when C6 cells were transfected with GFP-PKCII and treated with 5-HT for 2 h, sequestration of cPKC to the pericentrion was also observed (Fig. 2B). When cells were co-transfected with GFP-tagged Rab11 and PKCII-mCherry, colocalization of PKC with Rab11 compartment was observed (Fig. Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro Objective. The content of exotoxin A, rhamnolipid, and elastase secreted by P. aeruginosa was measured to evaluate the effect of negative pressure on the main virulence factors. Exotoxin A in the negative pressure group was significantly less than that in the control group ( < 0.01) (Figure 2(a)). A similar effect was observed for rhamnolipid and elastase ( < 0.01 and < 0.05, resp.) (Figures 2(b) and 2(c)).
Biofilm Formation.
Biofilm formation was observed in both the atmospheric pressure (AP) group and the negative pressure (NP) group through fluorescence microscopy at 24 h. P. aeruginosa (green) were observed to be big aggregates with excessive biofilm (red) under atmospheric pressure (Figures 3(a)-3(c)). However, P. aeruginosa (green) were observed to be small aggregates with a small amount of biofilm (red) under negative pressure (Figures 3(d)-3(f)).
Negative Pressure Changes Virulence and Biofilm-Regulated Genes in P. aeruginosa.
To investigate the mechanism of negative pressure induction in reducing virulence of P. aeruginosa, quantitative real-time PCR was used to assess relative expression levels of ToxA, RhlA, LasB, LasI, and RhlI genes. Negative pressure was found to significantly inhibit the transcription of ToxA, RhlA, LasB, LasI, and RhlI and the expression of these genes in the negative pressure group was 0.3-, 0.7-, 0.68-, 0.21-, 0.11-fold that of the control group, respectively (Figure 4). The repression of these genes under negative pressure supports the observed reduction in virulence factors and biofilm formation of P. aeruginosa.
Discussion
In recent years, physical therapies have been increasingly popular in the management of contaminated wounds owing to their satisfying wound closure and low risk of microbial resistance [25]. In particular, NPWT has been shown to promote the healing rates and prevent wound infections by multiple mechanisms, including decreasing edema, removal of wound exudates, and translating physical stimulation to signal transduction in cells [26,27]. Previous studies indicated that negative pressure conditions caused by NPWT could alter the gene expression and the function of host cells in vitro, such as bone marrow MSCs and keratinocytes [19,20,28]. However, its potential effects on P. aeruginosa and virulence factors have not been studied yet. In this study, we investigated the effect of negative pressure on the proliferation, virulence factor secretion, and virulenceregulated gene expression of P. aeruginosa, which is one of the most frequently isolated pathogens during wound infections [3].
In this study, the negative pressure value (−125 mmHg) was consistent with the clinical use of negative pressure in NPWT, and the O 2 tension was kept at 20% during bacterial culture in order to reduce interference from low oxygenation [29]. aeruginosa.
Authors' Contributions
Guo-Qi Wang, Tong-Tong Li, and Zhi-Rui Li contributed equally to this work.
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Domain: Biology Medicine
|
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Novel Host Proteins and Signaling Pathways in Enteropathogenic E. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation-mimicking SEPT9 mutant rescued these effects. Collectively, this study provides the first global analysis of phosphorylation-mediated processes during infection with an extracellular, diarrheagenic bacterial pathogen.
Diarrheagenic E. coli are a major global health burden and cause much morbidity and mortality worldwide. Enteropathogenic E. coli (EPEC) 1 is the causative agent of potentially fatal infantile diarrhea and remains an endemic health threat for children in developing countries. EPEC and the closely related enterohemorrhagic E. coli (EHEC) belong to the group of at-taching and effacing (A/E) pathogens that form distinct A/E lesions on the surface of intestinal epithelial cells causing the loss of the characteristic intestinal brush border architecture (1).
Upon attachment to intestinal cells, EPEC uses a syringelike molecular apparatus, the type III secretion system (T3SS), to inject at least 25 unique bacterial effector proteins into the host cell (2)(3)(4). Once translocated into mammalian cells, these effectors manipulate a wide range of host signaling pathways, thereby subverting host cell function and promoting virulence (5). The bacterial translocated intimin receptor (Tir) is one of the first and most abundant effectors injected into the host cell: it mediates intimate attachment of EPEC to the enterocyte apical surface via its interaction with the bacterial surface adhesin intimin (6,7). In concert with other effectors, Tir also provokes an expansive cytoskeletal rearrangement leading to the formation of actin-rich protrusions, termed pedestals, beneath the site of bacterial attachment (8). Besides altering the host cell cytoskeleton, EPEC effectors also manipulate cellular trafficking, host immune response and ion and water homeostasis to cause disease (5). Although significant effort in recent years has led to the identification of multiple key players in both the host and the pathogen, the complex interactions between EPEC and the epithelial host cell, and the underlying molecular mechanisms, are still collectively not well understood.
There is increasing evidence that hijacking host post-translational mechanisms such as protein phosphorylation is a key strategy for bacterial pathogens to efficiently subvert host cell function (9) and there are several indications that this may be the case for EPEC. For example, Tir is phosphorylated upon insertion into the host cell membrane and this event plays a role in the rearrangement of the actin cytoskeleton (10). Another EPEC-encoded effector, NleH, contains a functional kinase domain suggesting the potential of directly phosphorylating host cell targets (11). Moreover, the phosphorylation profiles of a few specific host proteins such as cortactin, CT10 regulator of kinase (CRK) adaptors, focal adhesion kinase (FAK) and mitogen-activated protein kinase 1 (MAPK1), as well as alterations in tyrosine phosphorylation of host proteins, are impacted in an EPEC effector-dependent manner (12)(13)(14)(15)(16)(17)(18). These are selective observations though. Thus, a more comprehensive, system-level analysis is needed to better understand how and to what extent EPEC hijacks host cell phosphorylation to cause disease.
EXPERIMENTAL PROCEDURES
Plasmids-The coding region of human septin-9 isoform a (SEPT9(a); NP_001106963; alternative nomenclature: SEPT9 transcript variant 1) was amplified by PCR and cloned into the EcoRI and XhoI sites of pCMV-Tag 2B to generate a mammalian expression plasmid encoding N-terminally FLAG-tagged SEPT9(a). Site-directed mutagenesis (QuikChange, Agilent Technologies, Santa Clara, CA) was performed to replace Ser-30 of FLAG-SEPT9(a) with glutamate (FLAG-SEPT9(a)-S30E) and alanine (FLAG-SEPT9(a)-S30A), respectively. We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine<|endoftext|>Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro Objective. When the skin or tissue is compromised, bacteria can easily access the underlying tissues, which are believed to be the optimal places for colonization and growth of bacteria. It is reported that the infection rate was as much as 12% in acute wounds and 38% in chronic wounds [2], posing a challenge to clinical doctors. Pseudomonas aeruginosa (P. aeruginosa), a kind of gramnegative bacteria, is one of the most common pathogens isolated from wound infections [3]. It has been widely used in wound infection-related studies [4][5][6] owing to its virulence factor secretion and biofilm formation. P. aeruginosa can secrete various exotoxins, such as exotoxin A, rhamnolipid, and elastase, which play an important role in impeding wound healing and inflammatory reaction [7][8][9]. Moreover, exotoxin A and elastase are encoded by ToxA and LasB and the RhlA gene encodes a rhamnolipid synthase involved in the biosynthetic pathway [10,11]. P. aeruginosa expresses two types of quorum sensing (QS) systems, LasI and RhlI, which contribute to the pathology of cutaneous wound infections [12,13]. Based on this fact, the search for measures to inhibit toxin production and biofilm formation is an active area of clinical research. Recently, as an effective management of contaminated wounds, negative pressure wound therapy (NPWT) has been widely used in clinical laboratories [14,15]. However, whether NPWT could reduce the bacterial load of wounds is still controversial. Weed reported that bacterial colonization increased significantly with NPWT 2 BioMed Research International [16]. Lalliss found that NPWT showed a significant and sustained decrease in the P. aeruginosa levels compared to WTD dressings [17]. However, the mechanism underlying the action of NPWT in the reduction of P. aeruginosa levels is still unknown. It is well known that both the immune status of host and bacterial invasiveness play important roles in the infection process [18]. Thus, the mechanism explaining the change in P. aeruginosa.
Authors' Contributions
Guo-Qi Wang, Tong-Tong Li, and Zhi-Rui Li contributed equally to this work.
==
Domain: Biology Medicine<|endoftext|>Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions*
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. FOXO1 phosphorylation in the cortex and nuclear phosphorylation decrease after ERK1/2 inhibition. A, representative immunoblot of phosphorylated FOXO1 and ERK1/2, AKT, and p38 kinases following trametinib administration. p, phosphorylated; MW, molecular weight. B, densitometry analysis of phosphorylated FOXO1 when compared with total FOXO1. C, densitometry analysis of phosphorylated AKT to total AKT, and phosphorylated p38 to total p38 after trametinib treatment. D, representative immunoblot of nuclear phosphorylated FOXO1 and ERK1/2 after trametinib treatment in mouse kidney cortex. E, densitometry analysis of phosphorylated FOXO1 when compared with total FOXO1 after inhibition of ERK1/2 in the nucleus. F, mRNA expression of FOXO1, SOD2, and catalase at 4 h following trametinib administration. Data are represented as mean Ϯ S. E., n Ն 4. Different superscripts indicate statistically significant differences (p Ͻ 0.05). (34), but not in primary cultures of tracheobronchial epithelial cells or lung fibroblasts. A decrease in oxygen consumption through inhibition of complex I in HL-60 cells was found to occur by ERK1/2 inhibition (35). Overall, ERK1/2 decreases mitochondrial function by three mechanisms: 1) post-translational modifications that decrease electron transport chain activity, 2) down-regulation of PGC-1␣ and mitochondrial gene expression, and 3) decreased expression of mitochondrial protectants (i.e. SOD2). However, ERK1/2 appears to regulate mitochondrial functions differently in various cell types, and the differences may be based on the level of oxidative phosphorylation that occurs in each cell type. RPTC used by Nowak et al. (7) and in our studies derive all their energy from oxidative phosphorylation, similar to that found in kidney cortex.
It is not clear the exact pathways that regulate ERK1/2 during basal conditions in RPTC. In experiments using the specific EGFR inhibitor erlotinib, ERK1/2 phosphorylation was blocked and PGC-1␣ mRNA was increased. Thus, EGFR appears to be a key regulator of ERK1/2, which in turn regulates the basal level of PGC-1␣ mRNA and its downstream mitochondrial genes. was responsible for the funding acquisition.
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Domain: Biology Medicine
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Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro Objective. Based on this fact, the search for measures to inhibit toxin production and biofilm formation is an active area of clinical research. Recently, as an effective management of contaminated wounds, negative pressure wound therapy (NPWT) has been widely used in clinical laboratories [14,15]. However, whether NPWT could reduce the bacterial load of wounds is still controversial. Weed reported that bacterial colonization increased significantly with NPWT 2 BioMed Research International [16]. Lalliss found that NPWT showed a significant and sustained decrease in the P. aeruginosa levels compared to WTD dressings [17]. However, the mechanism underlying the action of NPWT in the reduction of P. aeruginosa levels is still unknown. It is well known that both the immune status of host and bacterial invasiveness play important roles in the infection process [18]. Thus, the mechanism explaining the change in P. aeruginosa levels could not be confirmed under NPWT in vivo. Besides, few studies have reported the bacteria in wounds, secondary to negative pressure treatment, particularly with regard to P. aeruginosa proliferation, virulence, and gene expression. Previous studies have indicated that negative pressure induced by NPWT could alter the gene expression and proliferation of bone marrow mesenchymal stem cells [19,20]. Our previous work had shown that negative pressure had an effect on the growth, secretion, and biofilm formation of Staphylococcus aureus [21].
The aim of this study was to evaluate the influence of negative pressure on the proliferation, virulence factor secretion, biofilm formation, and the virulence-regulated gene expression of P. aeruginosa in vitro.
Bacterial Strain and Preparation
. P. aeruginosa laboratory strain PAO1 carrying the gene encoding the green fluorescent protein (GFP) was obtained from the laboratory of the Chinese PLA Institute for Disease Control and Prevention (Beijing, China). P. aeruginosa.
Authors' Contributions
Guo-Qi Wang, Tong-Tong Li, and Zhi-Rui Li contributed equally to this work.
==
Domain: Biology Medicine<|endoftext|>
In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines.
The above differences between maternal and paternal ICRs are accompanied by an asymmetric influence on mammalian development. Pioneering work in constructing uniparental conceptuses by nuclear transfer in the mouse showed that parthenogenetic embryos with two maternal genomes died before 8.5dpc (days post-coitum) with severely reduced extraembryonic structures, while diploid androgenetic embryos of strictly paternal origin died earlier, with a small embryonic contribution and hyperproliferative extraembryonic structures [10,11]. However, nuclear transplantation studies cannot define the net influence of maternal and paternal imprints on development because these create two sets of either maternal or paternal genomes, with a compounding effect of imprint excess of one parental origin and lack of imprints from the other parent. Next generation models of imprinting deficiency demonstrated the earlier requirement of maternal imprints for development: a specific lack of maternal imprints compromises embryonic viability at 9.5dpc, while the absence of paternal germline imprints leads to a later lethality, at 13.5dpc [3,6,12]. In both cases, the development of extraembryonic tissues is severely altered, in agreement with the proposed evolutionary link between placentation and genomic imprinting in eutherian mammals [13]. However, despite the key role of genomic imprinting for mammalian physiology, the overall effects that maternal and paternal imprints exert on the early embryo transcriptome are unknown, especially at the key developmental time when placentation and vascularization occur (around 8dpc in mouse). This stage represents a crucial transition, where after a period of autonomous growth, the continued embryonic development becomes strictly dependent on maternal resources allocation. Paternal imprints do not seem to be essential for the early embryo to make this transition, but it cannot be excluded that they exert some effects at this stage that will only become apparent later, at 13.5dpc.
Here, we gain insight into the importance of genomic imprinting for the early mammalian embryo (8.5dpc) by a functional dissection of the global gene regulatory impact of maternal versus paternal ICRs at the time of establishment of the fetal-maternal interface through the chorioallantoic placenta. Biological processes under the control of maternal versus paternal ICRs were defined by comparing the transcription profiles of fully imprinted embryos versus maternal imprint-free and completely imprint-fee embryos derived from Dnmt3L mutant mice. Overall, we found that maternal and paternal ICRs have a similar quantitative impact on the transcriptome of the early embryo. However, at 8.5dpc, only the effects of maternal ICRs were focused on biological pathways related to the fetal-maternal interface. In contrast, paternal ICRs elicited, in terms of biological processes, a broad and shallow effect.
We previously hypothesized that the different methylation histories of the two parental germlines may underlie the numerical imbalance between maternal and paternal ICRs [5,6]. Deamination of 5-methylcytosine occurs at a 10-fold higher rate than other transitions, leading to frequent CpG to TpG/CpA mutations in mammalian genomes despite a dedicated repair pathway [14][15][16][17].
Here, we test this hypothesis by a systematic assessment of the sequence evolution of ICRs in different mammalian lineages and in comparison to other sequence categories. In doing so, we provide evidence that paternal ICRs have lost CpG sites and therefore their methylation targets at a significantly higher rate than maternal ICRs, while the latter in fact exhibit a relative gain of CpG motifs compared to similar but non-imprinted genomic regions. We propose that a combination of high mutational pressures at paternal ICRs together with functional selective pressure reinforcing methylation-dependent repression of ICRs, has led to the oocyte dominating the control of the fetal-maternal interface through genomic imprinting in eutherian mammals. Our results provide the first comprehensive view of the forces acting upon the regulatory sequences governing genomic imprinting in mammals.
Developmental and epigenetic characterization of imprint-free embryos
The impact of imprinted gene expression on development prior to mid-gestation has never been investigated on a genome-wide scale. To understand which biological pathways are regulated by maternal and paternal ICRs, respectively, we compared the developmental potential and transcription profiles of 8.5dpc embryos that differ in their imprinting status but have an otherwise normal genome. Three different imprinting states were investigated: fully-imprinted (MP) embryos, maternal imprint-free (0P) embryos and completely imprint-free (00) embryos. Here, M and P denote a normally imprinted set of respectively maternal and paternal chromosomes, and 0 denotes a chromosome set without imprints.
Author Summary
In mammals, a subset of genes is expressed from only one chromosomal copy, depending on its parental origin. In the maternal case for example, 470 probes sets detected a decrease of expression in the 0P and 00 samples to 80% (-0.322 log2-ratio) or less relative to the MP sample.
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Domain: Biology Medicine
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Herpes Simplex Virus gD Forms Distinct Complexes with Fusion Executors gB and gH/gL in Part through the C-terminal Profusion Domain*
Herpes simplex virus entry into cells requires a multipartite fusion apparatus made of glycoprotein D (gD), gB, and heterodimer gH/gL. The aa sequence of gL C terminus was changed from SRRL to SRRLLAGLPIPNPLLGLDSTRTVHH-HHHHH. gC V5 was generated as follows. The gC ORF was PCR-amplified from HSV-5(F) DNA with primers encoding the V5 epitope 5Ј-AGA TCT AGG CCT ATG GCC CCG GGG CGG GTG GGC CTT GCC G TG GTC CTG TGG AGC CTG- 3Ј and 5Ј-GCA CGG GGC GGC CGC TTA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG GAT AGG CTT ACC GGC TAG CCG ATG ACG CTG CCG CGA CTG TGA TGT GCG G-3Ј. The StuI-NotI-digested gC amplimer was ligated to MTS vector. For all plasmids, the ORF was sequenced.
Genetic Engineering of HSV1(BAC)-gD st -HSV1(BAC)-gD st was generated by bacterial artificial chromosome (BAC) "galK recombineering," employing the Escherichia coli strain SW102 and galK (galactokinase) positive/negative selectable marker, kindly provided by Dr. N. G. Copeland) (45). pYEbac102 HSV-BAC was provided by Mireia Hudson and carries the BAC sequences inserted between UL3 and UL4 genes. The galK cassette was recombined in HSV-1(BAC) to replace the gD stop codon. To this end, the cassette was PCR-amplified from pGalK plasmid with primers that annealed in their 5Ј end to sequences upstream and downstream of gD stop codon. The oligonucleotides used for amplification were 5Ј- CCC ACA TCC GGG AAG ACG ACC AGC CGT CCT CGC ACC AGC CCT TGT TTT ACC CTG GTG ACA ATT AAT CAT CGGCA-3Ј and 5Ј-CAT CCC AAC CCC GCA GAC CTG ACC CCC CCG CAC CCA TTA AGG GGG GGT ATT CAG CAC TGT CCT GCT CCTT-3Ј. The amplimer was recombined into the HSV-5 BAC, and the recombinant bacteria were selected on minimal media containing galactose as the only carbon source. Next, the galK cassette was substituted with sequences encoding the One-streptag by means of a double-stranded oligonuocletide that carried flanking sequences homologous to gD sequences upstream and downstream of the stop codon. The double-stranded oligonucleotide was generated by annealing-extension of two synthetic oligonucleotides 5Ј- CCC ACA TCC GGG AAG ACG ACC AGC CGT CCT CGC ACC AGC CCT TGT TTT ACA GCG CTT GGA GCC ACC CGC AGT TCG AGA AAG GTG GAG GTT CCG GAG GTG GAT CCG GAG GT- 3Ј and 5Ј-CAT CCC AAC CCC GCA GAC CTG ACC CCC CCG CAC CCA TTA AGG GGG GGT ATT CAT TTT TCG AAC TGC GGG TGG CTC CAC GAT CCA CCT CCG GAT CCA CCT CCG GAA CC-3Ј. Its homologous recombination into the HSV-BAC was achieved by selecting against the galK cassette, i.e. by resistance to 2-deoxy-galactose on minimal plates containing glycerol as carbon source. Chloramphenicol selection was kept throughout, to maintain the HSV-BAC sequences. The recombinant HSV1(BAC)-gD st DNA was extracted from bacteria and transfected into 293 T cells to reconstitute the virus. The 3Ј of gD gene was sequenced for accuracy in DNA extracted from bacteria and in virus stock.
gD Constructs-The linear map of the gD constructs employed in this study is shown in Fig. The resolution of function of the various complexes will ultimately require cell-free assembly as well as identification and characterization of the cellular partners with which the glycoproteins interact.
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Domain: Biology Medicine<|endoftext|>This below document has 2 paragraphs that end with 'significant at (P < 0.05)'. It has approximately 607 words, 28 sentences, and 14 paragraph(s).
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DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice
Background Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health.
Statistical analysis
All statistical analyses were performed by Graphpad Prism 5. Ink software. The differences of the data between all the groups were compared by one-way ANOVA. Survival rate of the mice was compared using the Kaplan-Meier method. The results in comparisons between groups were considered different if P < 0.05.
TgDPA recombinant plasmids expression
As for the eukaryotic plasmid pTgDPA, a separate trial was conducted to screen in vitro expression. Lysates of the BHK cells transfected with pTgDPA were probed with anti-rTgDPA polyclonal antibodies revealing successful expression of the protein ( Figure 2C), while cells transfected with empty pVAX1 exposed no specific bands and remained negative ( Figure 2D).
Antibody response in immunized mice and subclass determination
The titers of total IgG, beside subclasses IgG 1 and IgG 2a were measured prior to and after immunization, using standard ELISA. As shown in Figure 3A, specific total IgG antibodies were detected in the experimental group vaccinated with pTgDPA. There was a significant difference at (p < 0.05) between pTgDPA group after first immunization (0.651 ± 0.04) and 2 nd immunization (0.752 ± 0.03), compared to the control groups of pVAX1 (0.073 ± 0.011), PBS (0.050 ± 0.07) and Blank (0.07 ± 0.03).
IgG isotype determination revealed that, both IgG 1 (1.33 ± 0.485) and IgG 2a (0.506 ± 0.029) were significantly (P < 0.05) stimulated after delivering the antigen ( Figure 3B and C). Moreover, the difference between the levels of these isotypes was found to be significant at (P < 0.05), for the advantage of IgG 1 ( Figure 3G). Regarding IgA, IgM and IgE, and when compared to the control groups, dynamics of the first two antibody types demonstrated high OD values (P < 0.05) in the immunized group (0.974 ± 0.33) and (1.55 ± 0.26) respectively ( Figure 3D and E). However, IgE activity showed no significant changes at the time of evaluation (Figure 3 F).
Cytokine production
IL-4 and IL-17 of the pTgDPA group showed a significant difference (P < 0.05) against the control groups ( Figure 4B and C). The peak of production was also at 2 weeks after the booster dose (91.6 ± 1.34) for IL-4 and (62.2 ± 2.83) for IL-17. Additionally, TGF-β1 ( Figure 4D) displayed a different activity. Immunized groups showed a significant peak after the first immunization (70.4 ± 6.66), which was dramatically decreased (34.2 ± 2.26) two weeks after the last immunization. Compared to the control groups both time points were significant at (P < 0.05).
After both prime and booster immunizations, MHC-I molecules of the immunized group displayed sustained high significant readings (33. Figures 5C & 6C). Concerning MHC-II molecules, a gradually increasing pattern was noticed in the vaccinated group ( Figures 5D & 6D) starting at week 2 of the experiment reaching a peak point (5.81 ± 0.87) at week 4. Compared to control groups (1.85 ± 0.74), (1.74 ± 0.65) and (1.99 ± 0.82) the difference between these values was found significant at (P < 0.05).
Protection of vaccinated mice against challenge with T. gondii RH strain
In order to evaluate the protective effect of pTgDPA DNA vaccine against acute toxoplasmosis, vaccinated and control mice groups were challenged with lethal T. gondii tachyzoites within the second week after booster immunization. This finding may encourage more investigations in evaluating the immunogenicity of DPA based vaccines against Toxoplasmosis.
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Domain: Biology Medicine
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Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner* Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. 7B). Importantly the protective effects of PMA and 5-HT were inhibited by 1-butanol (Fig. 7B), whereas the secondary alcohol (2-butanol) did not have any effect (data not shown).
PAR-1 provides an example of a GPCR that is rapidly internalized and degraded following its activation (17,18), and thus, it was chosen for additional study. First we confirmed that treatment with the PAR-1 agonist peptide (SFLLRN) led to significant degradation of PAR-1, whereas PMA caused sequestration of PAR-1 as analyzed by confocal microscopy (Fig. 8A). Most interestingly, however, treatment of cells with 5-HT led to co-sequestration of both receptors in a cPKCand PLD-dependent manner, and when cells were pretreated with 5-HT, SFLLRN did not induce degradation (Fig. 8B). Taken together, these results indicate that sustained stimulation with 5-HT changes the cellular fate of membrane receptors that are normally targeted for degradation.
DISCUSSION
The results from this study reveal dramatic effects of sustained stimulation of cPKC by GPCRs on the trafficking of several proteins leading to their sequestration in the perinuclear region. It is shown that sustained stimulation of G q -coupled GPCRs leads to their sequestration into a subset of the Rab11-positive recycling compartment. This sequestration was cPKC-dependent and required continuous cPKC activation and PLD activity. Importantly receptor stimulation and sequestration globally affected cellular trafficking by sequestration of recycling endosomes and changed the cellular fate of EGFR and PAR-1 by diverting their trafficking and thus protecting them from degradation (Fig. 9).
Many studies have implicated PKC in the internalization of specific receptors, transporters, and channels (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). Some of these studies have focused on direct phosphorylation of these molecules by PKC. Although the current studies do not rule out a role for direct phosphorylation by PKC in mediating specific effects, the results point to a more global cPKC-dependent process by which sustained activation of cPKC regulates endosomal trafficking. Sustained agonist treatment led to activation of cPKC and resulted in receptor and cPKC sequestration in a PKC-dependent subset of the Rab11 compartment, the pericentrion. This sequestration was dependent on continuous cPKC activity. Pretreatment with cPKC inhibitor prevented receptors sequestration, whereas posttreatment led to dispersal of sequestered receptors. It needs to be emphasized that internalization of receptors was not completely blocked because some receptors were still observed in early endosomes. Similarly internalization of the biotinylated receptor was only partially inhibited when cells were pretreated with cPKC inhibitor.
The observed process also requires PLD as a further downstream target of cPKC activation, suggesting that either phosphatidic acid (the product of the PLD reaction) or subsequent lipid metabolites are required for this novel endosomal trafficking. It was shown previously that PLD2 was required for endocytosis of angiotensin receptor (33). Agonist-induced sequestration of recycling components may have profound effects on their function and could explain pathologies resulting from sustained receptor activation.
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Domain: Biology Medicine<|endoftext|>Novel Host Proteins and Signaling Pathways in Enteropathogenic E. Primers are listed in the supplemental Table S1. All plasmids were verified by sequencing.
Cell Culture and SILAC Labeling-HeLa cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) high glucose (Thermo Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids (NEAA) (Gibco) and 1% GlutaMax (Gibco, Thermo Scientific, Waltham, MA). Cells were grown at 5% (v/v) CO 2 Bacterial Strains and Infection-The prototypical EPEC O127:H6 strain E2348/69 and the T3SS-deficient strain EPEC ⌬escN were used in this study (23,24). Prior to infection, EPEC grown on Luria-Bertani (LB) agar plates were used to inoculate LB broth. The bacterial culture was incubated overnight at 37°C in a shaking incubator. The bacterial cultures were centrifuged at 2300 ϫ g for 5 min at room temperature. The bacterial pellet was resuspended in prewarmed serum-and antibiotic-free cell culture medium to a density of 8 ϫ 10 7 bacteria/ml and incubated in a 37°C incubator with 5% (v/v) CO 2 for 3-4 h. Upon reaching 80 -90% confluence HeLa cells were first washed and then incubated with prewarmed serum-and antibioticfree cell culture medium 2 h prior to infection with EPEC at a multiplicity of infection (MOI) of 20:1. Small volumes of serum-and antibiotic-free cell culture medium were used during infection to accelerate EPEC attachment and promote more synchronized infection conditions. Uninfected cells were treated with prewarmed serumand antibiotic-free cell culture medium only.
Sample Preparation for Phosphoproteome Analysis-At specified time points postinfection, HeLa cells were washed twice with ice-cold phosphate-buffered saline (PBS) and harvested on ice. Cell lysis was performed similarly to what has been previously described (21). Briefly, cells were resuspended in 50 mM ammonium bicarbonate and 1% (w/v) sodium deoxycholate and boiled for 5 min. MgCl 2 was added to a final concentration of 1.5 mM and DNA was digested by Benzonase endonuclease (Santa Cruz Biotechnology, Dallas, TX) at room temperature for 30 min. After combining the lysates of light, medium and heavy labeled cells (1:1:1), proteins were reduced with 10 mM DTT at room temperature for 30 min and alkylated with 55 mM iodoacetamide at room temperature for 20 min in the dark. Trypsin (Promega, Fitchburg, WI) digestion was performed at a trypsin-toprotein ratio of 1:100 (w/w) under constant agitation at 37°C for 16 h. Peptides were desalted with C18 StAGE Tips (25). Phosphopeptides were enriched using lactic acid-modified titanium dioxide (GL Sciences, Tokyo, Japan) and consecutively eluted by 5% (v/v) ammonium hydroxide and 0.5% (v/v) pyrrolidine as previously reported (26,27). Eluted phosphopeptides were desalted with C18 StAGE Tips (25) prior to analysis by LC-MS/MS. NanoLC-MS/MS Analysis-Phosphopeptides were separated using an 1100 series nanoflow high-performance liquid chromatography (HPLC) instrument (Agilent) equipped with a two column set up. Samples were loaded onto a 2 cm long, 100 m inner diameter fused silica trap column containing 5.0 m Aqua C18 beads (Phenomenex, Torrance, CA) for 10 min in buffer A (0.5% acetic acid) at a flow rate of 5 l/min prior to analytical separation. We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine<|endoftext|>Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions*
Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. Different superscripts indicate statistically significant differences (p Ͻ 0.05). (34), but not in primary cultures of tracheobronchial epithelial cells or lung fibroblasts. A decrease in oxygen consumption through inhibition of complex I in HL-60 cells was found to occur by ERK1/2 inhibition (35). Overall, ERK1/2 decreases mitochondrial function by three mechanisms: 1) post-translational modifications that decrease electron transport chain activity, 2) down-regulation of PGC-1␣ and mitochondrial gene expression, and 3) decreased expression of mitochondrial protectants (i.e. SOD2). However, ERK1/2 appears to regulate mitochondrial functions differently in various cell types, and the differences may be based on the level of oxidative phosphorylation that occurs in each cell type. RPTC used by Nowak et al. (7) and in our studies derive all their energy from oxidative phosphorylation, similar to that found in kidney cortex.
It is not clear the exact pathways that regulate ERK1/2 during basal conditions in RPTC. In experiments using the specific EGFR inhibitor erlotinib, ERK1/2 phosphorylation was blocked and PGC-1␣ mRNA was increased. Thus, EGFR appears to be a key regulator of ERK1/2, which in turn regulates the basal level of PGC-1␣ mRNA and its downstream mitochondrial genes. Nevertheless, it remains to be determined what is activating the EGFR under these conditions. Possible candidates FIGURE 6. ERK1/2 inhibition during AKI attenuates an increase in serum creatinine and increases PGC-1␣ and TFAM proteins. A, representative immunoblot of phosphorylated ERK1/2, total ERK1/2, TFAM, and PGC-1␣ following IR AKI. p, phosphorylated; MW, molecular weight. B, mRNA expression of PGC-1␣ and NRF1 following IR AKI at 3 h after IR in the kidney cortex. C and D, densitometry analysis of PGC-1␣ (C) and TFAM (D) proteins following 3 h IR. E, serum creatinine was assessed 3 h after IR AKI. Data are represented as mean Ϯ S. E., n Ն 5. was responsible for the funding acquisition.
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Domain: Biology Medicine
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Novel Host Proteins and Signaling Pathways in Enteropathogenic E. Analytical separation was accomplished using a 200 mm in length and 75 m of inner diameter column containing 3 m ReproSil-Pur C18-AQ material (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) at a flow rate of 250 nL/min. Both trap and analytical columns were prepared in-house. Peptides were eluted from the analytical column by altering the gradient from 100% buffer A (0.5% acetic acid) to 40% buffer B (0.5% acetic acid, 80% acetonitrile) over 90 min. The eluting peptides were directly infused into an LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific) via ESI. MS and MS/MS information was collected in a data-dependent manner using the following settings: one full scan (resolution 60,000; m/z 300 -1600) followed by top 15 MS/MS scans using collision-induced dissociation in the linear ion trap mass spectrometer (ITMS; min. signal required: 200, isolation width: 3 Th, normalized collision energy: 35, activation Q: 0.25, activation time: 10 ms) using dynamic exclusion (repeat count: 1, repeat duration: 30 s, exclusion list size: 500, exclusion duration: 60 s). AGC targets of 1 ϫ 10 6 and 3 ϫ 10 4 with maximum fill times of 250 and 100 ms were used for MS and MS-MS scans, respectively.
Because of higher sample complexity, peptides for whole proteome analysis were analyzed on the Q Exactive mass spectrometer (ThermoFisher Scientific) coupled to a two-column EASY-nLC1000 system. Trap and analytical column set up were similar as described above, with the analytical column being 400 mm in length and containing 1.9 m ReproSil-Pur C18-AQ material (Dr. Maisch GmbH). Buffer A (0.1% formic acid) was used for the trap column. Peptides were eluted from the analytical column using 240 min gradient with following conditions: 100% buffer A (0.1% formic acid) to 32% buffer B (0.1% formic acid, 80% acetonitrile) in 210 min, then 32% to 40% buffer B in 10 min followed by increase to 100% buffer B in 5 min and 100% buffer B for an additional 15 min. MS and MS/MS information was collected in a top-10 data-dependent approach switching between MS (resolution 70,000; AGC target of 3 ϫ 10 6 ), and HCD MS-MS events (resolution 17,500, 4.5% underfill ratio, AGC target of 1 ϫ 10 5 with a maximum injection time of 40 ms, an isolation width of 2.2 Th, and an NCE of 28 with 20% stepping).
SILAC ratios were transformed into log 2 scale and p values for changes from the null hypothesis (SILAC ratio 1:1) were calculated. Only protein and phosphopeptide measurements observed in a minimum of two out of three biological replicates were considered for analysis. To provide a broad overview of regulated phosphorylation site and protein candidates during EPEC infection, following criteria were applied: all phosphorylation sites and proteins that showed a statistically significant (p Ͻ 0.05) change compared with the uninfected control at least at one time point and either an increase or a decrease of 1.5-fold in at least one experiment at that time point were considered to be "regulated" by EPEC (if a SILAC ratio could only be determined in one of the three experiments then that protein was not considered at all). All bioinformatic analysis was performed using this set of regulated phosphosites and proteins. T3SS-dependence for regulated sites and proteins was determined by comparing their H/L SILAC ratios (EPEC WT-infected versus uninfected) with their M/L SILAC ratios (EPEC ⌬escN-infected versus uninfected) at each time point based on a minimum of two out of three biological replicates. Phosphosites and proteins were defined as being "T3SS-regulated" when the impact of EPEC WT-and EPEC ⌬escN-infected on the host phosphoproteome or proteome were significantly different in at least one time point, i.e. when a significant difference between H/L and M/L SILAC ratios was observed (p Ͻ 0.05). A significant increase of H/L SILAC ratios compared with M/L SILAC ratios was considered T3SSdependent up-regulation, whereas a decrease was defined as T3SSdependent down-regulation. We identified SEPT9 as a novel host target in EPEC pathogenesis, established the biological relevance of SEPT9 Ser-30 phosphorylation, and provide mechanistic insights into EPEC's strategy to hijack host signaling.
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Domain: Biology Medicine<|endoftext|>New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. The transcript encoding the GRIK2 subunit of the high affinity kainite (KA) receptor is edited at the Q/R site in transmembrane segment 1 (TM1) and also at the I/V and Y/C sites in TM2 [80]. Editing at these positions affects the Ca 2+ permeability of the KA receptor channel. In contrast to the AMPA receptors, editing at the Q/R site of the GRIK2 transcript increases the Ca 2+ permeability of the KA receptor [80]. Burns and colleagues demonstrated that transcripts encoding the G-protein-coupled 5-HT2C undergo editing at sites within the second intracellular loop which results in 3 amino acid changes of isoleucine to valine (I/V), asparagine to serine (N/S) and isoleucine to valine (I/V) [81]. RNA editing at these sites generates several 5-HT2C isoforms which are expressed at various levels in different brain regions [81]. The fully edited 5-HT2C isoform has a reduced affinity for the G-protein PLC which inhibits the downstream phosphatidylinositol signal pathway [81]. Therefore, RNA editing regulates the efficacy of receptor-G-protein coupling. The Kv1.1 transcript encoded by the voltage gated potassium channel gene is edited at a single site and results in an isoleucine-to-valine (I/V) recoding event [82]. The I/V site is located in a highly conserved region of the channel pore that is involved in modulating potassium ion flow and RNA editing at this site results in the fast inactivation of the Kv1.1 channel [82]. The Gabra-3 transcript encoding the α3 subunit of the GABAA receptor undergoes site-specific editing, resulting in an isoleucine to methionine (I/M) amino acid change [83]. RNA editing at this site increases during development and editing levels are inversely related to expression of the α3 subunit containing GABAA receptor [84].
Overall, the highest levels of site-specific editing events in mammals occur within transcripts expressed in the CNS, affecting the functional properties of the proteins. However, the mechanism through which ADARs is able to identify a specific adenosine within these transcripts is not yet understood.
RNA Editing in Alu Repeated Sequences
The continuing development of high throughput sequencing methods has led to major improvements in the detection levels of genome-wide editing events. To date it has been estimated that there are over one hundred million editing sites in the human genome [85], and the whole transcriptome analysis has revealed that A-to-G changes account for greater than 90% of the total mismatches identified in humans [64,86], editing that result in recoding account are rare amongst the total number of editing sites identified in the human transcriptome. However the level of editing at site-specific sites is high whereas editing of repeat sequences is general low but widespread.
Alu elements are primate specific non autonomous short interspersed nuclear elements (SINEs) which make up approximately 10% of the human genome [87]. The Alu element is approximately 300 bp comprising of an A-rich region. Alu repeats are generally clustered in gene rich regions, within UTRs and introns [88]. Due to their repetitive nature Alus are capable of forming long intramolecular duplex structures through complementary pairing between antisense repeats found within a single pre-mRNA. Often two Alu elements are present, adjacent to each other in reverse orientation in the genome which are ideal substrates for ADARs. SINE families in mice are not as evolutionary conserved as the primate SINE Alu elements so they do not base pair as well. Perhaps they play a role in both, as in the words of Tennessee Williams, "If I got rid of my demons I'd lose my angels".
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Domain: Biology Medicine
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Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. Mutations of Glu-9 and Glu-115 reduced the Mg(II) binding stoichiometry significantly after equilibrium dialysis against buffer containing 0.4 mM MgCl 2 . This might have resulted from one of the multiple coordination sites for Mg(II) in the enzyme being removed or replaced by a ligand of lower affinity to Mg(II). The effects of the mutations of Glu-9, Asp-111, Asp-113, and Glu-115 to alanine on the relaxation activity were less severe than those observed for mutations of carboxylates proposed to be Mg(II) coordination sites involved in nucleotidyl transfer in other enzyme systems (11,14,30,31). It is possible that for E. coli DNA topoisomerase I, water or a DNA phosphate could substitute for the mutated carboxylate in Mg(II) coordination in the relaxation reaction to provide partial enzymatic activity, especially at Mg(II) concentration significantly above the minimal concentrations needed for relaxation activity. The effect of mutation of Asp-111 on the enzyme activity and Mg(II) binding was significantly smaller than mutations of the other carboxylates, so it is unlikely that Asp-111 is a catalyticly important residue.
The Glu-9, Asp-113, Glu-115, and Arg-321 mutants also had reduced DNA binding activity. This could be due to the participation of the residue in direct protein-DNA interaction which is a plausible role for Arg-321. Alternatively, the reduced DNA binding could be due to the effect of the mutation on the protein folding. It also cannot be ruled out that the lower Mg(II) binding stoichiometries observed for the Glu-9 and Glu-115 mutants might be due to the effect of the mutations on the protein structure and not due to loss of a Mg(II) coordination site. A Mg(II) binding site distinct from the catalytic center has been proposed for the EcoRV restriction endonuclease (32). E. coli DNA topoisomerase I may also have a second Mg(II) binding site away from the active site region that is required for relaxation activity because of its effect on the protein conformational changes that take place during the relaxation reaction cycle (28). The carboxylates at the active site may be conserved for their roles in protein structure instead of catalytic functions. The magnitude of the effect of a single mutation of these catalytically non-essential residues on the overall protein stability may depend on the E. coli strain background, but nevertheless be of sufficient significance to account for the evolutionary conservation.
The steps of DNA cleavage and religation may or may not involve the same catalytic residues in the activation of nucleophiles as well as the stabilization of transition states and leaving groups. Presently there is a lack of data to address this question, and it remains unclear what these catalytic residues may be. Among the mutants tested, the Glu-9 mutant had the greatest loss of DNA cleavage activity with only a modest reduction in non-covalent DNA binding. Besides a possible role in binding Mg(II), this residue might be involved in the catalytic step of DNA cleavage. In contrast, the Arg-136 mutant had normal DNA cleavage activity but no relaxation activity. It might be needed in the DNA strand passage step since it could perform the inter-molecular rejoining of DNA.
There is also a lack of information on the orientation of the DNA substrate when it binds to the active site. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
==
Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. However, the mammalian enzyme exhibits additional hydrophobic properties (37). Sequence analysis revealed that mammalian ValRS has conserved the positively charged N-terminal extension (residues 200 -298) that distinguishes yeast ValRS from its bacterial counterparts while acquiring an additional hydrophobic domain (residues 1-199) that is responsible for interacting with the four subunits of elongation factor EF-1H (␣, , ␥, and ␦ subunits) (38). A very recent report showed that, like the Ad of yeast ValRS, the positively charged N-terminal extension (residues 200 -298) of the mammalian enzyme is also a nonspecific TRBD (15).
However, regardless of the detailed interpretation, the most interesting findings reported here are the capability of a nonspecific TRBD to enhance the formation of an active synthetase-tRNA complex and the conversion of an E. coli aaRS gene to a dual functional yeast gene encoding both cytoplasmic and mitochondrial activities. To our knowledge, this appears to be the first example wherein an engineered bacterial aaRS gene can provide both cytoplasmic and mitochondrial aminoacylation activities in yeast. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
==
Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. coli ValRS and its fusions were all properly expressed in yeast. Fusion of Arc1p or the Ad of yeast GlnRS to E. coli ValRS significantly enhanced its expression level (2-and 7-fold, respectively), whereas fusion of the Ad of yeast ValRS to the E. coli enzyme slightly reduced its expression level (1.5-fold). Evidently, not all TRBDs enhanced the protein expression level of E. coli ValRS in yeast. This observation also reinforces the idea that the negative phenotype of E. coli ValRS in the complementation assay (Fig. 2, number 3) was not caused by a lower level of protein expression.
Converting E. coli ValRS into a Functional Yeast Mitochondrial Enzyme-In contrast to the cytoplasmic aaRSs, the mitochondrial enzymes are generally believed to be more bacterium-like in terms of their genetic origin and sequence homology. To investigate whether the mitochondrial activity of yeast VAS1 can be functionally substituted with E. coli valS, a DNA segment coding for the MTS of the mitochondrial precursor form of yeast ValRS (base pairs ϩ1 to ϩ138 relative to the ATG1 initiator codon) was inserted in-frame at the 5Ј end of E. coli valS to facilitate the mitochondrial import of the encoded bacterial protein, and the mitochondrial complementation activity of the resulting construct was tested. As shown in Fig. 3, although the E. coli enzyme per se (without fusion of an MTS) failed to restore the growth phenotype of CW1 on YPG, fusion of an MTS to the bacterial enzyme enabled it to rescue the mitochondrial defect of the knock-out strain (Fig. 3B, numbers 3 and 5). Interestingly, ValRS of B. subtilis, a Gram-positive bacterium that is genetically highly divergent from E. coli, could also be made a functional yeast mitochondrial enzyme by fusion of the MTS (Fig. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
==
Domain: Biology Chemistry Medicine
|
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PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. It is thus clear that PA28 does not act simply by expending the fraction of proteasomal products that can be accommodated in the groove of MHC class I molecules directly or after trimming. In this regard, it is worth noting that both 20S and 26S immunoproteasomes display a similar high propensity to release 8-10 residue long products, although 20S has also an increased capacity to generate longer fragments [101]. However, correct evaluation of the overall efficiency of different immunoproteasome species in producing peptides with a size potentially suitable for class I antigen presentation requires normalization of their rates of generation with those of substrate hydrolysis. Therefore, since in vitro 26S immunoproteasomes were found to degrade unfolded proteins at 10-fold higher rates than 20S and PA28Įȕ-20S [84,101,114], it is evident that 26S holoenzymes are potentially the most efficient immunoproteasomal species in terms of generating higher amounts of peptides with the correct size to serve in MHC class I antigen presentation either directly or after aminopeptidase trimming.
PA28 as a Smart Sieve
Surprisingly, PA28Įȕ-20S immunoproteasomes were found to display a reduced ability to generate longer products that, in principle, might depend upon conformational changes in proteasomal active sites. This hypothesis has already been suggested to explain the biochemical properties of PA28 [36,72,75], and specifically its ability to stimulate coordinated dual cleavages of short synthetic peptides (typically 19-25 residues long) by 20S particles [82,83]. This possibility, however, seems unlikely for PA28Įȕ-20S immunoproteasomes since, as already pointed out, association with PA26 does not induce any structural modification of proteasomal catalytic ȕ subunits [71]. Alternatively, PA28Įȕ might primarily act as a molecular sieve that retains longer protein fragments inside the 20S proteolytic chamber until they are cleaved to peptides that are small enough to diffuse to the outside. This later model would be consistent with detailed kinetic analyses showing that PA28 exerts its activating influence by enhancing bi-directional passage of short (3-4 residues) peptides [52] and with an important in vitro/in silico study that identified one of the major factors involved in the enhancement of double cut efficiency induced by PA28 in a reduced efflux of longer peptides out of the 20S particle [115]. Furthermore, it was recently shown that a PA28Įȕ complex lacking the unstructured and highly mobile PA28Į loops surrounding the central pore of the heptameric ring cleaves substrates longer than a nonpeptide more efficiently than wild type PA28. On these bases, it was hypothesized that the flexible loops of PA28 might act as gatekeepers that block the exit of longer peptides from the proteolytic chamber [44]. Selectivity based exclusively on peptide size, however, cannot account for the overall effects of PA28 on the patterns of proteasome products observed by Raule et al. [101]. In fact, quantitation of products demonstrated that several individual peptides with a length of 8-23 residues are released in much higher amounts by PA28Įȕ-20S than by 20S or 26S immunoproteasomes [101]. Therefore, PA28 appears to act as a selective filter that promotes preferential passage of only subset of specific long products through its central channel, presumably based on sequence.
At present, the properties that might allow specific longer peptides to evade the constraint imposed by PA28 towards their efflux are not completely clear. However, the finding that products longer than seven residues whose generation is strongly enhanced in the presence of PA28 are, on average, more hydrophilic than those preferentially released by 20S alone [101], strongly suggests that the passage of polar/charged long peptides thorough PA28 might be favored. In this model, PA28 would act as a selective "smart" sieve that strictly controls the exit of products from proteasomes on the basis of size and sequence ( Figure 2). As a result, PA28Įȕ would promote preferential efflux from the 20S proteolytic cavity of only a reduced number of individual peptides longer than 6-7 amino acids, while the majority of the other proteasomal products are retained inside where they are further cleaved to smaller pieces before they diffuse outside. In accordance with this model, the central channel of PA28Į ring is almost completely lined by charged or polar residues [34], and is thus well suited for permitting the passage of water soluble peptides. Importantly, this molecular model would be also consistent with our findings on 19S-20S-PA28 immunoproteasomes. In this case, the absence of a clear difference in size distribution [21] argues that in hybrid proteasomes (as in 26S canonical particles) the main route of exit of peptides from the inner proteolytic chamber is regulated by the 19S cap, while PA28 would exert its major effect by allowing preferential sorting of selected products through its central channel.
Other Potential Biological Functions
A full understanding of the exact biological functions of PA28 would undoubtedly represent an important achievement, especially in the light of the observation that mammalian cells contain significant amounts of PA28Įȕ-20S immunocomplexes [22,24,26,[58][59][60][61] whose abundance further increases upon INF-Ȗ stimulation [31,55,62]. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
==
Domain: Biology Chemistry Medicine<|endoftext|>Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. S. C. Section 1734 solely to indicate this fact. before use. Plasmid DNA was purified by cesium chloride centrifugation.
Enzyme Expression and Purification-Wild-type E. coli DNA topoisomerase I enzyme was expressed from E. coli MV1190 cells transformed with plasmid pJW312. The R136A and R321A mutants were expressed in E. coli AS17 (topA am PLL1(Tc r supD ts ), from R. E. Depew, Northeastern Ohio University). The E9A, D111A, D113A, and E115A mutants were expressed in E. coli GP200 (gyrA(Nal r ) gyrB225⌬ (topAcysB)204) (22). Enzyme purification was carried out using the previously described procedures (23) with an additional hydroxyapatite chromatography step between the phosphocellulose and singlestranded DNA-agarose columns.
Relaxation Activity Assay-Wild-type and mutant enzymes were serially diluted and assayed for relaxation activity in 20 l with 0.5 g of negatively supercoiled plasmid DNA, 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/ml gelatin, and 6 mM MgCl 2 unless indicated otherwise. Incubation was at 37°C for 30 min. The reactions were stopped by the addition of 5 l of 50% glycerol, 50 mM EDTA, and 0.5% (v/v) bromphenol blue. After electrophoresis in a 0.7% agarose gel with TAE buffer (40 mM Tris acetate, pH 8.1, 2 mM EDTA), the DNA was stained with ethidium bromide and photographed over UV light.
Covalent Complex with Oligonucleotide Substrate-The singlestranded oligonucleotide 5Ј-CAATGCGCT-3Ј, containing the sequence of a strong cleavage site (24), was labeled at the 3Ј end with [␣-32 P]dATP and terminal deoxynucleotidyl transferase. After incubation of 0.15 g of the labeled oligonucleotide with 0.4 g of the enzyme in 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 20 mM potassium phosphate at 37°C for 5 min, the reaction was stopped with the addition of 1% SDS. The enzyme was separated from the non-covalently bound oligonucleotides by electrophoresis in a 10% SDS-polyacrylamide gel. The labeled covalent complex was visualized by autoradiography of the dried gel.
tion was carried out using the Molecular Dynamics PhosphorImager.
Intrinsic Tryptophan Fluorescence Measurements-Fluorescence measurements were performed with the Perkin-Elmer LS-5B spectrometer with excitation at either 280 or 295 nm at either 42°C or room temperature (ϳ25°C). DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
==
Domain: Biology Chemistry Medicine
|
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Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. coli AS17 comparable with that of the wild-type enzyme. The 7. Effect of the mutations on the formation of non-covalent complex as assayed by the gel mobility shift assay using a 5-end-labeled 36-mer.
other mutant proteins were accumulated at a reduced or unobservable level. In contrast, mutations in the Zn(II) binding domain resulted in mutant proteins that were accumulated at higher levels than the wild-type enzyme (27). In the E. coli strain GP200 where the Glu-9, Asp-111, Asp-113, and Glu-115 mutants were stably expressed, these mutants were able to confer some degree of thermoresistance to compensate for the absence of chromosomal topA activity.
In the crystal structure of the 67-kDa N-terminal fragment, Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 are part of an extensive network of hydrogen bonds and salt bridges that are responsible for the domain-domain interactions (8). Mutation of these residues to alanines might abolish some of these interactions and destabilize the protein structure. This potential effect of the mutation on the enzyme structure and stability is consistent with the protein fluorescence data shown in Table IV, with the structure of Arg-321 mutant particularly sensitive to the increase in temperature.
Mg(II) is required for the relaxation of DNA by E. coli DNA topoisomerase I. It is not required for the cleavage of singlestranded DNA although the rate of cleavage of small oligonucleotide substrates can be enhanced by the presence of Mg(II) (28,29). Mutations of Glu-9 and Glu-115 reduced the Mg(II) binding stoichiometry significantly after equilibrium dialysis against buffer containing 0.4 mM MgCl 2 . This might have resulted from one of the multiple coordination sites for Mg(II) in the enzyme being removed or replaced by a ligand of lower affinity to Mg(II). The effects of the mutations of Glu-9, Asp-111, Asp-113, and Glu-115 to alanine on the relaxation activity were less severe than those observed for mutations of carboxylates proposed to be Mg(II) coordination sites involved in nucleotidyl transfer in other enzyme systems (11,14,30,31). It is possible that for E. coli DNA topoisomerase I, water or a DNA phosphate could substitute for the mutated carboxylate in Mg(II) coordination in the relaxation reaction to provide partial enzymatic activity, especially at Mg(II) concentration significantly above the minimal concentrations needed for relaxation activity. The effect of mutation of Asp-111 on the enzyme activity and Mg(II) binding was significantly smaller than mutations of the other carboxylates, so it is unlikely that Asp-111 is a catalyticly important residue.
The Glu-9, Asp-113, Glu-115, and Arg-321 mutants also had reduced DNA binding activity. This could be due to the participation of the residue in direct protein-DNA interaction which is a plausible role for Arg-321. Alternatively, the reduced DNA binding could be due to the effect of the mutation on the protein folding. It also cannot be ruled out that the lower Mg(II) binding stoichiometries observed for the Glu-9 and Glu-115 mutants might be due to the effect of the mutations on the protein structure and not due to loss of a Mg(II) coordination site. A Mg(II) binding site distinct from the catalytic center has been proposed for the EcoRV restriction endonuclease (32). DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
==
Domain: Biology Chemistry Medicine<|endoftext|>PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. However, lymphoid cells and cells exposed to cytokines such as IFN-Ȗ alternatively express three homologous subunits (ȕ5i/LMP7, ȕ2i/MECL-1, ȕ1i/LMP2), which replace the constitutive ones in newly assembled so-called immunoproteasome particles [11]. A variety of studies have demonstrated that incorporation of these INF-Ȗ-induced subunits quantitatively modifies proteasomal cleavage preferences and enhances the production of a significant number of antigenic peptides, although there are examples of epitopes that are generated with lower efficiency, or which are not released, by immunoproteasomes [12]. In any case, the recent generation of transgenic mice lacking all three proteasomal catalytic ȕ-immune subunits clearly demonstrated the pivotal role that immunoproteasomes play in the generation of MHC class I ligands [13]. Additionally, immunoproteasomes have been shown to be important for efficient cytokine production [14] and have been implicated in a number of pathological disorders such as cancer and neurodegenerative and autoimmune diseases [15][16][17]. Finally, it has been recently demonstrated that 26S immunoproteasomes possess the capacity to hydrolyze basic proteins (such as Histones and Myelin Basic Protein) at greatly increased rates compared to constitutive proteasomes [18].
Structure
Another INF-Ȗ-inducible UPS component that affects MHC class I antigen presentation is PA28 (also termed REG or 11S), a ring-shaped 180 kDa multimeric complex that binds, in an ATP-independent manner, to the two ends of the 20S proteasome and substantially enhances its ability to degrade short peptide substrates, but not proteins or ubiquitin-conjugated proteins [19,20]. In addition, PA28 can also associate with the free end of asymmetric 26S proteasomes (19S-20S) to form so-called "hybrid proteasomes" (19S-20S-PA28) [21][22][23][24] that hydrolyze tri-and tetra-peptides at higher rates than canonical 26S particles [21,23]. In mammals, PA28 is composed of two homologous subunits, namely PA28Į (REGĮ or PSME1) and PA28ȕ (REGȕ or PSME2), both of which are induced by Ȗ-interferon [25][26][27][28][29][30][31]. A third member of PA28 family is PA28Ȗ (also known as REGȖ, 11SȖ or Ki antigen), which is a nuclear antigen that is not induced by INF-Ȗ and not involved in MHC class I antigen presentation. The biochemical and biological properties of PA28Ȗ has been reviewed elsewhere [32], and consequently will not be considered in detail.
PA28Į and PA28ȕ have an apparent molecular weight of 28 kDa on SDS-PAGE electrophoresis and share a nearly 50% amino acid sequence identity [25,29,33]. Based on the crystal structure of PA28Į [34], the overall secondary structure of PA28 proteins is composed of four long Į-helices of 33-45 residues in length that are involved in intra-and intermolecular interactions. The linker sequence between helices 2 and 3, which is highly conserved in PA28 Į and ȕ subunits, is designated the "activation loop" since it is responsible for stimulation of proteasome peptidase activities [35]. A second region of the molecule involved in the activation of 20S proteasome is the 10 residue C-terminal tail, which provides binding energy for PA28-proteasome association [36][37][38]. Finally, the linker between helices 1 and 2 is composed of sequences that are highly divergent between PA28 Į and ȕ subunits and for this reason are known as "homolog specific inserts" [39]. Although these inserts are not resolved in the X-ray structure of PA28Į, presumably since they are flexible, it is almost certain that they protrude from the upper surface of the PA28Į ring [30].
PA28 has been reported to be phosphorylated in vivo on serine residues [40,41] and phosphorylation was believed to be indispensable to activate proteasome peptidase activities [41]. Considering, however, that nearly all in vitro studies concerning the biochemical properties of PA28 have been performed using the recombinant protein expressed in E. coli (which in the vast majority of cases are not competent for phosphorylation of recombinant proteins), it is clear that phosphorylation is not essential for the stimulatory activity of PA28, while in vivo it may exert more subtle and yet unidentified regulatory functions. Moreover, binding of PA28 to calcium has also been described although the physiological significance of this observation remains an open question [42].
While it is clear that at least in vitro recombinant PA28Į can form a heptameric ring [34,43] and recombinant PA28ȕ is a monomer [35,39], the subunit stoichiometry of native PA28Įȕ has been initially reported [26,38] to be a hexamer (3Į3ȕ). Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
==
Domain: Biology Chemistry Medicine
|
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|
PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. Based on the crystal structure of PA28Į [34], the overall secondary structure of PA28 proteins is composed of four long Į-helices of 33-45 residues in length that are involved in intra-and intermolecular interactions. The linker sequence between helices 2 and 3, which is highly conserved in PA28 Į and ȕ subunits, is designated the "activation loop" since it is responsible for stimulation of proteasome peptidase activities [35]. A second region of the molecule involved in the activation of 20S proteasome is the 10 residue C-terminal tail, which provides binding energy for PA28-proteasome association [36][37][38]. Finally, the linker between helices 1 and 2 is composed of sequences that are highly divergent between PA28 Į and ȕ subunits and for this reason are known as "homolog specific inserts" [39]. Although these inserts are not resolved in the X-ray structure of PA28Į, presumably since they are flexible, it is almost certain that they protrude from the upper surface of the PA28Į ring [30].
PA28 has been reported to be phosphorylated in vivo on serine residues [40,41] and phosphorylation was believed to be indispensable to activate proteasome peptidase activities [41]. Considering, however, that nearly all in vitro studies concerning the biochemical properties of PA28 have been performed using the recombinant protein expressed in E. coli (which in the vast majority of cases are not competent for phosphorylation of recombinant proteins), it is clear that phosphorylation is not essential for the stimulatory activity of PA28, while in vivo it may exert more subtle and yet unidentified regulatory functions. Moreover, binding of PA28 to calcium has also been described although the physiological significance of this observation remains an open question [42].
While it is clear that at least in vitro recombinant PA28Į can form a heptameric ring [34,43] and recombinant PA28ȕ is a monomer [35,39], the subunit stoichiometry of native PA28Įȕ has been initially reported [26,38] to be a hexamer (3Į3ȕ). However, subsequent studies indicated that heteromeric PA28 is a heptamer formed by the association of three Į subunits and four ȕ subunits [44,45]. Electron microscopy images show that PA28Įȕ forms a cap on the end of 20S particle by interacting with proteasome Į-subunits [46]. This cap is about 10-11 nm wide at the base, where it attaches to proteasome Į subunits, and 7-8 nm long from the base to the tip. EM data also indicate that PA28 contains a central channel that apparently traverses it entirely to the central pore of proteasome Į-ring. Accordingly, the crystal structure of recombinant PA28Į at 2.8 Å resolution reveals a heptameric ring traversed by a central aqueous channel with a diameter of 30 Å on the face contacting the proteasome and 20 Å on the other [34].
Role in MHC Class I Antigen Presentation
The effects of PA28Įȕ on antigen processing and CD8 + T-cell responses are still unclear and controversial [2,30]. Professional antigen presenting cells generally express PA28Įȕ at high levels, which is in agreement with a possible function of this complex in MHC class I antigen processing [47,48]. Accordingly, expression of PA28Į alone [49,50] or PA28Įȕ [50][51][52][53][54] has been reported to enhance MHC class I-presentation of some, but not all, antigens. Furthermore, cells lacking this complex have a reduced ability to generate certain antigens [55], and recent studies identified the second most important UPS component in PA28 (surpassed only by ȕ5i/LMP7) for production of MHC class I ligands [56], although its effects seem to be restricted to specific MHC class I alleles [57].
Although concerted expression of PA28 Į and ȕ and proteasomal ȕ1i, ȕ2i and ȕ5i subunits following INF-Ȗ induction leads to the formation of PA28Įȕ-20S immunoproteasomes in vitro and in vivo [22,24,26,31,55,[58][59][60][61][62][63][64], however PA28Įȕ can also be detected in cells, tissues and organs that lack immunoproteasomes like erythrocytes, muscle and brain [19,20,[58][59][60][61]65,66]. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
==
Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The various MBP-VP16 mutant fusion proteins were linked to amylose resin and the beads were incubated with purified VCAF-1. In preparing the affinity matrix, care was taken to secure that the concentration of the immobilized mutants was similar for each case. Bound and nonbound material was collected and assayed for VIC formation in the presence of wt MBP-VP16 and GST-Oct-1. As shown in Fig. 4, beads coupled with MBP-VP16, R155A, R162A, R164A, C176A, and R360A retained VCAF-1 activity (lanes c, d, e, f, h, and i, respectively), whereas VCAF-1 activity was not retained on beads coupled with MBP or to beads coupled with R169A, R366A, R368A, or K370A (lanes b, g, j, k, and l, respectively). These results demonstrate that prior and independent interaction between VP16 and VCAF-1 is not essential for VIC formation.
Interaction of VP16 with Vhs-We have recently identified the HSV-1 virion host shutoff protein vhs as a novel ligand for VP16 (30). Preliminary mapping studies indicated that stable interaction with Vhs requires several regions present in the amino-terminal 369 amino acids of VP16, suggesting that the overall conformation of VP16 is important for VP16-Vhs complex formation. We therefore determined whether the point mutant derivatives of VP16 were compromised in their ability to interact with Vhs. For this purpose, [ 35 S]methionine-labeled Vhs was prepared by transcription and translation in vitro. The different MBP-VP16 point mutants were coupled to amylose beads as described above and incubated with labeled Vhs. The beads were recovered, washed extensively, and the bound material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown in Fig. 5, Vhs bound to all of the mutant VP16 derivatives as effectively as it did to MBP-VP16. Labeled Vhs did not bind to the beads alone (lane h) or to beads complexed with MBP (lane b). Thus, none of the VP16 point mutants were defective in interaction with Vhs. These findings show that the targeted mutations are not important for Vhs interaction and, moreover, that the introduced mutations did not grossly affect the overall conformation of VP16.
Transactivation Properties of VP16 Mutant Derivatives-There is a strict correlation with the ability of VP16 to assembly into the VIC complex with subsequent transactivation in vivo. It is thought that assembly of this multicomponent complex is required only to bring the activation domain of VP16 to the promoter; however, additional VP16-DNA, VP16-Oct-1 and VP16-VCAF-1 interactions in the full complex may influence the overall conformation of the complex and thus contribute directly to transactivation. We therefore cloned the different VP16 derivatives into a mammalian expression vector that restored the carboxyl-terminal acidic activation domain and tested their abilities to transactivate gene expression in transient transfection assays using a CAT reporter gene that contained the promoter/regulatory region from the ICP4 gene. As shown in Fig. 6, co-transfection of Vero cells with the fulllength wild type VP16 expression plasmid induced expression of the reporter gene approximately 30-fold when 0.5 g of effector plasmid was used. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine
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### Q: Fill in the missing text.
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<missing text> second Mg(II) binding site away from the active site region that is required for relaxation activity because of its effect on the protein conformational changes that take place during the relaxation reaction cycle (28). The carboxylates at the active site <missing text>
### Answer:
Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. E. coli DNA topoisomerase I may also have a second Mg(II) binding site away from the active site region that is required for relaxation activity because of its effect on the protein conformational changes that take place during the relaxation reaction cycle (28). The carboxylates at the active site may be conserved for their roles in protein structure instead of catalytic functions. The magnitude of the effect of a single mutation of these catalytically non-essential residues on the overall protein stability may depend on the E. coli strain background, but nevertheless be of sufficient significance to account for the evolutionary conservation.
The steps of DNA cleavage and religation may or may not involve the same catalytic residues in the activation of nucleophiles as well as the stabilization of transition states and leaving groups. Presently there is a lack of data to address this question, and it remains unclear what these catalytic residues may be. Among the mutants tested, the Glu-9 mutant had the greatest loss of DNA cleavage activity with only a modest reduction in non-covalent DNA binding. Besides a possible role in binding Mg(II), this residue might be involved in the catalytic step of DNA cleavage. In contrast, the Arg-136 mutant had normal DNA cleavage activity but no relaxation activity. It might be needed in the DNA strand passage step since it could perform the inter-molecular rejoining of DNA.
There is also a lack of information on the orientation of the DNA substrate when it binds to the active site. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. 2).
Effect of the Mutations on Enzyme Activities-Purified mutant enzymes were diluted serially and compared with the wild-type topoisomerase I for relaxation of negatively supercoiled plasmid DNA in relaxation buffer containing 6 mM MgCl 2 (Fig. 3A). The results showed that the Arg-136 mutant was totally inactive in the relaxation assay. No relaxation activity was observable even with 400 ng of the enzyme, a 50-fold excess over the amount needed to observe relaxation by the wild-type enzyme. The other mutants had varying degrees of loss of relaxation activity. Examination of the effect of dilutions and time course of relaxation (Fig. 3B) showed that the Glu-9 mutant had the greatest reduction in catalytic activity (Ͼ90% reduction). The Glu-115, Asp-113, and Arg-321 mutants had about 80 -90% reduction in activity. The Asp-111 mutant was closest to the wild-type enzyme in activity (Ͻ50% reduction).
The E. coli strain AS17 does not grow at 42°C because of the temperature sensitivity of the suppressor for the chromosomal topA am mutation. The pJW312 plasmid carrying the wild-type topoisomerase I gene can complement this chromosomal mutation (21). Each one of the active site mutations tested here was found to abolish this in vivo complementation even though they showed varying degrees of loss of in vitro relaxation (Table I).
The inability of the Glu-9, Glu-111, Asp-113, and Glu-115 mutants to complement efficiently in E. coli AS17 may be due to their low level of expression in this E. coli strain. To evaluate the in vivo activities of these mutants, the thermosensitivities of GP200 expressing these mutants were examined (Table II). Loss of topA activity in E. coli can lead to a lower rate of survival when the temperature was raised to 52°C (reviewed in Ref. 25). The rate of survival of E. coli GP200 was increased ϳ50-fold when plasmid encoded wild-type topA activity was present (Table II). DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. 2B, numbers 4 -6). Constructs pCW355 and pCW349 served as positive and negative controls, respectively, in the assay (Fig. 2B, numbers 1 and 2). Construct pCW355 carries a wild-type VAS1 gene that expresses both cytoplasmic and mitochondrial forms of yeast ValRS, whereas construct pCW349 carries an initiator residues that is absent from its E. coli counterpart. The polypeptide sequence of the mitochondrial (mit) precursor form of yeast ValRS is essentially identical to that of its cytoplasmic isoform except for a mitochondrial targeting signal attached at its N terminus. To examine whether E. coli ValRS and its fusions are properly expressed in yeast, we next examined the relative expression levels of these constructs by Western blotting using an anti-FLAG tag antibody. As shown in Fig. 2C, E. coli ValRS and its fusions were all properly expressed in yeast. Fusion of Arc1p or the Ad of yeast GlnRS to E. coli ValRS significantly enhanced its expression level (2-and 7-fold, respectively), whereas fusion of the Ad of yeast ValRS to the E. coli enzyme slightly reduced its expression level (1.5-fold). Evidently, not all TRBDs enhanced the protein expression level of E. coli ValRS in yeast. This observation also reinforces the idea that the negative phenotype of E. coli ValRS in the complementation assay (Fig. 2, number 3) was not caused by a lower level of protein expression.
Converting E. coli ValRS into a Functional Yeast Mitochondrial Enzyme-In contrast to the cytoplasmic aaRSs, the mitochondrial enzymes are generally believed to be more bacterium-like in terms of their genetic origin and sequence homology. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. In most cases, cytoplasmic and mitochondrial synthetase activities are encoded by two distinct nuclear genes, regardless of the cell compartments to which they are confined. However, in some cases, cytoplasmic and mitochondrial forms of a tRNA synthetase with a given amino acid specificity are encoded by the same nuclear gene through alternative initiation of translation, examples of which include ALA1 (coding for alanyl-tRNA synthetase) (6,7), GRS1 (coding for glycyl-tRNA synthetase) (8), HTS1 (coding for histidyl-tRNA synthetase) (9), and VAS1 (coding for valyl-tRNA synthetase (ValRS)) (10). Because the isozymes are targeted to different compartments, the two isoforms of ValRS, for example, cannot be substituted for each other in vivo. A similar scenario has been observed for genes encoding mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase, threonyl-tRNA synthetase, and ValRS (11).
Many yeast cytoplasmic tRNA synthetases contain an N-or C-terminal polypeptide extension that is absent from their bacterial homologs (12). A well studied example is the appended domain (Ad) of yeast glutaminyl-tRNA synthetase (GlnRS), which binds crude yeast tRNAs, single-stranded RNA, and pseudoknot RNA with comparable affinities; the K d values are ϳ0. 6 M (13, 14). Similar examples have been reported in yeast ValRS (15) and tRNA synthetases of higher eukaryotes, such as the EMAPII-like domain of plant methionyl-tRNA synthetase (16), the repeat domain of human methionyl-tRNA synthetase (17), and the N-terminal domain of mammalian lysyl-tRNA synthetase (18,19). In addition to serving as a cis-acting tRNAbinding domain (TRBD), the Ads of some yeast tRNA synthetases were found to participate in protein-protein interactions, such as those of yeast glutamyl-, methionyl- (20), and seryl-tRNA synthetases (21). These interactions were shown to enhance their tRNA binding and aminoacylation (20,21).
Interestingly, many of the Ads of yeast tRNA synthetases contain one or several canonical nuclear localization signals (22) that are thought to play a role in the nuclear import of these otherwise "cytoplasmic" proteins. Nuclear aminoacylation of tRNAs is thought to serve as a functional checkpoint for the maturity of tRNAs before they are exported from the nucleus (23,24). By contrast, in higher eukaryotes, nine aminoacyl-tRNA synthetases and three auxiliary proteins (p43, p38, and p18) form a multienzyme complex through interactions of their hydrophobic Ads (25). In addition, ValRS from mammalian cells is exclusively isolated as a high molecular mass complex with the elongation factor EF-1H (26 -28). Recently, several tRNA synthetases from prokaryotes and eukaryotes have been shown to take part in functions beyond aminoacylation, including roles in mitochondrial RNA splicing, transcriptional and translational regulation, cytokine-like activity, and amino acid biosynthesis (29,30).
In this report, we focused on the cross-species and crosscompartmental complementation activities of a bacterial tRNA synthetase in an attempt to understand further the evolutionary pathway that has converted a bacterial tRNA synthetase gene into a dual functional yeast gene possessing both cytoplasmic and mitochondrial activities. Our results show that although Escherichia coli ValRS per se cannot substitute for the cytoplasmic activity of yeast VAS1, fusion of a nonspecific TRBD (such as Arc1p or the Ads of yeast GlnRS and ValRS) to the bacterial enzyme enabled the otherwise nonfunctional enzyme to rescue the growth defect of a VAS1 knock-out strain on 5-fluoroorotic acid (5-FOA). In contrast, the E. coli enzyme, when targeted to mitochondria, could substitute for the mitochondrial activity of VAS1 without the assistance of a cis-acting nonspecific TRBD. These results, together with others, suggest that acquiring a cis-or trans-acting TRBD might be necessary and sufficient for functioning by a yeast cytoplasmic tRNA synthetase, which might explain why so many yeast cytoplasmic aaRSs contain an N-or C-terminal Ad (12). In contrast, obtaining a cis-or trans-acting TRBD does not appear to be necessary for functioning of most yeast mitochondrial aaRSs.
EXPERIMENTAL PROCEDURES
Construction of Plasmids-Cloning of the wild-type yeast VAS1 gene into the high copy number yeast shuttle vector pADH was previously described (31). A short DNA duplex coding for a FLAG (for Western blotting) or His 6 tag (for protein purification) was inserted in-frame at the 3Ј end of the VAS1 gene. To clone the E. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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This below document has 2 sentences that start with 'Further studies will be required'. It has approximately 891 words, 27 sentences, and 16 paragraph(s).
<<<<>>>>
PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. However, subsequent studies indicated that heteromeric PA28 is a heptamer formed by the association of three Į subunits and four ȕ subunits [44,45]. Electron microscopy images show that PA28Įȕ forms a cap on the end of 20S particle by interacting with proteasome Į-subunits [46]. This cap is about 10-11 nm wide at the base, where it attaches to proteasome Į subunits, and 7-8 nm long from the base to the tip. EM data also indicate that PA28 contains a central channel that apparently traverses it entirely to the central pore of proteasome Į-ring. Accordingly, the crystal structure of recombinant PA28Į at 2.8 Å resolution reveals a heptameric ring traversed by a central aqueous channel with a diameter of 30 Å on the face contacting the proteasome and 20 Å on the other [34].
Role in MHC Class I Antigen Presentation
The effects of PA28Įȕ on antigen processing and CD8 + T-cell responses are still unclear and controversial [2,30]. Professional antigen presenting cells generally express PA28Įȕ at high levels, which is in agreement with a possible function of this complex in MHC class I antigen processing [47,48]. Accordingly, expression of PA28Į alone [49,50] or PA28Įȕ [50][51][52][53][54] has been reported to enhance MHC class I-presentation of some, but not all, antigens. Furthermore, cells lacking this complex have a reduced ability to generate certain antigens [55], and recent studies identified the second most important UPS component in PA28 (surpassed only by ȕ5i/LMP7) for production of MHC class I ligands [56], although its effects seem to be restricted to specific MHC class I alleles [57].
Although concerted expression of PA28 Į and ȕ and proteasomal ȕ1i, ȕ2i and ȕ5i subunits following INF-Ȗ induction leads to the formation of PA28Įȕ-20S immunoproteasomes in vitro and in vivo [22,24,26,31,55,[58][59][60][61][62][63][64], however PA28Įȕ can also be detected in cells, tissues and organs that lack immunoproteasomes like erythrocytes, muscle and brain [19,20,[58][59][60][61]65,66]. It is, therefore, conceivable that PA28Įȕ can exert its biological function(s) also in association with constitutive proteasomes, probably related to its capacity to substantially modify the pattern of proteasomal peptides products. Further studies will be required to assess whether the changes in the patterns of peptides generated from proteins following binding of PA28Įȕ with 20S and 26S constitutive are similar to those already identified for its association with 20S and 26S immunoproteasomes [18].
Hydrolysis of Fluorogenic Peptide Substrates
As pointed out above, PA28Įȕ was originally identified due to its capability to markedly stimulate peptidase activities of the 20S proteasome, as measured using short fluorogenic model substrates.
Gate Opening Mechanism
In the crystal structure of PA26 solved by Hill and coworkers [71], the binding of PA26 was found to open the gate on the channel in the proteasome Į-ring through which substrates enter [94] and products exit [70]. Specifically, PA26 C-terminal residues dock into pockets between adjacent proteasome Į subunits and, by forming hydrogen bonds and a salt bridge between the C-terminal carboxylate of the activator and a highly conserved proteasome lysine side chain (Lys 66), provide binding energy for PA26-20S complexes [71,95]. Binding to the C-termini of PA26, however, is not sufficient to activate the 20S proteasome, which requires the activation loop that forms a seven-fold symmetric circular array that interacts with the base of the N-terminal gating residues of the seven proteasomal Į subunits. In particular, a glutamate side chain (Glu 102) in each PA26 subunit activation loop contacts and repositions a proline residue (Pro 17) of 20S Į subunits located above the surface of the proteasome. This interaction triggers gate opening by disrupting packing and hydrogen bonding interactions of the asymmetrical closed conformation and by widening the pore opening to a more symmetrical arrangement that allows a belt of intersubunit contacts to form around the circumference of the opening [96,97].
On the basis of these structural observations, PA28 was predicted to lead to attenuation of proteasomal processivity and consequent release of peptide products of greater mean length [71], as occurs on deletion of the Į-gate [70]. Since most peptides released by proteasomes are too short to bind to MHC class I molecules [84,[98][99][100][101], the generation of larger products would be expected to enhance the fraction of products capable of serving in antigen presentation, either directly or after trimming by aminopeptidases in the cytosol [89,102] or endoplasmic reticulum [86][87][88]90,103]. However, since not only PA28 but also the 19S regulator can induce the opening of the 20S core particle central gate [104], it remains unclear whether the above-mentioned structural observations can explain the specific effects of PA28 on substrate cleavage and, consequently, on antigen presentation.
Effects of PA28Įȕ on Protein Degradation
In this respect, it is clear that a full understanding of the specific biochemical functions of PA28Įȕ requires quantitative information on the rates of protein substrate hydrolysis and generation of peptide products by PA28Įȕ-containing immunoproteasomal species. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
==
Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. 4 -14; referred to as VCAF-1 in this paper). The fully assembled functional complex, referred to as the VP16-induced complex (VIC), is thought to position the strong carboxyl-terminal acidic activation domain of VP16 in the correct spatial arrangement for functional interaction with downstream target proteins, among which include several basal transcription factors and adapter proteins (15,16).
The ordered assembly of VP16-containing protein-DNA complexes involves cooperative sequence-specific protein-DNA interactions as well as specific and selective protein-protein associations with Oct-1 and VCAF-1. Oct-1 binds independently to TAATGARAT elements and directs the recruitment of VP16 via determinants present in the POU homeodomain (17)(18)(19)(20)(21). The prior binding of Oct-1 to TAATGARAT elements is a prerequisite to the formation of VIC; however, the efficient incorporation of VP16 into the complex requires the auxiliary component VCAF-1, a large cellular factor which can interact directly with VP16 in the absence of Oct-1 and DNA (5,7,8,13,14,17). There is no evidence for the formation of Oct-1-VP16 or Oct-1-VCAF-1-VP16 heteromeric complexes in the absence of DNA; however, it has been demonstrated that VP16 is able to bind directly to DNA or to generate a complex with DNA-bound Oct-1 in the absence of VCAF-1 when high concentrations of VP16 are used in binding assays (17,19,22).
VP16 is a modular protein and contains separable domains that are important for complex formation and transactivation (19,(23)(24)(25)(26)(27)(28)(29). The carboxyl-terminal acidic activation domain is essential for transcriptional activation but not for complex assembly. The residual 400 amino-terminal amino acids are necessary and sufficient for VIC formation as well as for interaction with VCAF-1 and binding to DNA (14,24,25,27). More recently, it has been demonstrated that this region also contains determinants for interaction with the virion host shutoff protein (Vhs), a viral structural protein that is responsible for the cessation of host protein synthesis following viral infection (30). At least two subregions within the amino-terminal domain are involved in complex assembly with mammalian factors in vitro; region 1, spanning residues 140 -250, and region 2, encompassing residues 335-390 (19,24,25,27). Both subregions are involved in VIC formation, binding to VCAF-1, interaction with DNA-bound Oct-1, and contribute to the weak DNA binding activity associated with VP16 (19,24,27,(31)(32)(33).
Despite a enceinte numeral of mutational studies, it is not known whether the individual interactions attributed to VP16 are essential for the assembly of transcriptionally active complexes. For instance, while VCAF-1 is essential for VIC formation, it is not known whether the intrinsic ability of VP16 to bind independently to VCAF-1 is a prerequisite for multicomponent complex assembly. Also, the weak DNA binding activity associated with VP16, or VCAF-1-independent interaction with DNA-bound Oct-1, is only observed when very high concentrations of VP16 are used in binding assays (17,19); thus, the physiological significance of these interactions is not clear. Most mutational studies of VP16 have been carried out using large deletions or linker insertions and these might be expected to cause conformational changes in VP16 that could have secondary effects on complex assembly and protein-protein interactions. The only point mutational analysis so far described assessed the overall effects of specific point mutants on VIC formation and transactivation (25). In order to determine if individual protein-protein and protein-DNA interactions involving VP16 can be uncoupled from VP16-induced complex formation and transactivation, we converted selected charged residues in regions 1 and 2 into alanine residues by site-directed mutagenesis. Mutant proteins were analyzed in vitro for protein-DNA complex formation with Oct-1 and interaction with VCAF-1 and in vivo for transactivation of a VP16-responsive reporter gene. Among the mutants generated were those that were defective in direct VCAF-1 interaction and DNA binding but were still capable of generating the VIC complex and directing transcriptional activation in vivo. Our findings indicate that the assembly of functional VP16-containing multicomponent complexes does not necessarily require the prior interaction of VP16 with VCAF-1 or the capability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1, suggesting that the cooperative assembly of VP16-dependent complexes can occur by different pathways.
MATERIALS AND METHODS
Site-directed Mutagenesis-Site-directed mutagenesis was carried out by the Kunkle method (34) using a commercially available kit (Bio-Rad). Single-stranded M13mp19 containing the antisense strand of the VP16 open reading frame was used as the template for mutagenesis. The following mutagenic oligonucleotides, designed to convert the indicated wild type amino acids into alanine residues, were used. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine
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PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. Selectivity based exclusively on peptide size, however, cannot account for the overall effects of PA28 on the patterns of proteasome products observed by Raule et al. [101]. In fact, quantitation of products demonstrated that several individual peptides with a length of 8-23 residues are released in much higher amounts by PA28Įȕ-20S than by 20S or 26S immunoproteasomes [101]. Therefore, PA28 appears to act as a selective filter that promotes preferential passage of only subset of specific long products through its central channel, presumably based on sequence.
At present, the properties that might allow specific longer peptides to evade the constraint imposed by PA28 towards their efflux are not completely clear. However, the finding that products longer than seven residues whose generation is strongly enhanced in the presence of PA28 are, on average, more hydrophilic than those preferentially released by 20S alone [101], strongly suggests that the passage of polar/charged long peptides thorough PA28 might be favored. In this model, PA28 would act as a selective "smart" sieve that strictly controls the exit of products from proteasomes on the basis of size and sequence ( Figure 2). As a result, PA28Įȕ would promote preferential efflux from the 20S proteolytic cavity of only a reduced number of individual peptides longer than 6-7 amino acids, while the majority of the other proteasomal products are retained inside where they are further cleaved to smaller pieces before they diffuse outside. In accordance with this model, the central channel of PA28Į ring is almost completely lined by charged or polar residues [34], and is thus well suited for permitting the passage of water soluble peptides. Importantly, this molecular model would be also consistent with our findings on 19S-20S-PA28 immunoproteasomes. In this case, the absence of a clear difference in size distribution [21] argues that in hybrid proteasomes (as in 26S canonical particles) the main route of exit of peptides from the inner proteolytic chamber is regulated by the 19S cap, while PA28 would exert its major effect by allowing preferential sorting of selected products through its central channel.
Other Potential Biological Functions
A full understanding of the exact biological functions of PA28 would undoubtedly represent an important achievement, especially in the light of the observation that mammalian cells contain significant amounts of PA28Įȕ-20S immunocomplexes [22,24,26,[58][59][60][61] whose abundance further increases upon INF-Ȗ stimulation [31,55,62]. In this respect, it seems extremely unlikely that PA28 functions in vivo to stimulate degradation of cytosolic oligopeptides by proteasomes, because such peptides are very rapidly hydrolyzed by other cellular exo-and endo-peptidases [116]. Moreover, it has been shown in vitro that peptides released by proteasomes are further cleaved by these proteolytic particles at extremely low rates [117], and they are thus unlikely to efficiently compete for degradation with proteins, which are much more preferred substrates [118]. Importantly, the advantage of PA28-containing proteasomes that generate highly divergent patterns of potential antigenic peptides, characterized by profound qualitative and quantitative differences, might become relevant under specific pathophysiological conditions especially if favoring a non-canonical, ubiquitin-independent, protein hydrolysis pathway. In fact, most of the in vitro studies addressing the production of antigenic peptides have focused on degradation by proteasomes of denatured, non-ubiquitinated proteins since ubiquitinated proteins are not hydrolyzed by 20S and PA28-20S complexes [10,19]. This is likely due to the fact that these particles lack the enzymatic activities necessary to remove and/or to unfold polyubiquitin chains that otherwise would sterically block translocation of substrates into the proteolytic chamber [119][120][121]. Although further studies will be required to clarify whether the patterns of peptides released are different if a protein substrate initially binds to the 19S regulatory particle through a polyubiquitin chain or directly through an unstructured domain, several lines of evidence indicate that in vivo denatured proteins are important, physiological substrates for intracellular proteolysis. A large fraction of MHC class I epitopes is derived from rapid degradation of DriPs [122][123][124] in a process that has been shown to be at least partially ubiquitin-independent [124][125][126]. Furthermore, several recent reports indicate that some loosely-folded, short-lived regulatory, viral and oxidized proteins are degraded in vivo by the 20S proteasome in an ATP-and ubiquitin-independent manner [126][127][128][129][130][131][132][133]. In this regard, a contribution of PA28 in this alternative pathway of protein catabolism pathway seems plausible. Moreover, immunoproteasomes were recently reported to be strongly induced under conditions of oxidative stress [134], and according to several lines of evidence, oxidized proteins are preferentially degraded without ubiquitination by 20S proteasomes [131] in a process that was reported to be stimulated by PA28Įȕ [135][136][137]. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. Our results presented herein argue that a bacterial aaRS gene can be converted into a dual functional yeast gene by fusion of a DNA sequence coding for both an MTS and a TRBD (Fig. 5). In addition, our results underline the necessity of obtaining a nonspecific TRBD for efficient binding and aminoacylation of yeast cytoplasmic tRNA by the bacterial enzyme ( Fig. 4 and Table 1).
ValRS variant
The binding affinity of a tRNA synthetase for its cognate tRNAs is generally characterized by dissociation constants on the order of 0.1-1 M under physiological conditions (34). This relatively poor affinity ensures that the synthetases (or tRNAs) turn over rapidly during aminoacylation. In this sense, it is interesting to point out that the binding affinities of the Ads of yeast GlnRS and ValRS for tRNA also fall into this range, making them useful as a cis-acting tRNA-binding cofactor during aminoacylation (15). Although the Ads of GluRS and MetRS are also rich in positively charged residues and important for aminoacylation, they do not function as a TRBD. Instead, these Ads specifically interact with a tRNA-binding cofactor, Arc1p, which, in turn, recruits tRNA to the vicinity of the enzymes for aminoacylation (35). A functionally similar tRNA-recruiting domain was identified in an auxiliary protein associated with the mammalian multisynthetase complex (36).
ValRS from mammalian cells is exclusively isolated as a high molecular mass complex with the elongation factor EF-1H (26 -28). Like yeast ValRS, the mammalian enzyme also contains a strong affinity for the polyanionic carrier, heparin-Ultrogel. However, the mammalian enzyme exhibits additional hydrophobic properties (37). Sequence analysis revealed that mammalian ValRS has conserved the positively charged N-terminal extension (residues 200 -298) that distinguishes yeast ValRS from its bacterial counterparts while acquiring an additional hydrophobic domain (residues 1-199) that is responsible for interacting with the four subunits of elongation factor EF-1H (␣, , ␥, and ␦ subunits) (38). A very recent report showed that, like the Ad of yeast ValRS, the positively charged N-terminal extension (residues 200 -298) of the mammalian enzyme is also a nonspecific TRBD (15).
However, regardless of the detailed interpretation, the most interesting findings reported here are the capability of a nonspecific TRBD to enhance the formation of an active synthetase-tRNA complex and the conversion of an E. coli aaRS gene to a dual functional yeast gene encoding both cytoplasmic and mitochondrial activities. To our knowledge, this appears to be the first example wherein an engineered bacterial aaRS gene can provide both cytoplasmic and mitochondrial aminoacylation activities in yeast. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. A number of residues found in the active site, including Glu-9, Asp-111, Asp-113, and Glu-115, and Arg-321, are strictly conserved among type IA DNA topoisomerase sequences (Fig. 1). This evolutionary conservation extends from the archaeal (Methanococcus jannaschii) topoisomerase I to human topoisomerase III. There is a lower degree of conservation for Arg-136 which is also near the active site tyrosine. The acidic and basic functional groups on these residues may be important for the enzyme catalytic function. For DNA polymerases, two divalent metal ions coordinated by carboxylates are essential for the enzyme activity (10 -14). One or two metal ion mechanisms utilizing carboxylates for coordination have been proposed for the EcoRI and EcoRV restriction endonucleases (15,16). Certain bacterial transposases and retroviral integrases have a conserved DDE motif that has been proposed to be involved directly in catalysis (17)(18)(19). In a mechanism proposed for the yeast site-specific recombinases Flp and R, invariant arginines either facilitate the phosphoryl transfer by stabilizing the leaving group or the partial charge on the phosphorus in the transition state during the phosphodiester cleavage or exchange reactions (20). These recombinases do not require divalent ions for their activity. Their mechanisms may also share some similarities with topoisomerases. It should be noted that Mg(II) is not absolutely required for the DNA cleavage activity of E. coli DNA topoisomerase I.
To investigate the roles of the carboxylates and arginine residues in the active site region of E. coli topoisomerase I, they were altered by site-directed mutagenesis to alanines, abolishing the acidic or basic functional groups. The mutant enzymes were expressed and purified. Different enzymatic assays were carried out to determine how the mutation affected the interaction of the topoisomerase enzyme with DNA and/or Mg(II).
EXPERIMENTAL PROCEDURES
Materials-All chemical reagents used were ultrapure or Baker analyzed reagent grade. Solutions were prepared with water first deionized with the Barnstead Nanopure system and then passed over a Bio-Rad Chelex 100 resin (100 -200 mesh sodium form) to remove any remaining contaminating metal ions. Tubes, spectrophotometric cells, and glassware for metal ion-sensitive experiments were first washed with 10 mM EDTA and then rinsed extensively with metal-free water * This work was supported by Grant GM54226 from NIGMS, National Institutes of Health, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. A number of distinct properties of VP16 have been implicated in the assembly of the VP16-induced complex (VIC). These include its independent association with VCAF-1 and, under appropriate conditions, its ability to bind to DNA or to DNA-bound Oct-1 in the absence of VCAF-1. In order to probe the requirements of these individual interactions in the functional asembly of VIC, we mutated selected charged amino acids in two subdomains of VP16 previously shown to be important in protein-DNA complex formation. Purified VP16 proteins were analyzed for their ability to direct protein-DNA complex formation and to interact directly with VCAF-1. Several classes of mutants that were differentially compromised in VCAF-1 interaction, direct DNA binding, and/or association with DNA-bound Oct-1 were obtained. Interestingly, all of the derivatives were still capable of generating the VIC complex in vitro and activating transcription in vivo. Our findings indicate that the cooperative assembly of functional VP16-containing complexes can occur by pathways that do not necessarily require the prior interaction of VP16 with VCAF-1 or the ability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1.
Transcriptional regulation of the herpes simplex virus immediate early (IE) 1 genes by the viral transactivator VP16 (also called Vmw65 or ␣TIF) has provided a valuable model system to investigate how multicomponent protein-protein and protein-DNA assemblies orchestrate specific gene regulatory patterns (reviewed in Refs. [1][2][3]. VP16 is an abundant 490-amino acid-long structural phosphoprotein which, when delivered into the host cell by the infecting virus particle, strongly stimulates the transcription of the viral IE genes through recognition of cis-regulatory TAATGARAT (R ϭ purine) target elements that are present in one or more copies in the upstream regions of responsive genes. VP16, however, has only weak intrinsic DNA binding activity and efficient binding to target sites requires the assembly of VP16 into a multicomponent complex along with at least two cellular factors, the ubiquitously expressed POU homeodomain protein Oct-1, and an additional cellular factor variously called VCAF-1, HCF, C1, or CCF (Refs. 4 -14; referred to as VCAF-1 in this paper). The fully assembled functional complex, referred to as the VP16-induced complex (VIC), is thought to position the strong carboxyl-terminal acidic activation domain of VP16 in the correct spatial arrangement for functional interaction with downstream target proteins, among which include several basal transcription factors and adapter proteins (15,16).
The ordered assembly of VP16-containing protein-DNA complexes involves cooperative sequence-specific protein-DNA interactions as well as specific and selective protein-protein associations with Oct-1 and VCAF-1. Oct-1 binds independently to TAATGARAT elements and directs the recruitment of VP16 via determinants present in the POU homeodomain (17)(18)(19)(20)(21). The prior binding of Oct-1 to TAATGARAT elements is a prerequisite to the formation of VIC; however, the efficient incorporation of VP16 into the complex requires the auxiliary component VCAF-1, a large cellular factor which can interact directly with VP16 in the absence of Oct-1 and DNA (5,7,8,13,14,17). There is no evidence for the formation of Oct-1-VP16 or Oct-1-VCAF-1-VP16 heteromeric complexes in the absence of DNA; however, it has been demonstrated that VP16 is able to bind directly to DNA or to generate a complex with DNA-bound Oct-1 in the absence of VCAF-1 when high concentrations of VP16 are used in binding assays (17,19,22).
VP16 is a modular protein and contains separable domains that are important for complex formation and transactivation (19,(23)(24)(25)(26)(27)(28)(29). The carboxyl-terminal acidic activation domain is essential for transcriptional activation but not for complex assembly. The residual 400 amino-terminal amino acids are necessary and sufficient for VIC formation as well as for interaction with VCAF-1 and binding to DNA (14,24,25,27). More recently, it has been demonstrated that this region also contains determinants for interaction with the virion host shutoff protein (Vhs), a viral structural protein that is responsible for the cessation of host protein synthesis following viral infection (30). At least two subregions within the amino-terminal domain are involved in complex assembly with mammalian factors in vitro; region 1, spanning residues 140 -250, and region 2, encompassing residues 335-390 (19,24,25,27). Both subregions are involved in VIC formation, binding to VCAF-1, interaction with DNA-bound Oct-1, and contribute to the weak DNA binding activity associated with VP16 (19,24,27,(31)(32)(33).
Despite a enceinte numeral of mutational studies, it is not known whether the individual interactions attributed to VP16 are essential for the assembly of transcriptionally active complexes. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The values represent the average (ϮS. D.) from at least three separate transfections carried out in duplicate and normalized in each case to the value obtained from transfection of the reporter gene alone (control), which was taken as 1. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine
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Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. The transformants evicted the maintenance plasmid with a URA3 marker in the presence of 5-FOA and thus could not grow on the selection medium unless a functional cytoplasmic ValRS was encoded by the test plasmid.
Complementation Assays for Mitochondrial Function-CW1 was cotransformed with a test plasmid (carrying a LEU2 marker) and a second maintenance plasmid (carrying a HIS3 marker) that expresses only the cytoplasmic form of ValRS (due to a mutation in the ATG1 initiator codon). In the presence of 5-FOA, the first maintenance plasmid (carrying a URA3 marker) was evicted from the cotransformants, whereas the second maintenance plasmid was retained. Thus, all cotransformants survived 5-FOA selections due to the presence of the cytoplasmic ValRS derived from the second maintenance plasmid. The mitochondrial phenotypes of the cotransformants were further tested on YPG plates at 30°C, with results documented on day 3 after plating. Because a yeast cell cannot survive on glycerol without functional mitochondria, the cotransformants did not grow on the YPG plates unless a functional mitochondrial ValRS was generated from the test plasmid.
Western Blot Analysis-The protein expression patterns of the constructs used in the complementation assays were determined by a chemiluminescence-based Western blot analysis. INVSc1 (Invitrogen) was first transformed with the constructs of interest, and total protein extracts were prepared from each transformant. Aliquots of the protein extracts (40 g) were loaded onto a mini gel (8 ϫ 10 cm) containing 10% polyacrylamide and electrophoresed at 100 V for 1-2 h. After electrophoresis, the resolved proteins were transferred using a semidry transfer device to a polyvinylidene fluoride membrane in a buffer containing 30 mM glycine, 48 mM Tris base (pH 8.3), 0.037% SDS, and 20% methanol. The membrane was probed with a horseradish peroxidase-conjugated anti-FLAG tag antibody (Sigma) and then exposed to x-ray film after the addition of the appropriate substrates.
Aminoacylation Assay-Aminoacylation reactions were carried out at 25°C in a buffer containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 Purification of the His 6 -tagged proteins was as described previ-ously (32). Determination of active protein concentrations by active-site titration was as described (33). The final concentration of ValRS used in the reaction was 5, 10, or 50 nM. Reactions were quenched by spotting 10-l aliquots of the reaction mixture onto Whatman filters soaked in 5% trichloroacetic acid and 1 mM valine. The filters were washed three times, for 15 min each, in ice-cold 5% trichloroacetic acid before liquid scintillation counting. Data were obtained from at least three independent experiments and averaged. Determination of the kinetic parameters, K m and k cat , of the purified enzymes for yeast tRNA Val was as described (15).
Converting E. coli ValRS into a Functional Yeast Cytoplasmic Enzyme-Previous studies showed that a single VAS1
gene specifies both the mitochondrial and cytoplasmic forms of ValRS through alternative use of two in-frame AUG initiator codons, AUG1 and AUG47 (10,31). Hence, the mitochondrial precursor form essentially possesses the same polypeptide sequence as its cytoplasmic counterpart, except for an N-terminal MTS that is subsequently cleaved away by a matrix-processing peptidase upon being imported into mitochondria. Comparison of E. coli and yeast (the cytoplasmic form or the mature mitochondrial form) ValRSs showed that the catalytic cores of these two enzymes are significantly homologous to each another (ϳ42% identity), but the yeast enzyme has an N-terminal polypeptide extension of ϳ97 residues that is absent from its E. coli counterpart (Fig. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine<|endoftext|>PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. In cells, PA28 also exists in larger complexes along with the 19S particle, which allows ATP-dependent degradation of proteins; although in vivo a large fraction of PA28 is present as PA28αβ-20S particles whose exact biological functions are largely unknown. Although several lines of evidence strongly indicate that PA28αβ plays a role in MHC class I antigen presentation, the exact molecular mechanisms of this activity are still poorly understood. Herein, we review current knowledge about the biochemical and biological properties of PA28αβ and discuss recent findings concerning its role in modifying the spectrum of proteasome’s peptide products, which are important to better understand the molecular mechanisms and biological consequences of PA28αβ activity.
MHC Class I Antigen Presentation
The continual presentation of intracellular proteins fragments on major histocompatibility complex (MHC) class I molecules is a process that allows cytotoxic CD8 + T lymphocytes (CTLs) to identify and selectively eliminate cells that synthesize foreign (e.g., viral) or abnormal (e.g., oncogene products) proteins [1,2]. The vast majority of MHC class I-presented peptides (known as antigenic peptides or epitopes) are generated during the degradation of mature proteins or defective ribosomal products (DRiPs) by the ubiquitin-proteasome system (UPS) [3,4]. These peptides are then translocated through TAP transporters [5] to the endoplasmic reticulum (ER) where they bind to MHC class I heterodimers and are delivered to the cell surface [6]. The active form of the proteasome, which appears to degrade OPEN ACCESS most cellular proteins, is the 26S proteasome [7,8]. This large (2.4 MDa) and abundant multi-subunit proteolytic complex consists of the 20S proteasome, in which proteins are degraded, capped at one or both ends by the 19S regulatory particle, which is responsible for recognizing, unfolding, and translocating polyubiquitinated (and some non-ubiquitinated, e.g., denatured) substrates into the 20S internal proteolytic cavity [9]. The 20S proteasome is a barrel-shaped structure composed of four stacked heptameric rings. The two outer rings consist of Į-subunits, while the two central rings are made up of ȕ-subunits [10]. In most cells, the proteolytic activity of the 20S proteasome is located at subunits ȕ5 (X), ȕ2 (Z), and ȕ1 (Y) of the core particle. However, lymphoid cells and cells exposed to cytokines such as IFN-Ȗ alternatively express three homologous subunits (ȕ5i/LMP7, ȕ2i/MECL-1, ȕ1i/LMP2), which replace the constitutive ones in newly assembled so-called immunoproteasome particles [11]. A variety of studies have demonstrated that incorporation of these INF-Ȗ-induced subunits quantitatively modifies proteasomal cleavage preferences and enhances the production of a significant number of antigenic peptides, although there are examples of epitopes that are generated with lower efficiency, or which are not released, by immunoproteasomes [12]. In any case, the recent generation of transgenic mice lacking all three proteasomal catalytic ȕ-immune subunits clearly demonstrated the pivotal role that immunoproteasomes play in the generation of MHC class I ligands [13]. Additionally, immunoproteasomes have been shown to be important for efficient cytokine production [14] and have been implicated in a number of pathological disorders such as cancer and neurodegenerative and autoimmune diseases [15][16][17]. Finally, it has been recently demonstrated that 26S immunoproteasomes possess the capacity to hydrolyze basic proteins (such as Histones and Myelin Basic Protein) at greatly increased rates compared to constitutive proteasomes [18].
Structure
Another INF-Ȗ-inducible UPS component that affects MHC class I antigen presentation is PA28 (also termed REG or 11S), a ring-shaped 180 kDa multimeric complex that binds, in an ATP-independent manner, to the two ends of the 20S proteasome and substantially enhances its ability to degrade short peptide substrates, but not proteins or ubiquitin-conjugated proteins [19,20]. In addition, PA28 can also associate with the free end of asymmetric 26S proteasomes (19S-20S) to form so-called "hybrid proteasomes" (19S-20S-PA28) [21][22][23][24] that hydrolyze tri-and tetra-peptides at higher rates than canonical 26S particles [21,23]. In mammals, PA28 is composed of two homologous subunits, namely PA28Į (REGĮ or PSME1) and PA28ȕ (REGȕ or PSME2), both of which are induced by Ȗ-interferon [25][26][27][28][29][30][31]. A third member of PA28 family is PA28Ȗ (also known as REGȖ, 11SȖ or Ki antigen), which is a nuclear antigen that is not induced by INF-Ȗ and not involved in MHC class I antigen presentation. The biochemical and biological properties of PA28Ȗ has been reviewed elsewhere [32], and consequently will not be considered in detail.
PA28Į and PA28ȕ have an apparent molecular weight of 28 kDa on SDS-PAGE electrophoresis and share a nearly 50% amino acid sequence identity [25,29,33]. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
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Domain: Biology Chemistry Medicine
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Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. The survival rates of GP200 transformed with plasmid encoding the Glu-9, Glu-111, Asp-113, and Glu-115 mutants correlated with their in vitro activity, with the Glu-111 mutant conferring close to wild-type thermoresistance and the Glu-9 mutant conferring the least amount of thermoresistance.
The Arg-136 Mutant Could Cleave DNA and Form the Covalent Complex-The cleavage activities of the mutant enzymes were examined using 5Ј-end-labeled single-stranded DNA. Even though the Arg-136 mutant was totally inactive in the relaxation assay, the amount of cleaved DNA formed was comparable with that from the wild-type enzyme (Fig. 4). For the other mutants, the amounts of cleavage products observed were decreased, with the Glu-9 mutant having the lowest cleavage activity, so that the reduction of relaxation activity seen with these other mutants might be due to the decrease in the amount of cleaved complex formed by these mutants. The Asp-111 mutant had the same DNA cleavage efficiency as the wild-type enzyme.
A 3Ј-end-labeled oligonucleotide substrate was used to form a covalent complex with the enzyme. At 5 min after the addition of the enzyme to the oligonucleotides, the amounts of the covalent complex observed for the wild-type and the Arg-136 mutant were identical (Fig. 5). The amount of covalent complex formed by the Asp-111 mutant was also close to that of the wild type while the other mutant enzymes gave lower levels of This experiment also demonstrated that the Arg-136 mutation affected a step in the enzyme relaxation mechanism that took place after DNA cleavage. This could be the strand passage or the DNA religation step. The inter-molecular strand transfer activity of the Arg-136 mutant was compared with the wild type enzyme using the covalent complex formed with this 3Ј-end-labeled oligonucleotide substrate and HindIII-digested DNA as acceptor molecules (26). The result from one experiment is shown in Fig. 6. Data from several experiments indicated that the inter-molecular religation activity is about the same for the wild-type and the Arg-136 mutants. The efficiency of this reaction is low for both the wild-type and Arg-136 mutant enzymes. It may or may not reflect the relative activity of the intra-molecular religation that takes place during relaxation of supercoiled DNA.
Effect of the Glu-9, Asp-113, Glu-115, and R321 Mutations on Non-covalent DNA Binding-The decreased amount of cleaved complex formed by some of these mutants could be due to the effect of the mutation on non-covalent binding to DNA. The gel mobility shift assay was used to evaluate the non-covalent topoisomerase-DNA complex formation. The results (Fig. 7) showed that the Arg-321 and Glu-9 mutants had about 50% of the DNA binding activity as that of the wild-type enzyme. The Glu-115 mutant had slightly less binding activity (40%) while the Asp-113 mutant had the lowest DNA binding activity (25%).
The Glu-9 and Glu-115 Mutations Altered the Mg(II) Binding-The mutant enzymes were tested for Mg(II) binding by equilibrium dialysis against buffer containing 0.4 mM MgCl 2 . The Mg(II) binding stoichiometry was determined by inductively coupled plasma analysis (Table III). DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. To investigate whether the mitochondrial activity of yeast VAS1 can be functionally substituted with E. coli valS, a DNA segment coding for the MTS of the mitochondrial precursor form of yeast ValRS (base pairs ϩ1 to ϩ138 relative to the ATG1 initiator codon) was inserted in-frame at the 5Ј end of E. coli valS to facilitate the mitochondrial import of the encoded bacterial protein, and the mitochondrial complementation activity of the resulting construct was tested. As shown in Fig. 3, although the E. coli enzyme per se (without fusion of an MTS) failed to restore the growth phenotype of CW1 on YPG, fusion of an MTS to the bacterial enzyme enabled it to rescue the mitochondrial defect of the knock-out strain (Fig. 3B, numbers 3 and 5). Interestingly, ValRS of B. subtilis, a Gram-positive bacterium that is genetically highly divergent from E. coli, could also be made a functional yeast mitochondrial enzyme by fusion of the MTS (Fig. 3B, numbers 4 and 6).
Because the MTS of the fusion enzymes was cleaved away upon being imported into mitochondria (Fig. 3C, numbers 3-6), it is therefore likely that the MTS did not participate in the aminoacylation reactions catalyzed by the bacterial enzymes. Constructs pCW355 and pCW327 served as positive and negative controls, respectively, in the assay (Fig. 3B, numbers 1 and 2). Construct pCW355 carries a wildtype VAS1 gene that expresses both cytoplasmic and mitochondrial forms of yeast ValRS, whereas construct pCW327 carries an initiator mutant of VAS1 (ATG1 to GCG) that expresses only the cytoplasmic form of yeast ValRS.
Enhancing the Aminoacylation Activity of E. coli ValRS with a TRBD-To investigate whether the TRBDs shown in Fig. 2A enhance the aminoacylation activity of E. coli ValRS in vitro, His 6 -tagged E. coli ValRS, Ad(ScValRS)-EcValRS, and yeast ValRS were purified to homogeneity using nickel-nitrilotriacetic acid column chromatography and then assayed using unfractionated E. coli and yeast tRNAs as the substrates. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The introduced mutations are shown in Fig. 1A. These include alanine substitutions of arginines at positions 155, 162, 164, 169, 360, 366, and 368. Cys-176 was also mutated to examine the role of this sulfhydryl group. Also, Lys-379 was mutated, since the region surrounding this amino acid has been shown to be surface exposed and to be involved in protein-DNA complex formation (32).
VP16 and the various mutant derivatives were cloned into an MBP expression vector, and the proteins were purified by affinity chromatography on amylose resin ( Fig. 1B; all MBP-VP16 derivatives encoded residues 4 -411 and are thus missing the acidic activation domain). The purified proteins were used to monitor the following properties of VP16: direct DNA binding to the TAATGARAT elements, interaction with DNA-bound Oct-1, direct interaction with VCAF-1, VIC formation, and association with Vhs.
Direct Interaction of VP16 with DNA-Kristie and Sharp (17) first showed that if sufficiently large amounts of VP16 are used in DNA binding assays, VP16 can bind independently and sequence specifically to TAATGARAT elements. Fig. 2A shows the results of DNA binding assays with VP16 carried out in the absence of Oct-1 and VCAF-1 using the labeled ICP0 octa ϩ TAATGARAT element. A 10 -20-fold excess of VP16, compared with the amount sufficient to form VIC in the presence of Oct-1 and VCAF-1 (see below), was used in this case in order to observe direct DNA binding. As can be seen, MBP-VP16 (referred to as wild type protein unless noted otherwise), forms a distinct protein DNA complex with the probe (lane b). This interaction is sequence-specific, since it was not observed with mutant oligonucleotides that contained alterations in the GA-RAT portion (17, 37; data not presented). By way of comparison, a protein A-VP16 fusion protein (PA-VP16), which is larger than MBP-VP16, generates a slower migrating complex as expected (lane a). Mutant derivatives R155A, R164A, R169A, C176A, R368A, and K370A bound to DNA with efficiencies comparable with MBP-VP16 (compare lanes b with lanes c, e, f, g, j, and k, respectively). In contrast, R162A, R366A, and R368A were unable to bind to the probe (lanes d, h, and i, respectively), even when 3-fold higher concentrations of protein were used in the assays (not presented).
Interaction of VP16 with DNA-bound Oct-1-Oct-1 does not interact with VP16 in solution; however, under appropriate conditions, VP16 can interact weakly with DNA-bound Oct-1 in the absence of VCAF-1 (19,22). In order to examine the upshot of the introduced mutations on this interaction, mobility shift assays were carried out in the presence of purified GST-Oct-1 and the various VP16 derivatives. As shown in Fig. 2B, incubation of MBP-VP16 with GST-Oct-1 results in the formation of a weak Oct-1-MBP-VP16 complex that migrates slightly faster than the complex formed with MBP-VP16, which is also generated under these conditions with comparable efficiency (lane b). The reason this complex migrates slightly faster than the VP16-DNA complex is not clear; however, it may be due to conformation of the ternary complex that results in faster electrophoretic mobility compared with the VP16-DNA complex alone. A complex with similar mobility to wild type MBP-VP16-Oct-1 was observed with all the region 1 mutants (R155A, R162A, R164A, R169A, C176A; lanes c-g) but not with any of the region 2 mutants (R360A, R366A, R368A, K370A; lanes h-k). Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The altered amino acid codons are underlined. The notation refers to the type and position of the wild type amino acid (single letter code) targeted for alteration to an alanine residue. Purified phage DNA containing the desired mutations were screened by DNA sequence analysis and the replicative form DNA was prepared (34).
Gel Retardation Analysis-Protein-DNA mobility shift assays were carried out as before (31). Briefly, standard 20-l reactions contained 5 mM HEPES, pH 7.9, 0.1 mM dithiothreitol, 0.5 mM EDTA, 25 mM KCl, 4 g of nonspecific competitor DNA (a 1:2 mixture of salmon sperm DNA:poly(dI-dC)) and combinations of 0.05 g of purified GST-Oct-1, 2-4 l of affinity-purified VCAF-1, and various amounts of MBP-VP16 fusion proteins as described in the figure legends. Protein concentration in each reaction was normalized to 20 g with BSA. Reactions were preincubated for 5 min prior to the addition of 32 P-end-labeled probe DNA corresponding to the promoter proximal TAATGARAT element from the HSV-1 ICP0 gene, 5Ј GATCCCGTGCATGCTAATGATAT-TCTTT. Reactions were incubated for 20 min at 30°C and resolved on 3.5% polyacrylamide gels run at 4°C in 0.25 ϫ Tris borate-EDTA (34).
VCAF-1 Binding Assays-VCAF-1 binding assays were carried out essentially as described using purified MBP or MBP-VP16 fusion proteins adsorbed onto amylose resin (31). Briefly, 20 l of settled beads coupled with the various fusion proteins (2.5 mg of protein/ml of settled beads) were incubated for 2 h at 4°C with an equal volume of affinitypurified VCAF-1 with constant rotation. After low speed centrifugation, the supernatant containing the nonbound material was collected, and the beads were washed three times with 10 volumes of buffer D containing 100 g/ml BSA and 0.5% Nonidet P-40, followed by three washes with buffer D alone. Bound material was eluted from the settled beads with 20 l of buffer D containing 0.6 M KCl. Equivalent volumes of bound and nonbound material (6 l) were assayed for VCAF-1 activity by mobility shift analysis with GST-Oct-1 and wild type MBP-VP16. Buffer components and protein concentration in the binding assays were normalized as appropriate.
Vhs Binding Assays-Full-length [ 35 S]methionine-labeled HSV-1 vhs protein was prepared by transcription and translation of the in vitro expression plasmid pSPUTKvhs using rabbit reticulocyte lysates (Promega) as described previously (30). For binding assays, amylose beads adsorbed with equivalent amounts of MBP or the various MBP-VP16 fusion proteins were washed sequentially with 10 volumes of CB, CB ϩ 0.2% BSA, and CB ϩ 0.2% BSA ϩ 0.05% Nonidet P-40. 50 l of settled beads were recovered by low speed centrifugation and incubated with radiolabeled Vhs protein (200 l of a 1:100 dilution of programmed reticulocyte lysate). The beads were incubated for 1 h at 4°C with continuous rotation and recovered by centrifugation. Beads were washed once with 10 volumes of CB, three times with 10 volumes of CB ϩ 0.2% BSA, and 3 times with 10 volumes of CB ϩ 0.2% BSA ϩ 0.5% Nonidet P-40. Bound material was eluted by boiling with an equal volume of twice concentrated sodium dodecyl sulfate sample buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
RESULTS
Previous studies have shown that amino acids 140 -250 and 335-390 of VP16 contain determinants that are important for VIC formation and VCAF-1 interaction (19,24,25,27,31). Both regions 1 and 2 are enriched in positively charged residues, in particular arginine residues, relative to the whole protein. Since positively charged amino acids are important in both protein-protein and protein-DNA interactions, we decided to alter selected arginine residues to alanine residues by sitedirected mutagenesis. Alanine residues were chosen, since these would not be expected to alter the conformation of the polypeptide backbone (36). Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine
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Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. coli ValRS was not (Fig. 2). It should be noted that although the fusion enzyme had a significantly higher aminoacylation activity against yeast tRNA than did the E. coli enzyme, this level of activity was still far lower than that of the yeast enzyme (Fig. 4B).
To gain further insights into the molecular mechanism of enzyme catalysis, the K m and k cat values of E. coli ValRS and Ad(ScValRS)-EcValRS for yeast tRNA Val were determined. As it turned out, fusion of the Ad of yeast ValRS to the E. coli enzyme significantly enhanced its tRNA-binding affinity (ϳ5.3-fold increase) and had little effect on its turnover number. Ad(ScValRS)-EcValRS has a k cat /K m value 5-fold higher than that of E. coli ValRS (see Table 1). In sum, these results suggest that TRBDs such as the Ad of yeast ValRS can significantly improve the aminoacylation activity of E. coli ValRS against yeast tRNA.
Merging Cytoplasmic and Mitochondrial ValRS Activities into a Single Gene-Because E. coli ValRS can be converted to a functional yeast cytoplasmic or mitochondrial enzyme, we wondered whether the E. coli ValRS gene can be converted into a dual functional yeast gene specifying both cytoplasmic and mitochondrial activities. To investigate this possibility, a DNA segment (base pairs Ϫ300 to ϩ432 relative to the ATG1 initiator codon of yeast VAS1) that contains the VAS1 promoter and the sequence coding for the MTS (residues 1-46) and Ad (residues 47-144) of yeast ValRS was fused in-frame to the 5Ј end of the open reading frame of E. coli valS, and the complementation activities of the resultant construct were tested. As shown in Fig. 5, the fusion construct successfully rescued the growth defects of the VAS1 knock-out strain on both 5-FOA and YPG, suggesting that, as with VAS1, the fusion construct specifies both activities, with the mitochondrial activity being provided by the longer form (MTS-Ad(ScValRS)-EcValRS) and the cytoplasmic activity by the shorter form (Ad(ScValRS)-EcValRS). Because the MTS of the mitochondrial precursor form (the longer form) was cleaved away upon being imported into mitochondria, making the processed mitochondrial form indistinguishable in size from its cytoplasmic counterpart, only one protein band with a molecular mass close to Ad(ScValRS)-EcValRS (ϳ119 kDa) was identified by Western blotting (Fig. 5D).
DISCUSSION
For a given amino acid specificity, there are generally two distinct nuclear aaRS genes in yeast, one encoding the cytoplas-
TABLE 1 Kinetic parameters for aminoacylation of tRNA Val by EcValRS and Ad(ScValRS)-EcValRS
Each value is determined from a hyperbolic fit of two independent data sets. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine<|endoftext|>Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. 1-7) catalyze the interconversion of different DNA topological isomers by first forming a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl on the DNA phosphodiester linkage. After strand passage through the break, religation involving nucleophilic attack of the displaced DNA hydroxyl group on the phosphotyrosine linkage takes place. Type IA and type II DNA topoisomerases are linked to the 5Ј-phosphoryl end of the cleaved DNA while type IB DNA topoisomerases are linked to the 3Ј-phosphoryl end. Mg(II) is required for the relaxation activities of both type IA and type II DNA topoisomerases but not for the type IB enzymes. The detailed catalytic mechanism of DNA cleavage and religation by topoisomerases remains to be elucidated. The mechanism of the type IA and type II topoisomerase may share similarities with other enzymes that also require Mg(II) for nucleotidyl transfer activity.
Tyr-319 of Escherichia coli DNA topoisomerase I is the catalytic residue that provides the hydroxyl group for forming the covalent intermediate with DNA. The three-dimensional struc-ture of the 67-kDa N-terminal domain of this enzyme has been determined by x-ray crystallography (8). In this structure, Tyr-319 is present in the interface between domains I and III. It has been pointed out (8) that the spatial arrangement of the three acidic residues Asp-111, Asp-113, and Glu-115 in the active site region is similar to the acidic residues that coordinate two divalent cations in the exonuclease catalytic site of Klenow fragment (9). However, the structure observed has to undergo additional conformational changes before there is sufficient space in the active site region for DNA and possibly Mg(II) to bind. A number of residues found in the active site, including Glu-9, Asp-111, Asp-113, and Glu-115, and Arg-321, are strictly conserved among type IA DNA topoisomerase sequences (Fig. 1). This evolutionary conservation extends from the archaeal (Methanococcus jannaschii) topoisomerase I to human topoisomerase III. There is a lower degree of conservation for Arg-136 which is also near the active site tyrosine. The acidic and basic functional groups on these residues may be important for the enzyme catalytic function. For DNA polymerases, two divalent metal ions coordinated by carboxylates are essential for the enzyme activity (10 -14). One or two metal ion mechanisms utilizing carboxylates for coordination have been proposed for the EcoRI and EcoRV restriction endonucleases (15,16). Certain bacterial transposases and retroviral integrases have a conserved DDE motif that has been proposed to be involved directly in catalysis (17)(18)(19). In a mechanism proposed for the yeast site-specific recombinases Flp and R, invariant arginines either facilitate the phosphoryl transfer by stabilizing the leaving group or the partial charge on the phosphorus in the transition state during the phosphodiester cleavage or exchange reactions (20). These recombinases do not require divalent ions for their activity. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. As can be seen, MBP-VP16 (referred to as wild type protein unless noted otherwise), forms a distinct protein DNA complex with the probe (lane b). This interaction is sequence-specific, since it was not observed with mutant oligonucleotides that contained alterations in the GA-RAT portion (17, 37; data not presented). By way of comparison, a protein A-VP16 fusion protein (PA-VP16), which is larger than MBP-VP16, generates a slower migrating complex as expected (lane a). Mutant derivatives R155A, R164A, R169A, C176A, R368A, and K370A bound to DNA with efficiencies comparable with MBP-VP16 (compare lanes b with lanes c, e, f, g, j, and k, respectively). In contrast, R162A, R366A, and R368A were unable to bind to the probe (lanes d, h, and i, respectively), even when 3-fold higher concentrations of protein were used in the assays (not presented).
Interaction of VP16 with DNA-bound Oct-1-Oct-1 does not interact with VP16 in solution; however, under appropriate conditions, VP16 can interact weakly with DNA-bound Oct-1 in the absence of VCAF-1 (19,22). In order to examine the upshot of the introduced mutations on this interaction, mobility shift assays were carried out in the presence of purified GST-Oct-1 and the various VP16 derivatives. As shown in Fig. 2B, incubation of MBP-VP16 with GST-Oct-1 results in the formation of a weak Oct-1-MBP-VP16 complex that migrates slightly faster than the complex formed with MBP-VP16, which is also generated under these conditions with comparable efficiency (lane b). The reason this complex migrates slightly faster than the VP16-DNA complex is not clear; however, it may be due to conformation of the ternary complex that results in faster electrophoretic mobility compared with the VP16-DNA complex alone. A complex with similar mobility to wild type MBP-VP16-Oct-1 was observed with all the region 1 mutants (R155A, R162A, R164A, R169A, C176A; lanes c-g) but not with any of the region 2 mutants (R360A, R366A, R368A, K370A; lanes h-k). The findings that R368A and K320A were still able to bind independently to DNA but did not form the Oct-1/VP16 complex, whereas R162A was unable to bind to DNA (Fig. 2A, lane d) but could generate the Oct-1/VP16 complex (Fig. 2B, lane d), indicates that interaction of VP16 with DNA-bound Oct-1 does not require its intrinsic DNA binding activity.
Mutant VP16 Derivatives Direct VIC Formation-Assembly of the full multicomponent VIC complex requires the presence of VCAF-1 in addition to Oct-1 and VP16 (Fig. 3, compare lanes 2, 3, and 6, respectively). All of the mutant VP16 derivatives were capable of directing the formation of VIC when reactions were supplemented with VCAF-1 and Oct-1, although there was some variation in the amount of VIC formed with different mutants. Under the experimental conditions used in this experiment, an excess of VP16 was used in order to visualize both its independent DNA binding and interaction with Oct-1 in the same experiment (under standard conditions, only the VIC complex is observed; see Fig. 4). The findings indicate that neither the intrinsic DNA binding activity of VP16 nor its ability to directly associate with DNA-bound Oct-1 is required for higher order complex assembly directed by VCAF-1.
Independent Binding to VCAF-1 Is Not Essential for VIC Formation-VCAF-1 interacts directly with VP16 in the absence of target DNA or Oct-1; however, it is not known whether prior interaction of VP16 with VCAF-1 is a prerequisite for VIC formation. If this were the case, all of the VP16 derivatives should interact with VCAF-1, since they were able to direct VIC formation. To examine this directly, VCAF-1 microaffinity assays were performed. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. In this respect, it seems extremely unlikely that PA28 functions in vivo to stimulate degradation of cytosolic oligopeptides by proteasomes, because such peptides are very rapidly hydrolyzed by other cellular exo-and endo-peptidases [116]. Moreover, it has been shown in vitro that peptides released by proteasomes are further cleaved by these proteolytic particles at extremely low rates [117], and they are thus unlikely to efficiently compete for degradation with proteins, which are much more preferred substrates [118]. Importantly, the advantage of PA28-containing proteasomes that generate highly divergent patterns of potential antigenic peptides, characterized by profound qualitative and quantitative differences, might become relevant under specific pathophysiological conditions especially if favoring a non-canonical, ubiquitin-independent, protein hydrolysis pathway. In fact, most of the in vitro studies addressing the production of antigenic peptides have focused on degradation by proteasomes of denatured, non-ubiquitinated proteins since ubiquitinated proteins are not hydrolyzed by 20S and PA28-20S complexes [10,19]. This is likely due to the fact that these particles lack the enzymatic activities necessary to remove and/or to unfold polyubiquitin chains that otherwise would sterically block translocation of substrates into the proteolytic chamber [119][120][121]. Although further studies will be required to clarify whether the patterns of peptides released are different if a protein substrate initially binds to the 19S regulatory particle through a polyubiquitin chain or directly through an unstructured domain, several lines of evidence indicate that in vivo denatured proteins are important, physiological substrates for intracellular proteolysis. A large fraction of MHC class I epitopes is derived from rapid degradation of DriPs [122][123][124] in a process that has been shown to be at least partially ubiquitin-independent [124][125][126]. Furthermore, several recent reports indicate that some loosely-folded, short-lived regulatory, viral and oxidized proteins are degraded in vivo by the 20S proteasome in an ATP-and ubiquitin-independent manner [126][127][128][129][130][131][132][133]. In this regard, a contribution of PA28 in this alternative pathway of protein catabolism pathway seems plausible. Moreover, immunoproteasomes were recently reported to be strongly induced under conditions of oxidative stress [134], and according to several lines of evidence, oxidized proteins are preferentially degraded without ubiquitination by 20S proteasomes [131] in a process that was reported to be stimulated by PA28Įȕ [135][136][137]. Importantly, oxidative damage of DNA is recognized as an important cause of malignant transformation and cancer development [138], which emphasizes the importance of efficient MHC class I immune surveillance in the presence of oxidative stress. Of note, PA28Įȕ was shown to be highly induced in a naturally occurring tumor [15]. Under conditions of altered redox homeostasis, therefore, the pool of peptides specifically or preferentially released by PA28Įȕ-20S immunoproteasomes might be critical in eliciting an effective CTL response. Furthermore, by enhancing fragmentation of the large majority of proteasomal products, but at the same time, by promoting release from the 20S particle of specific peptides with a length of eight or more residues, PA28 is likely to exert a profound influence on the immunodominance hierarchy of CD8 + responses. Finally, it is also conceivable that by promoting release of peptides that apparently cannot serve in class I antigen presentation [101], PA28 might exert a regulatory function aimed at blunting excessive cytotoxic responses against antigens of self-origin, thus preventing the risk of potentially harmful autoimmune reactions.
Although PA28 has been discovered more than 20 years ago, its precise biological functions have remained somewhat elusive, and despite that several in vitro and in vivo studies have attempted to clarify its effects on MHC class I antigen presentation pathway, its role in adaptive immunity is still quite unclear. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
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Domain: Biology Chemistry Medicine
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Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. The result from one experiment is shown in Fig. 6. Data from several experiments indicated that the inter-molecular religation activity is about the same for the wild-type and the Arg-136 mutants. The efficiency of this reaction is low for both the wild-type and Arg-136 mutant enzymes. It may or may not reflect the relative activity of the intra-molecular religation that takes place during relaxation of supercoiled DNA.
Effect of the Glu-9, Asp-113, Glu-115, and R321 Mutations on Non-covalent DNA Binding-The decreased amount of cleaved complex formed by some of these mutants could be due to the effect of the mutation on non-covalent binding to DNA. The gel mobility shift assay was used to evaluate the non-covalent topoisomerase-DNA complex formation. The results (Fig. 7) showed that the Arg-321 and Glu-9 mutants had about 50% of the DNA binding activity as that of the wild-type enzyme. The Glu-115 mutant had slightly less binding activity (40%) while the Asp-113 mutant had the lowest DNA binding activity (25%).
The Glu-9 and Glu-115 Mutations Altered the Mg(II) Binding-The mutant enzymes were tested for Mg(II) binding by equilibrium dialysis against buffer containing 0.4 mM MgCl 2 . The Mg(II) binding stoichiometry was determined by inductively coupled plasma analysis (Table III). At this Mg(II) concentration, each molecule of the wild-type enzyme has been found to bind around 2 Mg(II) (27). A higher Mg(II) binding stoichiometry was observed for the Arg-136 and Arg-321 mutants. The replacement of the positively charged arginine residues by an alanine might make available an additional Mg(II) binding site in the enzyme, but this higher Mg(II) binding did not compensate for the mutation in the enzyme. The Asp-111 mutant had only a slight reduction in the amount of bound Mg(II) when compared with the wild-type enzyme. The Glu-9 and Glu-115 mutants had the greatest reduction in the binding of Mg(II).
Change in Topoisomerase I Fluorescence from Mutation-The fluorescence spectra of the wild-type and mutant topoisomerase I mutants were first compared at room temperature ( Fig. 8 and Table IV). The Glu-9 and Glu-115 mutants were found to have an ϳ30% drop in maximal fluorescence intensity, indicating a change in the protein conformation influencing the environment of the tryptophan residues in the enzyme. The decreases in fluorescence intensities were to a lesser extent for the Arg-136 and Arg-321 mutants (around 20%) while the Asp-111 and Asp-113 mutants had an ϳ25% drop in maximal fluorescence intensity. The fluorescence measurements were repeated at 42°C. The wild-type enzyme fluorescence was not affected significantly by the temperature shift. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>This below document has 2 sentences that start with 'As shown in', 2 sentences that end with 'on 5-FOA (Fig'. It has approximately 364 words, 23 sentences, and 3 paragraph(s).
<<<<>>>>
Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. 1). This Ad has recently been shown to act in cis as a nonspecific TRBD to facilitate tRNA binding and aminoacylation by the enzyme (15).
To test whether the E. coli enzyme can functionally substitute for its yeast homolog in vivo, the wild-type E. coli valS gene (encoding ValRS) was cloned into a yeast shuttle vector, and the ability of the resultant construct to rescue the growth defect of a yeast vas1 Ϫ strain on 5-FOA was tested. As shown in Fig. 2, the construct containing E. coli valS failed to restore the growth phenotype of the knock-out strain CW1 on 5-FOA (Fig. 2B, number 3), suggesting that the cytoplasmic activity of yeast VAS1 cannot be substituted with the bacterial enzyme in vivo. However, fusion of a nonspecific TRBD, such as Arc1p or the Ads of yeast GlnRS and ValRS, to the E. coli enzyme enabled the otherwise nonfunctional bacterial enzyme to act as a yeast enzyme; each of these fusions effectively rescued the growth defect of the knock-out strain on 5-FOA (Fig. 2B, numbers 4 -6). Constructs pCW355 and pCW349 served as positive and negative controls, respectively, in the assay (Fig. 2B, numbers 1 and 2). Construct pCW355 carries a wild-type VAS1 gene that expresses both cytoplasmic and mitochondrial forms of yeast ValRS, whereas construct pCW349 carries an initiator residues that is absent from its E. coli counterpart. The polypeptide sequence of the mitochondrial (mit) precursor form of yeast ValRS is essentially identical to that of its cytoplasmic isoform except for a mitochondrial targeting signal attached at its N terminus. To examine whether E. coli ValRS and its fusions are properly expressed in yeast, we next examined the relative expression levels of these constructs by Western blotting using an anti-FLAG tag antibody. As shown in Fig. 2C, E. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine<|endoftext|>Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. In contrast, the fluorescence intensity of the Arg-321 mutant decreased by more than 40%, indicating lower stability. The Glu-9 mutant had a much smaller decrease in fluorescence intensity at the higher temperature (Ͻ14%) while the fluorescence intensities of the other mutants did not change significantly.
DISCUSSIONS
The site-directed mutagenesis study described here aimed at elucidating the function of several strictly conserved acidic or basic amino acid residues found at the proximity of the active site nucleophile Tyr-319. For Arg-136, mutation to alanine abolished relaxation activity totally. Mutations of the other residues produced enzymes with reduced but observable in vitro activities. Therefore, the strict conservation in evolution did not necessarily correlate with absolute requirement of the residue for in vitro activity. Nevertheless, all the mutants tested failed to complement E. coli AS17 for growth at 42°C. This might at least partly be due to effect of the mutations on the stability and thus expression level of the enzyme in E. coli AS17. The Arg-136 mutant was the only one among the active site mutants tested here found to have an expression level in E. coli AS17 comparable with that of the wild-type enzyme. The 7. Effect of the mutations on the formation of non-covalent complex as assayed by the gel mobility shift assay using a 5-end-labeled 36-mer.
other mutant proteins were accumulated at a reduced or unobservable level. In contrast, mutations in the Zn(II) binding domain resulted in mutant proteins that were accumulated at higher levels than the wild-type enzyme (27). In the E. coli strain GP200 where the Glu-9, Asp-111, Asp-113, and Glu-115 mutants were stably expressed, these mutants were able to confer some degree of thermoresistance to compensate for the absence of chromosomal topA activity.
In the crystal structure of the 67-kDa N-terminal fragment, Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 are part of an extensive network of hydrogen bonds and salt bridges that are responsible for the domain-domain interactions (8). Mutation of these residues to alanines might abolish some of these interactions and destabilize the protein structure. This potential effect of the mutation on the enzyme structure and stability is consistent with the protein fluorescence data shown in Table IV, with the structure of Arg-321 mutant particularly sensitive to the increase in temperature.
Mg(II) is required for the relaxation of DNA by E. coli DNA topoisomerase I. It is not required for the cleavage of singlestranded DNA although the rate of cleavage of small oligonucleotide substrates can be enhanced by the presence of Mg(II) (28,29). DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine
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Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. 3B, numbers 4 and 6).
Because the MTS of the fusion enzymes was cleaved away upon being imported into mitochondria (Fig. 3C, numbers 3-6), it is therefore likely that the MTS did not participate in the aminoacylation reactions catalyzed by the bacterial enzymes. Constructs pCW355 and pCW327 served as positive and negative controls, respectively, in the assay (Fig. 3B, numbers 1 and 2). Construct pCW355 carries a wildtype VAS1 gene that expresses both cytoplasmic and mitochondrial forms of yeast ValRS, whereas construct pCW327 carries an initiator mutant of VAS1 (ATG1 to GCG) that expresses only the cytoplasmic form of yeast ValRS.
Enhancing the Aminoacylation Activity of E. coli ValRS with a TRBD-To investigate whether the TRBDs shown in Fig. 2A enhance the aminoacylation activity of E. coli ValRS in vitro, His 6 -tagged E. coli ValRS, Ad(ScValRS)-EcValRS, and yeast ValRS were purified to homogeneity using nickel-nitrilotriacetic acid column chromatography and then assayed using unfractionated E. coli and yeast tRNAs as the substrates. Aminoacylation was carried out at 25°C at a final concentration of 5, 10, or 50 nM ValRS. As shown in Fig. 4A, yeast ValRS could barely charge E. coli tRNA in vitro compared with the E. coli enzyme under the conditions used. Likewise, E. coli ValRS had a relatively poor charging activity against yeast tRNA in vitro compared with the yeast enzyme (Fig. 4B). This finding is not surprising, considering the fact that the E. coli and yeast enzymes have coevolved with their respective cognate tRNAs and therefore developed certain levels of species specificity or barriers in tRNA recognition. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The findings that R368A and K320A were still able to bind independently to DNA but did not form the Oct-1/VP16 complex, whereas R162A was unable to bind to DNA (Fig. 2A, lane d) but could generate the Oct-1/VP16 complex (Fig. 2B, lane d), indicates that interaction of VP16 with DNA-bound Oct-1 does not require its intrinsic DNA binding activity.
Mutant VP16 Derivatives Direct VIC Formation-Assembly of the full multicomponent VIC complex requires the presence of VCAF-1 in addition to Oct-1 and VP16 (Fig. 3, compare lanes 2, 3, and 6, respectively). All of the mutant VP16 derivatives were capable of directing the formation of VIC when reactions were supplemented with VCAF-1 and Oct-1, although there was some variation in the amount of VIC formed with different mutants. Under the experimental conditions used in this experiment, an excess of VP16 was used in order to visualize both its independent DNA binding and interaction with Oct-1 in the same experiment (under standard conditions, only the VIC complex is observed; see Fig. 4). The findings indicate that neither the intrinsic DNA binding activity of VP16 nor its ability to directly associate with DNA-bound Oct-1 is required for higher order complex assembly directed by VCAF-1.
Independent Binding to VCAF-1 Is Not Essential for VIC Formation-VCAF-1 interacts directly with VP16 in the absence of target DNA or Oct-1; however, it is not known whether prior interaction of VP16 with VCAF-1 is a prerequisite for VIC formation. If this were the case, all of the VP16 derivatives should interact with VCAF-1, since they were able to direct VIC formation. To examine this directly, VCAF-1 microaffinity assays were performed. The various MBP-VP16 mutant fusion proteins were linked to amylose resin and the beads were incubated with purified VCAF-1. In preparing the affinity matrix, care was taken to secure that the concentration of the immobilized mutants was similar for each case. Bound and nonbound material was collected and assayed for VIC formation in the presence of wt MBP-VP16 and GST-Oct-1. As shown in Fig. 4, beads coupled with MBP-VP16, R155A, R162A, R164A, C176A, and R360A retained VCAF-1 activity (lanes c, d, e, f, h, and i, respectively), whereas VCAF-1 activity was not retained on beads coupled with MBP or to beads coupled with R169A, R366A, R368A, or K370A (lanes b, g, j, k, and l, respectively). These results demonstrate that prior and independent interaction between VP16 and VCAF-1 is not essential for VIC formation.
Interaction of VP16 with Vhs-We have recently identified the HSV-1 virion host shutoff protein vhs as a novel ligand for VP16 (30). Preliminary mapping studies indicated that stable interaction with Vhs requires several regions present in the amino-terminal 369 amino acids of VP16, suggesting that the overall conformation of VP16 is important for VP16-Vhs complex formation. We therefore determined whether the point mutant derivatives of VP16 were compromised in their ability to interact with Vhs. For this purpose, [ 35 S]methionine-labeled Vhs was prepared by transcription and translation in vitro. The different MBP-VP16 point mutants were coupled to amylose beads as described above and incubated with labeled Vhs. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The fact that determinants important for VP16 interaction with VCAF-1 and Oct-1 overlap, or are in close proximity to each other, suggests that VCAF-1 also contacts Oct-1, as implied previously by mutation and peptide competition studies (19,32). Similarly, the observation that the intrinsic DNA binding activity of VP16 is dispensable for VIC formation does not necessarily mean that this interaction does not occur in the fully assembled complex.
In summary, our findings suggest versatility and flexibility in the assembly of VP16-containing complexes, whereby complexes can assemble by different pathways. This is compatible with recent findings that demonstrate that the conformation of Oct-1 POU homeodomain, and possibly the VP16-induced complex itself, is different on distinct TAATGARAT elements and that diverse response elements may have different functional properties in vivo (22, 38, 40 -42). This flexibility could provide a mechanism by which VP16 function can adapt to different physiological conditions in the host cell and thus modulate progression of the lytic cycle. For instance, the surprising observation that direct interaction of VCAF-1 with VP16 can be uncoupled from subsequent higher order protein-DNA complex assembly and transactivation does not necessarily mean that prior interaction between VCAF-1 and VP16 is dispensable for VIC formation under all conditions. Thus, in cells where VCAF-1 concentration is low, prior assembly of the VCAF-1-FIG. 6. Transactivation by VP16 mutants in vivo. The various VP16 mutants were cloned into a mammalian expression vector that restored the acidic activation domain and transfected, at the indicated concentrations, into Vero cells along with the VP16-responsive p175cat reporter gene. CAT activity was monitored 48 h post-transfection. The values represent the average (ϮS. D.) from at least three separate transfections carried out in duplicate and normalized in each case to the value obtained from transfection of the reporter gene alone (control), which was taken as 1. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. For instance, while VCAF-1 is essential for VIC formation, it is not known whether the intrinsic ability of VP16 to bind independently to VCAF-1 is a prerequisite for multicomponent complex assembly. Also, the weak DNA binding activity associated with VP16, or VCAF-1-independent interaction with DNA-bound Oct-1, is only observed when very high concentrations of VP16 are used in binding assays (17,19); thus, the physiological significance of these interactions is not clear. Most mutational studies of VP16 have been carried out using large deletions or linker insertions and these might be expected to cause conformational changes in VP16 that could have secondary effects on complex assembly and protein-protein interactions. The only point mutational analysis so far described assessed the overall effects of specific point mutants on VIC formation and transactivation (25). In order to determine if individual protein-protein and protein-DNA interactions involving VP16 can be uncoupled from VP16-induced complex formation and transactivation, we converted selected charged residues in regions 1 and 2 into alanine residues by site-directed mutagenesis. Mutant proteins were analyzed in vitro for protein-DNA complex formation with Oct-1 and interaction with VCAF-1 and in vivo for transactivation of a VP16-responsive reporter gene. Among the mutants generated were those that were defective in direct VCAF-1 interaction and DNA binding but were still capable of generating the VIC complex and directing transcriptional activation in vivo. Our findings indicate that the assembly of functional VP16-containing multicomponent complexes does not necessarily require the prior interaction of VP16 with VCAF-1 or the capability of VP16 to bind directly to DNA or associate with DNA-bound Oct-1, suggesting that the cooperative assembly of VP16-dependent complexes can occur by different pathways.
MATERIALS AND METHODS
Site-directed Mutagenesis-Site-directed mutagenesis was carried out by the Kunkle method (34) using a commercially available kit (Bio-Rad). Single-stranded M13mp19 containing the antisense strand of the VP16 open reading frame was used as the template for mutagenesis. The following mutagenic oligonucleotides, designed to convert the indicated wild type amino acids into alanine residues, were used. The altered amino acid codons are underlined. The notation refers to the type and position of the wild type amino acid (single letter code) targeted for alteration to an alanine residue. Purified phage DNA containing the desired mutations were screened by DNA sequence analysis and the replicative form DNA was prepared (34).
Gel Retardation Analysis-Protein-DNA mobility shift assays were carried out as before (31). Briefly, standard 20-l reactions contained 5 mM HEPES, pH 7.9, 0.1 mM dithiothreitol, 0.5 mM EDTA, 25 mM KCl, 4 g of nonspecific competitor DNA (a 1:2 mixture of salmon sperm DNA:poly(dI-dC)) and combinations of 0.05 g of purified GST-Oct-1, 2-4 l of affinity-purified VCAF-1, and various amounts of MBP-VP16 fusion proteins as described in the figure legends. Protein concentration in each reaction was normalized to 20 g with BSA. Reactions were preincubated for 5 min prior to the addition of 32 P-end-labeled probe DNA corresponding to the promoter proximal TAATGARAT element from the HSV-1 ICP0 gene, 5Ј GATCCCGTGCATGCTAATGATAT-TCTTT. Reactions were incubated for 20 min at 30°C and resolved on 3.5% polyacrylamide gels run at 4°C in 0.25 ϫ Tris borate-EDTA (34).
VCAF-1 Binding Assays-VCAF-1 binding assays were carried out essentially as described using purified MBP or MBP-VP16 fusion proteins adsorbed onto amylose resin (31). Briefly, 20 l of settled beads coupled with the various fusion proteins (2.5 mg of protein/ml of settled beads) were incubated for 2 h at 4°C with an equal volume of affinitypurified VCAF-1 with constant rotation. After low speed centrifugation, the supernatant containing the nonbound material was collected, and the beads were washed three times with 10 volumes of buffer D containing 100 g/ml BSA and 0.5% Nonidet P-40, followed by three washes with buffer D alone. Bound material was eluted from the settled beads with 20 l of buffer D containing 0.6 M KCl. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The beads were recovered, washed extensively, and the bound material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As shown in Fig. 5, Vhs bound to all of the mutant VP16 derivatives as effectively as it did to MBP-VP16. Labeled Vhs did not bind to the beads alone (lane h) or to beads complexed with MBP (lane b). Thus, none of the VP16 point mutants were defective in interaction with Vhs. These findings show that the targeted mutations are not important for Vhs interaction and, moreover, that the introduced mutations did not grossly affect the overall conformation of VP16.
Transactivation Properties of VP16 Mutant Derivatives-There is a strict correlation with the ability of VP16 to assembly into the VIC complex with subsequent transactivation in vivo. It is thought that assembly of this multicomponent complex is required only to bring the activation domain of VP16 to the promoter; however, additional VP16-DNA, VP16-Oct-1 and VP16-VCAF-1 interactions in the full complex may influence the overall conformation of the complex and thus contribute directly to transactivation. We therefore cloned the different VP16 derivatives into a mammalian expression vector that restored the carboxyl-terminal acidic activation domain and tested their abilities to transactivate gene expression in transient transfection assays using a CAT reporter gene that contained the promoter/regulatory region from the ICP4 gene. As shown in Fig. 6, co-transfection of Vero cells with the fulllength wild type VP16 expression plasmid induced expression of the reporter gene approximately 30-fold when 0.5 g of effector plasmid was used. Increasing the concentration of the wild type effector plasmid resulted in a reduction in activity as reported previously, likely as a result of squelching (24). Transfection of the mutant VP16 derivatives showed that all the mutants were capable of transactivating expression of the reporter gene, consistent with the observation that all of the mutant VP16 proteins remained capable of forming VIC. There were, however, variations in relative potency among the differ- In lane a, purified PA-VP16 (wild type VP16 fused to protein A) was used. PA-VP16 is larger than MBP-VP16, and thus the protein-DNA complex has a slower mobility. The probe used in this and subsequent experiments was the promoter proximal TAATGARAT element from the ICP0 promoter. B, gel retardation experiments were carried out with the indicated fusion proteins (6 g) in the presence of purified GST-Oct-1 POU homeodomain fusion protein (0.05 g; referred to as Oct-1 in all figures). Lane a is GST-Oct-1 alone. The positions of the VP16 DNA complex and the VP16-Oct-1 DNA complex are indicated. ent mutant proteins. At an effector plasmid dosage of 0.5 g, R164A and R169A behaved as wild type, while the remaining derivatives activated expression at an efficiency of 30 -40% compared with the wild type plasmid. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. The high concentration of VP16 necessary to observe these interactions is probably not physiologically significant. Indeed, transactivation of viral IE genes is detrimentally affected by large amounts of VP16 (24). Moreover, the source of VP16 may have an effect on the efficiency of these interactions. For instance, PA-VP16 has a higher affinity for DNA compared with MBP-VP16 ( Fig. 2A), 2 whereas GST-VP16 has only very weak intrinsic DNA binding activity (22). Thus, the carrier protein may have some influence on binding properties, perhaps by masking or exposing cryptic binding interfaces (17). It has been proposed that VP16 binds to the GARAT portion of the target site which, while not required for Oct-1 binding, is necessary for VIC assembly (5,17). More recent evidence indicates that the GARAT subregion serves to alter the conformation of the POU homeodomain and that this is necessary for subsequent recognition by VP16 (22). Our findings directly demonstrate that the independent DNA binding activity and interaction with DNA-bound Oct-1 is not absolutely essential for VIC assembly or for transcriptional activation in vivo (see below).
Region 2 is essential for VIC formation and also contains critical determinants for direct interaction with VCAF-1 (19,24,31). We, and others, have shown that the requirements for interaction with VCAF-1 can be uncoupled from VIC formation (19,31). Thus, VIC formation requires residues up to amino acid 388, whereas VCAF-1 can still interact with VP16 that is truncated to amino acid 379 (24,31). In addition, a linker insertion at position 379 abolished VIC formation but not interaction with VCAF-1 (19). Hayes previously, using limited proteolysis of VP16 and of VP16containing protein-DNA complexes, that the region surrounding Lys-370 is exposed on the surface of the protein and thus likely to be important for protein-protein interactions. Moreover, using peptide competition, these authors showed that a synthetic peptide encompassing amino acids 360 -373 (RE-HAYSRARTKNNY) or a truncated variant containing amino acids 360 -367 was able to inhibit VIC formation. The simplest conclusion was that this peptide interfered with the ability of VP16 to bind to VCAF-1, although this was not directly demonstrated. Our findings that mutation of arginine residues 366, 368, and lysine 370 disrupt VCAF-1 interaction is coherent with the conclusion that this subregion represents an important interface for VCAF-1 interaction. Furthermore, the finding that mutation of arginine 169 (this work) or a linker insertion at amino acid 177 (19) also disrupts association with VCAF-1 indicates that region 1 contributes to this interaction, further emphasizing that the overall conformation of VP16 is important. More significantly, all of the VP16 derivatives were capable of generating VIC in vitro and of transactivating expression of a VP16-responsive CAT reporter gene in vivo. Thus, the direct and independent interaction of VP16 with VCAF-1 is not absolutely required for the assembly of functionally active VIC complexes, suggesting that complex assembly can occur by different pathways. It is possible that multiple and additional layers of cooperativity occurring between VCAF-1, VP16, and Oct-1 in the full complex may compensate for deficiencies in individual interactions. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. As for the aminoacylation activity of the fusion enzyme (Ad(ScValRS)-EcValRS), our results showed that fusion of the Ad of yeast ValRS to the E. coli enzyme enhanced its aminoacylation activity against yeast tRNA by ϳ3.8-fold (Fig. 4B) but had only a minor effect on its aminoacylation activity against E. coli tRNA (ϳ1.4-fold increase) (Fig. 4A). To illustrate better the difference in aminoacylation activities against yeast tRNA between E. coli ValRS and the fusion enzyme, the protein concentration used for the assay was increased by 5-fold (from 10 to 50 nM). As shown in Fig. 4C, the fusion enzyme showed significantly higher aminoacylation activity against yeast tRNA than did E. coli ValRS, which might explain why the fusion enzyme was able to rescue the growth defect of CW1 on 5-FOA, whereas E. coli ValRS was not (Fig. 2). It should be noted that although the fusion enzyme had a significantly higher aminoacylation activity against yeast tRNA than did the E. coli enzyme, this level of activity was still far lower than that of the yeast enzyme (Fig. 4B).
To gain further insights into the molecular mechanism of enzyme catalysis, the K m and k cat values of E. coli ValRS and Ad(ScValRS)-EcValRS for yeast tRNA Val were determined. As it turned out, fusion of the Ad of yeast ValRS to the E. coli enzyme significantly enhanced its tRNA-binding affinity (ϳ5.3-fold increase) and had little effect on its turnover number. Ad(ScValRS)-EcValRS has a k cat /K m value 5-fold higher than that of E. coli ValRS (see Table 1). Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. To address this important issue, we recently performed a systematic analysis of the entire spectrum of peptides released during degradation of full-length proteins by PA28Įȕ-20S immunoproteasomes [101]. PA28Įȕ-20S particles were found to hydrolyze proteins at identical, slow rates compared to 20S proteasomes, and to generate higher amounts of very short products together with several longer peptides characterized by higher overall hydrophilicity.
The finding that unstructured polypeptides are hydrolyzed at rates that are nearly 10-fold higher by 26S compared to 20S and PA28Įȕ-20S immunoproteasomes is consistent with the notion that the free 20S particle is a relatively inactive protease since the N-terminal tails of its Į subunits obstruct the two opposite axial pores through which substrates access the internal catalytic lumen [94]. This autoinhibited state is relieved when the 20S core particle binds to activators such as 19S or PA28 that displace, in an ATP-dependent and independent manner, respectively, the N-terminal tails thereby opening an axial channel in the Į annulus [70,71]. However, the latency of unliganded 20S proteasome is not absolute since, even in the absence of artificial treatments (e.g., heating or presence of low concentrations of detergents or chaotropic agents) that are known to activate it [10], the 20S core particle degrades proteins at detectable and reproducible rates, probably involving transient and/or only partial channel opening [70,[76][77][78]. Surprisingly, despite the fact that the open-channel conformation of the gating residues induced by ATP-dependent and independent activators appears to be identical [105], our data show that PA28Įȕ-20S immunocomplexes hydrolyze proteins at the same rates as 20S immunoproteasomes, but much less efficiently than 26S immunoproteasomes. While on one hand this confirms initial reports on the biochemical properties of PA28 [19,20,65], it is difficult to reconcile with the known role of the proteasomal gate in controlling accessibility of substrates into the lumen of the 20S particle. In fact, opening of the axial channel by deletion of the N-terminal tails of eukaryotic [70,106] or archaeal [107] proteasomal Į subunits results in strong enhancement of the degradation rates of unfolded proteins, therefore suggesting that unstructured substrates can freely access the internal proteolytic chamber of the 20S particle simply by passive diffusion through a fully open gate. Furthermore, although it has been proposed that translocation of completely unstructured proteins might require a driving force produced by ATP hydrolysis [108], subsequent studies demonstrated that once the polypeptide chain is unfolded its transit through the ATPases ring can occur by passive diffusion, in which retrograde movement is probably prevented by a Brownian ratchet mechanism [109]. In light of these data, the inability of PA28Įȕ to enhance hydrolysis rates of loosely folded proteins is surprising. It can be speculated that the presence of a supplementary multimeric ring, sitting at the proteasomal outer Į surface, might impose an extra constraint to the completely free diffusion of large, although linear, polypeptide chains into the internal proteasomal lumen. From this point of view, transit thorough the pore of open gate mutants might not perfectly reflect passage across the PA26/28 central channel. In fact, the crystal structure of PA28Į shows that the aqueous channel through the heptamer has a diameter of 20 Å at its minimum, which is theoretically wide enough for passage of unfolded proteins [34]. However, the homolog-specific inserts present between helices 1 and 2, which are not resolved in the crystal structure, most likely form a ring-like collar on the upper, non-proteasome binding surface of the PA28 heptamer. Although several studies have shown that these loops do not restrict passage of tri-or tetra-peptide fluorogenic substrates [35,67], recent investigations have demonstrated that they can hinder the transit of longer peptides, and conceivably of proteins as well, through the PA28 channel [44]. In fact, we demonstrated that 20S and PA28Įȕ-20S immunoproteasomes hydrolyze proteins at exactly the same slow rates, which implies that denatured substrates transit through the partially or transiently open gate of unligated 20S and the fully open channel of PA28Įȕ-20S particles with comparable efficiency. However, it cannot be excluded that PA28Įȕ might selectively enhance degradation of some specific substrates that are yet to be identified, as has been unambiguously demonstrated for PA28Ȗ [110][111][112][113].
Effects of PA28Įȕ on Peptide Products Generation
Although PA28Įȕ is unable to enhance rates of protein degradation by proteasomes, its association with the 20S particle was found to lead to substantial changes in the patterns of peptides generated, which greatly differ from those produced by 20S and 26S immunoproteasomes [101]. In fact, only ~10% of peptides generated by 20S and 26S immunoproteasomes were found to be 8-10 residues long [101], which is the appropriate length to bind MHC class I heterodimers. Most importantly, association of PA28Įȕ with the ends of 20S immunoproteasomes does not increase the fraction of 8-10 residue peptides generated, but reduces it to 6% of the total. Moreover, the fraction of peptides longer than 10 amino acids, which might serve in MHC class I antigen presentation only after appropriate trimming by aminopeptidases in the cytosol or ER, is larger for 20S than for 26S immunoproteasomes; binding of PA28Įȕ to the 20S particle dramatically reduces the overall efficiency of generation of these longer products. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
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Domain: Biology Chemistry Medicine<|endoftext|>PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. Importantly, oxidative damage of DNA is recognized as an important cause of malignant transformation and cancer development [138], which emphasizes the importance of efficient MHC class I immune surveillance in the presence of oxidative stress. Of note, PA28Įȕ was shown to be highly induced in a naturally occurring tumor [15]. Under conditions of altered redox homeostasis, therefore, the pool of peptides specifically or preferentially released by PA28Įȕ-20S immunoproteasomes might be critical in eliciting an effective CTL response. Furthermore, by enhancing fragmentation of the large majority of proteasomal products, but at the same time, by promoting release from the 20S particle of specific peptides with a length of eight or more residues, PA28 is likely to exert a profound influence on the immunodominance hierarchy of CD8 + responses. Finally, it is also conceivable that by promoting release of peptides that apparently cannot serve in class I antigen presentation [101], PA28 might exert a regulatory function aimed at blunting excessive cytotoxic responses against antigens of self-origin, thus preventing the risk of potentially harmful autoimmune reactions.
Although PA28 has been discovered more than 20 years ago, its precise biological functions have remained somewhat elusive, and despite that several in vitro and in vivo studies have attempted to clarify its effects on MHC class I antigen presentation pathway, its role in adaptive immunity is still quite unclear. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
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Domain: Biology Chemistry Medicine
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Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. The spectral bandwidths were 3 and 10 nm, respectively, for excitation and emission. The wild-type or mutant topoisomerase I was present at 0.1 mg/ml in 20 mM potassium phosphate, pH 7.5, 0.1 M KCl, 0.2 mM dithiothreitol. All the measurements were corrected for the spectrum of the buffer used.
Equilibrium Dialysis to Determine Mg(II) Binding Stoichiometry-1 ml of topoisomerase I (0.4 mg/ml) was dialyzed against 400 ml of buffer (20 mM potassium phosphate, pH 7.5, 0.1 M KCl, 0.2 mM dithiothreitol, and 400 M MgCl 2 ) at room temperature for 7 h. The enzyme and dialysis buffer samples were submitted to Quantitative Technologies, Inc., NJ, for Mg(II) content analysis using the inductively coupled plasma method.
RESULTS
Expression and Purification of the Topoisomerase I Mutants-After site-directed mutagenesis of the plasmid pJW312 to produce the desired alanine substitutions, expression of the mutant proteins in E. coli strain AS17 was examined by SDSgel electrophoresis of the total soluble extract followed by Coomassie Blue staining of the gel. The expression level of the Arg-136 mutant was comparable with that of the wild-type topoisomerase I while expression of the Arg-321 mutant was detectable but lower than the wild type. Bands corresponding to the Glu-9, Asp-111, Asp-113, and Glu-115 mutants could not be identified in AS17 extracts (data not shown). Expression of these three mutant enzymes were then carried out in E. coli strain GP200. Detectable levels of expression of these mutant enzymes allowed the purification of the mutant enzymes to homogeneity (Fig. 2).
Effect of the Mutations on Enzyme Activities-Purified mutant enzymes were diluted serially and compared with the wild-type topoisomerase I for relaxation of negatively supercoiled plasmid DNA in relaxation buffer containing 6 mM MgCl 2 (Fig. 3A). The results showed that the Arg-136 mutant was totally inactive in the relaxation assay. No relaxation activity was observable even with 400 ng of the enzyme, a 50-fold excess over the amount needed to observe relaxation by the wild-type enzyme. The other mutants had varying degrees of loss of relaxation activity. Examination of the effect of dilutions and time course of relaxation (Fig. 3B) showed that the Glu-9 mutant had the greatest reduction in catalytic activity (Ͼ90% reduction). The Glu-115, Asp-113, and Arg-321 mutants had about 80 -90% reduction in activity. The Asp-111 mutant was closest to the wild-type enzyme in activity (Ͻ50% reduction).
The E. coli strain AS17 does not grow at 42°C because of the temperature sensitivity of the suppressor for the chromosomal topA am mutation. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. Depew, Northeastern Ohio University). The E9A, D111A, D113A, and E115A mutants were expressed in E. coli GP200 (gyrA(Nal r ) gyrB225⌬ (topAcysB)204) (22). Enzyme purification was carried out using the previously described procedures (23) with an additional hydroxyapatite chromatography step between the phosphocellulose and singlestranded DNA-agarose columns.
Relaxation Activity Assay-Wild-type and mutant enzymes were serially diluted and assayed for relaxation activity in 20 l with 0.5 g of negatively supercoiled plasmid DNA, 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1 mg/ml gelatin, and 6 mM MgCl 2 unless indicated otherwise. Incubation was at 37°C for 30 min. The reactions were stopped by the addition of 5 l of 50% glycerol, 50 mM EDTA, and 0.5% (v/v) bromphenol blue. After electrophoresis in a 0.7% agarose gel with TAE buffer (40 mM Tris acetate, pH 8.1, 2 mM EDTA), the DNA was stained with ethidium bromide and photographed over UV light.
Covalent Complex with Oligonucleotide Substrate-The singlestranded oligonucleotide 5Ј-CAATGCGCT-3Ј, containing the sequence of a strong cleavage site (24), was labeled at the 3Ј end with [␣-32 P]dATP and terminal deoxynucleotidyl transferase. After incubation of 0.15 g of the labeled oligonucleotide with 0.4 g of the enzyme in 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 20 mM potassium phosphate at 37°C for 5 min, the reaction was stopped with the addition of 1% SDS. The enzyme was separated from the non-covalently bound oligonucleotides by electrophoresis in a 10% SDS-polyacrylamide gel. The labeled covalent complex was visualized by autoradiography of the dried gel.
tion was carried out using the Molecular Dynamics PhosphorImager.
Intrinsic Tryptophan Fluorescence Measurements-Fluorescence measurements were performed with the Perkin-Elmer LS-5B spectrometer with excitation at either 280 or 295 nm at either 42°C or room temperature (ϳ25°C). The spectral bandwidths were 3 and 10 nm, respectively, for excitation and emission. The wild-type or mutant topoisomerase I was present at 0.1 mg/ml in 20 mM potassium phosphate, pH 7.5, 0.1 M KCl, 0.2 mM dithiothreitol. All the measurements were corrected for the spectrum of the buffer used.
Equilibrium Dialysis to Determine Mg(II) Binding Stoichiometry-1 ml of topoisomerase I (0.4 mg/ml) was dialyzed against 400 ml of buffer (20 mM potassium phosphate, pH 7.5, 0.1 M KCl, 0.2 mM dithiothreitol, and 400 M MgCl 2 ) at room temperature for 7 h. The enzyme and dialysis buffer samples were submitted to Quantitative Technologies, Inc., NJ, for Mg(II) content analysis using the inductively coupled plasma method.
RESULTS
Expression and Purification of the Topoisomerase I Mutants-After site-directed mutagenesis of the plasmid pJW312 to produce the desired alanine substitutions, expression of the mutant proteins in E. coli strain AS17 was examined by SDSgel electrophoresis of the total soluble extract followed by Coomassie Blue staining of the gel. The expression level of the Arg-136 mutant was comparable with that of the wild-type topoisomerase I while expression of the Arg-321 mutant was detectable but lower than the wild type. Bands corresponding to the Glu-9, Asp-111, Asp-113, and Glu-115 mutants could not be identified in AS17 extracts (data not shown). Expression of these three mutant enzymes were then carried out in E. coli strain GP200. Detectable levels of expression of these mutant enzymes allowed the purification of the mutant enzymes to homogeneity (Fig. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. Thus, VIC formation requires residues up to amino acid 388, whereas VCAF-1 can still interact with VP16 that is truncated to amino acid 379 (24,31). In addition, a linker insertion at position 379 abolished VIC formation but not interaction with VCAF-1 (19). Hayes previously, using limited proteolysis of VP16 and of VP16containing protein-DNA complexes, that the region surrounding Lys-370 is exposed on the surface of the protein and thus likely to be important for protein-protein interactions. Moreover, using peptide competition, these authors showed that a synthetic peptide encompassing amino acids 360 -373 (RE-HAYSRARTKNNY) or a truncated variant containing amino acids 360 -367 was able to inhibit VIC formation. The simplest conclusion was that this peptide interfered with the ability of VP16 to bind to VCAF-1, although this was not directly demonstrated. Our findings that mutation of arginine residues 366, 368, and lysine 370 disrupt VCAF-1 interaction is coherent with the conclusion that this subregion represents an important interface for VCAF-1 interaction. Furthermore, the finding that mutation of arginine 169 (this work) or a linker insertion at amino acid 177 (19) also disrupts association with VCAF-1 indicates that region 1 contributes to this interaction, further emphasizing that the overall conformation of VP16 is important. More significantly, all of the VP16 derivatives were capable of generating VIC in vitro and of transactivating expression of a VP16-responsive CAT reporter gene in vivo. Thus, the direct and independent interaction of VP16 with VCAF-1 is not absolutely required for the assembly of functionally active VIC complexes, suggesting that complex assembly can occur by different pathways. It is possible that multiple and additional layers of cooperativity occurring between VCAF-1, VP16, and Oct-1 in the full complex may compensate for deficiencies in individual interactions. The fact that determinants important for VP16 interaction with VCAF-1 and Oct-1 overlap, or are in close proximity to each other, suggests that VCAF-1 also contacts Oct-1, as implied previously by mutation and peptide competition studies (19,32). Similarly, the observation that the intrinsic DNA binding activity of VP16 is dispensable for VIC formation does not necessarily mean that this interaction does not occur in the fully assembled complex.
In summary, our findings suggest versatility and flexibility in the assembly of VP16-containing complexes, whereby complexes can assemble by different pathways. This is compatible with recent findings that demonstrate that the conformation of Oct-1 POU homeodomain, and possibly the VP16-induced complex itself, is different on distinct TAATGARAT elements and that diverse response elements may have different functional properties in vivo (22, 38, 40 -42). This flexibility could provide a mechanism by which VP16 function can adapt to different physiological conditions in the host cell and thus modulate progression of the lytic cycle. For instance, the surprising observation that direct interaction of VCAF-1 with VP16 can be uncoupled from subsequent higher order protein-DNA complex assembly and transactivation does not necessarily mean that prior interaction between VCAF-1 and VP16 is dispensable for VIC formation under all conditions. Thus, in cells where VCAF-1 concentration is low, prior assembly of the VCAF-1-FIG. 6. Transactivation by VP16 mutants in vivo. The various VP16 mutants were cloned into a mammalian expression vector that restored the acidic activation domain and transfected, at the indicated concentrations, into Vero cells along with the VP16-responsive p175cat reporter gene. CAT activity was monitored 48 h post-transfection. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. At this Mg(II) concentration, each molecule of the wild-type enzyme has been found to bind around 2 Mg(II) (27). A higher Mg(II) binding stoichiometry was observed for the Arg-136 and Arg-321 mutants. The replacement of the positively charged arginine residues by an alanine might make available an additional Mg(II) binding site in the enzyme, but this higher Mg(II) binding did not compensate for the mutation in the enzyme. The Asp-111 mutant had only a slight reduction in the amount of bound Mg(II) when compared with the wild-type enzyme. The Glu-9 and Glu-115 mutants had the greatest reduction in the binding of Mg(II).
Change in Topoisomerase I Fluorescence from Mutation-The fluorescence spectra of the wild-type and mutant topoisomerase I mutants were first compared at room temperature ( Fig. 8 and Table IV). The Glu-9 and Glu-115 mutants were found to have an ϳ30% drop in maximal fluorescence intensity, indicating a change in the protein conformation influencing the environment of the tryptophan residues in the enzyme. The decreases in fluorescence intensities were to a lesser extent for the Arg-136 and Arg-321 mutants (around 20%) while the Asp-111 and Asp-113 mutants had an ϳ25% drop in maximal fluorescence intensity. The fluorescence measurements were repeated at 42°C. The wild-type enzyme fluorescence was not affected significantly by the temperature shift. In contrast, the fluorescence intensity of the Arg-321 mutant decreased by more than 40%, indicating lower stability. The Glu-9 mutant had a much smaller decrease in fluorescence intensity at the higher temperature (Ͻ14%) while the fluorescence intensities of the other mutants did not change significantly.
DISCUSSIONS
The site-directed mutagenesis study described here aimed at elucidating the function of several strictly conserved acidic or basic amino acid residues found at the proximity of the active site nucleophile Tyr-319. For Arg-136, mutation to alanine abolished relaxation activity totally. Mutations of the other residues produced enzymes with reduced but observable in vitro activities. Therefore, the strict conservation in evolution did not necessarily correlate with absolute requirement of the residue for in vitro activity. Nevertheless, all the mutants tested failed to complement E. coli AS17 for growth at 42°C. This might at least partly be due to effect of the mutations on the stability and thus expression level of the enzyme in E. coli AS17. The Arg-136 mutant was the only one among the active site mutants tested here found to have an expression level in E. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine
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Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. Sequence comparison suggests that the yeast cytoplasmic (or mature mitochondrial) ValRS contains an N-terminal appendage that acts in cis as a nonspecific tRNA-binding domain (TRBD) and is absent from its bacterial relatives. We show here that Escherichia coli ValRS can substitute for the mitochondrial and cytoplasmic functions of VAS1 by fusion of a mitochondrial targeting signal and a TRBD, respectively. In addition, the bacterial ValRS gene can be converted into a dual functional yeast gene encoding both cytoplasmic and mitochondrial activities by fusion of a DNA sequence specifying both the mitochondrial targeting signal and TRBD. In vitro assays suggested that fusion of a nonspecific TRBD to the bacterial enzyme significantly enhanced its yeast tRNA-binding and aminoacylation activities. These results not only underscore the necessity of retaining a TRBD for functioning of a tRNA synthetase in yeast cytoplasm, but also provide insights into the evolution of tRNA synthetase genes.
Aminoacyl-tRNA synthetases (aaRSs) 2 are a group of ancient enzymes responsible for protein translation, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs, forming aminoacyl-tRNAs. These charged tRNAs are then delivered by elongation factor-1 to ribosomes for protein translation. In prokaryotes, there are typically 20 aminoacyl-tRNA synthetases, one for each amino acid (1)(2)(3)(4). In eukaryotes, protein synthesis occurs not only in the cytoplasm, but also in organelles, such as mitochondria and chloroplasts (5). Thus, eukaryotes, such as yeast, commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized in the cytoplasm and the other in the mitochondria. Each set aminoacylates the isoaccepting tRNAs within its respective cell compartment and is sequestered from the isoacceptors confined in other compartments. In most cases, cytoplasmic and mitochondrial synthetase activities are encoded by two distinct nuclear genes, regardless of the cell compartments to which they are confined. However, in some cases, cytoplasmic and mitochondrial forms of a tRNA synthetase with a given amino acid specificity are encoded by the same nuclear gene through alternative initiation of translation, examples of which include ALA1 (coding for alanyl-tRNA synthetase) (6,7), GRS1 (coding for glycyl-tRNA synthetase) (8), HTS1 (coding for histidyl-tRNA synthetase) (9), and VAS1 (coding for valyl-tRNA synthetase (ValRS)) (10). Because the isozymes are targeted to different compartments, the two isoforms of ValRS, for example, cannot be substituted for each other in vivo. A similar scenario has been observed for genes encoding mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase, threonyl-tRNA synthetase, and ValRS (11).
Many yeast cytoplasmic tRNA synthetases contain an N-or C-terminal polypeptide extension that is absent from their bacterial homologs (12). A well studied example is the appended domain (Ad) of yeast glutaminyl-tRNA synthetase (GlnRS), which binds crude yeast tRNAs, single-stranded RNA, and pseudoknot RNA with comparable affinities; the K d values are ϳ0. 6 M (13, 14). Similar examples have been reported in yeast ValRS (15) and tRNA synthetases of higher eukaryotes, such as the EMAPII-like domain of plant methionyl-tRNA synthetase (16), the repeat domain of human methionyl-tRNA synthetase (17), and the N-terminal domain of mammalian lysyl-tRNA synthetase (18,19). In addition to serving as a cis-acting tRNAbinding domain (TRBD), the Ads of some yeast tRNA synthetases were found to participate in protein-protein interactions, such as those of yeast glutamyl-, methionyl- (20), and seryl-tRNA synthetases (21). These interactions were shown to enhance their tRNA binding and aminoacylation (20,21).
Interestingly, many of the Ads of yeast tRNA synthetases contain one or several canonical nuclear localization signals (22) that are thought to play a role in the nuclear import of these otherwise "cytoplasmic" proteins. Nuclear aminoacylation of tRNAs is thought to serve as a functional checkpoint for the maturity of tRNAs before they are exported from the nucleus (23,24). Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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This below document has 2 sentences that start with 'Further studies will be required'. It has approximately 1055 words, 28 sentences, and 14 paragraph(s).
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PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. It is, therefore, conceivable that PA28Įȕ can exert its biological function(s) also in association with constitutive proteasomes, probably related to its capacity to substantially modify the pattern of proteasomal peptides products. Further studies will be required to assess whether the changes in the patterns of peptides generated from proteins following binding of PA28Įȕ with 20S and 26S constitutive are similar to those already identified for its association with 20S and 26S immunoproteasomes [18].
Hydrolysis of Fluorogenic Peptide Substrates
As pointed out above, PA28Įȕ was originally identified due to its capability to markedly stimulate peptidase activities of the 20S proteasome, as measured using short fluorogenic model substrates.
Gate Opening Mechanism
In the crystal structure of PA26 solved by Hill and coworkers [71], the binding of PA26 was found to open the gate on the channel in the proteasome Į-ring through which substrates enter [94] and products exit [70]. Specifically, PA26 C-terminal residues dock into pockets between adjacent proteasome Į subunits and, by forming hydrogen bonds and a salt bridge between the C-terminal carboxylate of the activator and a highly conserved proteasome lysine side chain (Lys 66), provide binding energy for PA26-20S complexes [71,95]. Binding to the C-termini of PA26, however, is not sufficient to activate the 20S proteasome, which requires the activation loop that forms a seven-fold symmetric circular array that interacts with the base of the N-terminal gating residues of the seven proteasomal Į subunits. In particular, a glutamate side chain (Glu 102) in each PA26 subunit activation loop contacts and repositions a proline residue (Pro 17) of 20S Į subunits located above the surface of the proteasome. This interaction triggers gate opening by disrupting packing and hydrogen bonding interactions of the asymmetrical closed conformation and by widening the pore opening to a more symmetrical arrangement that allows a belt of intersubunit contacts to form around the circumference of the opening [96,97].
On the basis of these structural observations, PA28 was predicted to lead to attenuation of proteasomal processivity and consequent release of peptide products of greater mean length [71], as occurs on deletion of the Į-gate [70]. Since most peptides released by proteasomes are too short to bind to MHC class I molecules [84,[98][99][100][101], the generation of larger products would be expected to enhance the fraction of products capable of serving in antigen presentation, either directly or after trimming by aminopeptidases in the cytosol [89,102] or endoplasmic reticulum [86][87][88]90,103]. However, since not only PA28 but also the 19S regulator can induce the opening of the 20S core particle central gate [104], it remains unclear whether the above-mentioned structural observations can explain the specific effects of PA28 on substrate cleavage and, consequently, on antigen presentation.
Effects of PA28Įȕ on Protein Degradation
In this respect, it is clear that a full understanding of the specific biochemical functions of PA28Įȕ requires quantitative information on the rates of protein substrate hydrolysis and generation of peptide products by PA28Įȕ-containing immunoproteasomal species. To address this important issue, we recently performed a systematic analysis of the entire spectrum of peptides released during degradation of full-length proteins by PA28Įȕ-20S immunoproteasomes [101]. PA28Įȕ-20S particles were found to hydrolyze proteins at identical, slow rates compared to 20S proteasomes, and to generate higher amounts of very short products together with several longer peptides characterized by higher overall hydrophilicity.
The finding that unstructured polypeptides are hydrolyzed at rates that are nearly 10-fold higher by 26S compared to 20S and PA28Įȕ-20S immunoproteasomes is consistent with the notion that the free 20S particle is a relatively inactive protease since the N-terminal tails of its Į subunits obstruct the two opposite axial pores through which substrates access the internal catalytic lumen [94]. This autoinhibited state is relieved when the 20S core particle binds to activators such as 19S or PA28 that displace, in an ATP-dependent and independent manner, respectively, the N-terminal tails thereby opening an axial channel in the Į annulus [70,71]. However, the latency of unliganded 20S proteasome is not absolute since, even in the absence of artificial treatments (e.g., heating or presence of low concentrations of detergents or chaotropic agents) that are known to activate it [10], the 20S core particle degrades proteins at detectable and reproducible rates, probably involving transient and/or only partial channel opening [70,[76][77][78]. Surprisingly, despite the fact that the open-channel conformation of the gating residues induced by ATP-dependent and independent activators appears to be identical [105], our data show that PA28Įȕ-20S immunocomplexes hydrolyze proteins at the same rates as 20S immunoproteasomes, but much less efficiently than 26S immunoproteasomes. While on one hand this confirms initial reports on the biochemical properties of PA28 [19,20,65], it is difficult to reconcile with the known role of the proteasomal gate in controlling accessibility of substrates into the lumen of the 20S particle. In fact, opening of the axial channel by deletion of the N-terminal tails of eukaryotic [70,106] or archaeal [107] proteasomal Į subunits results in strong enhancement of the degradation rates of unfolded proteins, therefore suggesting that unstructured substrates can freely access the internal proteolytic chamber of the 20S particle simply by passive diffusion through a fully open gate. Furthermore, although it has been proposed that translocation of completely unstructured proteins might require a driving force produced by ATP hydrolysis [108], subsequent studies demonstrated that once the polypeptide chain is unfolded its transit through the ATPases ring can occur by passive diffusion, in which retrograde movement is probably prevented by a Brownian ratchet mechanism [109]. In light of these data, the inability of PA28Įȕ to enhance hydrolysis rates of loosely folded proteins is surprising. It can be speculated that the presence of a supplementary multimeric ring, sitting at the proteasomal outer Į surface, might impose an extra constraint to the completely free diffusion of large, although linear, polypeptide chains into the internal proteasomal lumen. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
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Domain: Biology Chemistry Medicine
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<<< User: Continue the text. Add a conclusion.
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Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. In sum, these results suggest that TRBDs such as the Ad of yeast ValRS can significantly improve the aminoacylation activity of E. coli ValRS against yeast tRNA.
<<< Answer:
Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. In sum, these results suggest that TRBDs such as the Ad of yeast ValRS can significantly improve the aminoacylation activity of E. coli ValRS against yeast tRNA.
Merging Cytoplasmic and Mitochondrial ValRS Activities into a Single Gene-Because E. coli ValRS can be converted to a functional yeast cytoplasmic or mitochondrial enzyme, we wondered whether the E. coli ValRS gene can be converted into a dual functional yeast gene specifying both cytoplasmic and mitochondrial activities. To investigate this possibility, a DNA segment (base pairs Ϫ300 to ϩ432 relative to the ATG1 initiator codon of yeast VAS1) that contains the VAS1 promoter and the sequence coding for the MTS (residues 1-46) and Ad (residues 47-144) of yeast ValRS was fused in-frame to the 5Ј end of the open reading frame of E. coli valS, and the complementation activities of the resultant construct were tested. As shown in Fig. 5, the fusion construct successfully rescued the growth defects of the VAS1 knock-out strain on both 5-FOA and YPG, suggesting that, as with VAS1, the fusion construct specifies both activities, with the mitochondrial activity being provided by the longer form (MTS-Ad(ScValRS)-EcValRS) and the cytoplasmic activity by the shorter form (Ad(ScValRS)-EcValRS). Because the MTS of the mitochondrial precursor form (the longer form) was cleaved away upon being imported into mitochondria, making the processed mitochondrial form indistinguishable in size from its cytoplasmic counterpart, only one protein band with a molecular mass close to Ad(ScValRS)-EcValRS (ϳ119 kDa) was identified by Western blotting (Fig. 5D).
DISCUSSION
For a given amino acid specificity, there are generally two distinct nuclear aaRS genes in yeast, one encoding the cytoplas-
TABLE 1 Kinetic parameters for aminoacylation of tRNA Val by EcValRS and Ad(ScValRS)-EcValRS
Each value is determined from a hyperbolic fit of two independent data sets. Our results presented herein argue that a bacterial aaRS gene can be converted into a dual functional yeast gene by fusion of a DNA sequence coding for both an MTS and a TRBD (Fig. 5). In addition, our results underline the necessity of obtaining a nonspecific TRBD for efficient binding and aminoacylation of yeast cytoplasmic tRNA by the bacterial enzyme ( Fig. 4 and Table 1).
ValRS variant
The binding affinity of a tRNA synthetase for its cognate tRNAs is generally characterized by dissociation constants on the order of 0.1-1 M under physiological conditions (34). This relatively poor affinity ensures that the synthetases (or tRNAs) turn over rapidly during aminoacylation. In this sense, it is interesting to point out that the binding affinities of the Ads of yeast GlnRS and ValRS for tRNA also fall into this range, making them useful as a cis-acting tRNA-binding cofactor during aminoacylation (15). Although the Ads of GluRS and MetRS are also rich in positively charged residues and important for aminoacylation, they do not function as a TRBD. Instead, these Ads specifically interact with a tRNA-binding cofactor, Arc1p, which, in turn, recruits tRNA to the vicinity of the enzymes for aminoacylation (35). A functionally similar tRNA-recruiting domain was identified in an auxiliary protein associated with the mammalian multisynthetase complex (36).
ValRS from mammalian cells is exclusively isolated as a high molecular mass complex with the elongation factor EF-1H (26 -28). Like yeast ValRS, the mammalian enzyme also contains a strong affinity for the polyanionic carrier, heparin-Ultrogel. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. coli valS gene, the open reading frame of this gene was first amplified by PCR using bacterial genomic DNA as the template. The forward (with an inserted SpeI site, underlined) and reverse (with an inserted XbaI site, underlined) primers used contained sequences complementary to base pairs Ϫ15 to ϩ18 (5Ј-AACCTGGAAACTAGTATGGAAAA-GACATATAAC-3Ј) and ϩ2842 to ϩ2871 (5Ј-ATCACTGT-GTTTTCTAGACAGCGCGGCGAT-3Ј) of E. coli valS, respectively. After the PCR, the amplified DNA products were digested with the restriction enzymes SpeI and XbaI and then cloned into the appropriate sites of pADH for expression. Cloning of the Bacillus subtilis valS gene into pADH followed a similar protocol.
For fusion of the Ad of yeast ValRS (residues 1-97), the Ad of yeast GlnRS (residues 1-228), or Arc1p to E. coli ValRS, DNA sequence coding for the respective protein domain was PCRamplified as an SpeI-SpeI fragment and inserted in-frame into the SpeI site at the 5Ј end of the open reading frame of E. coli valS, yielding Ad(ScValRS)-EcValRS, Ad(ScGlnRS)-EcValRS, and Arc1p-EcValRS. For fusion of a mitochondrial targeting signal (MTS) to B. subtilis and E. coli ValRSs, DNA sequence coding for the MTS of the mitochondrial precursor form of yeast ValRS (residues 1-46) was PCR-amplified as an SpeI-SpeI fragment and inserted in-frame into the SpeI site at the 5Ј end of the open reading frames of the B. subtilis and E. coli valS genes. The orientation of the SpeI-SpeI fragment was subsequently verified by DNA sequencing. Expressions of these constructs were under the control of a constitutive ADH promoter. Fusion of the DNA segment containing the promoter and sequence coding for the MTS and Ad of yeast ValRS to E. coli valS followed a similar protocol, except that the vector used for cloning and expression was pRS425 in this case.
Complementation Assays for Cytoplasmic Function-The yeast VAS1 knock-out strain, CW1, was described previously (31). This strain is maintained by a plasmid containing the wildtype VAS1 gene and a URA3 marker. Complementation assays for cytoplasmic ValRS activity were carried out by introducing a test plasmid carrying the gene of interest and a LEU2 marker into CW1, and the ability of the transformants to grow in the presence of 5-FOA was determined. Starting from a cell density of 4.0 A 600 , cell cultures were 5-fold serially diluted, and 10-l aliquots of each dilution were spotted onto the designated plates containing 5-FOA. Plates were incubated at 30°C for 3-5 days. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. R155A was only 20% as active as the wild type plasmid at 0.5 g but was similar to wild type when 1 g of plasmid was used. The reduced potency observed with some of the derivatives did not correlate with the ability of VP16 to bind independently to DNA or to interact with VCAF-1 and thus may be related to differences in the relative stability of the proteins in vivo.
DISCUSSION
In this subject, we have analyzed the properties of specific point mutants of VP16 to examine the contributions of individual protein-protein and protein-DNA interactions of VP16 to the ordered assembly of VP16-containing complexes and VP16mediated transcriptional activation. We showed that specific point mutations in two regions of VP16 selectively affected the ability of VP16 to directly bind to DNA, to associate with DNA-bound Oct-1, and to form a complex with VCAF-1 (summarized in Table I). All of the mutants tested remained capable of forming VIC and transactivating gene expression in vivo, demonstrating that these individual interactions are not essential for the formation of a transcriptionally active VP16-containing protein-DNA complex.
VP16 derivatives in which arginines at position 162, 360, and 366 were altered failed to bind directly to DNA. This is consistent with previous mutational studies which showed that deletion of either region 1 or 2 destroyed direct DNA binding activity of VP16 (19). The putative DNA binding domain of VP16 has not been defined; however, Stern and Herr (19) showed that a synthetic peptide spanning a part of region 1 (amino acids residues 170 -202) bound to DNA, albeit in a nonsequence-specific manner. All of the region 1 mutants were capable of forming a weak complex with DNA-bound Oct-1; however, the region 2 mutants were defective. This is consistent with previous findings that region 2 contains determinants for direct interaction of VP16 with DNA bound-Oct-1 (19,24,31). Our findings that the R368A and K370A mutants cannot recognize DNA-bound Oct-1, yet are still able to bind to DNA indicate that direct DNA binding activity of VP16, is not required for this interaction.
The functional relevance of direct DNA binding activity of VP16 or interaction with DNA-bound Oct-1 is not clear, since there is no evidence that the VP16-Oct-1 binary complex or VP16 on its own is transcriptionally active in vivo. As demonstrated previously by others (17,19) and also shown here, the amount of VP16 required to observe these properties is significantly greater (10 -100-fold) than that which is sufficient to generate the VIC complex. The high concentration of VP16 necessary to observe these interactions is probably not physiologically significant. Indeed, transactivation of viral IE genes is detrimentally affected by large amounts of VP16 (24). Moreover, the source of VP16 may have an effect on the efficiency of these interactions. For instance, PA-VP16 has a higher affinity for DNA compared with MBP-VP16 ( Fig. 2A), 2 whereas GST-VP16 has only very weak intrinsic DNA binding activity (22). Thus, the carrier protein may have some influence on binding properties, perhaps by masking or exposing cryptic binding interfaces (17). It has been proposed that VP16 binds to the GARAT portion of the target site which, while not required for Oct-1 binding, is necessary for VIC assembly (5,17). More recent evidence indicates that the GARAT subregion serves to alter the conformation of the POU homeodomain and that this is necessary for subsequent recognition by VP16 (22). Our findings directly demonstrate that the independent DNA binding activity and interaction with DNA-bound Oct-1 is not absolutely essential for VIC assembly or for transcriptional activation in vivo (see below).
Region 2 is essential for VIC formation and also contains critical determinants for direct interaction with VCAF-1 (19,24,31). We, and others, have shown that the requirements for interaction with VCAF-1 can be uncoupled from VIC formation (19,31). Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. Increasing the concentration of the wild type effector plasmid resulted in a reduction in activity as reported previously, likely as a result of squelching (24). Transfection of the mutant VP16 derivatives showed that all the mutants were capable of transactivating expression of the reporter gene, consistent with the observation that all of the mutant VP16 proteins remained capable of forming VIC. There were, however, variations in relative potency among the differ- In lane a, purified PA-VP16 (wild type VP16 fused to protein A) was used. PA-VP16 is larger than MBP-VP16, and thus the protein-DNA complex has a slower mobility. The probe used in this and subsequent experiments was the promoter proximal TAATGARAT element from the ICP0 promoter. B, gel retardation experiments were carried out with the indicated fusion proteins (6 g) in the presence of purified GST-Oct-1 POU homeodomain fusion protein (0.05 g; referred to as Oct-1 in all figures). Lane a is GST-Oct-1 alone. The positions of the VP16 DNA complex and the VP16-Oct-1 DNA complex are indicated. ent mutant proteins. At an effector plasmid dosage of 0.5 g, R164A and R169A behaved as wild type, while the remaining derivatives activated expression at an efficiency of 30 -40% compared with the wild type plasmid. R155A was only 20% as active as the wild type plasmid at 0.5 g but was similar to wild type when 1 g of plasmid was used. The reduced potency observed with some of the derivatives did not correlate with the ability of VP16 to bind independently to DNA or to interact with VCAF-1 and thus may be related to differences in the relative stability of the proteins in vivo.
DISCUSSION
In this subject, we have analyzed the properties of specific point mutants of VP16 to examine the contributions of individual protein-protein and protein-DNA interactions of VP16 to the ordered assembly of VP16-containing complexes and VP16mediated transcriptional activation. We showed that specific point mutations in two regions of VP16 selectively affected the ability of VP16 to directly bind to DNA, to associate with DNA-bound Oct-1, and to form a complex with VCAF-1 (summarized in Table I). All of the mutants tested remained capable of forming VIC and transactivating gene expression in vivo, demonstrating that these individual interactions are not essential for the formation of a transcriptionally active VP16-containing protein-DNA complex.
VP16 derivatives in which arginines at position 162, 360, and 366 were altered failed to bind directly to DNA. This is consistent with previous mutational studies which showed that deletion of either region 1 or 2 destroyed direct DNA binding activity of VP16 (19). The putative DNA binding domain of VP16 has not been defined; however, Stern and Herr (19) showed that a synthetic peptide spanning a part of region 1 (amino acids residues 170 -202) bound to DNA, albeit in a nonsequence-specific manner. All of the region 1 mutants were capable of forming a weak complex with DNA-bound Oct-1; however, the region 2 mutants were defective. This is consistent with previous findings that region 2 contains determinants for direct interaction of VP16 with DNA bound-Oct-1 (19,24,31). Our findings that the R368A and K370A mutants cannot recognize DNA-bound Oct-1, yet are still able to bind to DNA indicate that direct DNA binding activity of VP16, is not required for this interaction.
The functional relevance of direct DNA binding activity of VP16 or interaction with DNA-bound Oct-1 is not clear, since there is no evidence that the VP16-Oct-1 binary complex or VP16 on its own is transcriptionally active in vivo. As demonstrated previously by others (17,19) and also shown here, the amount of VP16 required to observe these properties is significantly greater (10 -100-fold) than that which is sufficient to generate the VIC complex. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine
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Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. The pJW312 plasmid carrying the wild-type topoisomerase I gene can complement this chromosomal mutation (21). Each one of the active site mutations tested here was found to abolish this in vivo complementation even though they showed varying degrees of loss of in vitro relaxation (Table I).
The inability of the Glu-9, Glu-111, Asp-113, and Glu-115 mutants to complement efficiently in E. coli AS17 may be due to their low level of expression in this E. coli strain. To evaluate the in vivo activities of these mutants, the thermosensitivities of GP200 expressing these mutants were examined (Table II). Loss of topA activity in E. coli can lead to a lower rate of survival when the temperature was raised to 52°C (reviewed in Ref. 25). The rate of survival of E. coli GP200 was increased ϳ50-fold when plasmid encoded wild-type topA activity was present (Table II). The survival rates of GP200 transformed with plasmid encoding the Glu-9, Glu-111, Asp-113, and Glu-115 mutants correlated with their in vitro activity, with the Glu-111 mutant conferring close to wild-type thermoresistance and the Glu-9 mutant conferring the least amount of thermoresistance.
The Arg-136 Mutant Could Cleave DNA and Form the Covalent Complex-The cleavage activities of the mutant enzymes were examined using 5Ј-end-labeled single-stranded DNA. Even though the Arg-136 mutant was totally inactive in the relaxation assay, the amount of cleaved DNA formed was comparable with that from the wild-type enzyme (Fig. 4). For the other mutants, the amounts of cleavage products observed were decreased, with the Glu-9 mutant having the lowest cleavage activity, so that the reduction of relaxation activity seen with these other mutants might be due to the decrease in the amount of cleaved complex formed by these mutants. The Asp-111 mutant had the same DNA cleavage efficiency as the wild-type enzyme.
A 3Ј-end-labeled oligonucleotide substrate was used to form a covalent complex with the enzyme. At 5 min after the addition of the enzyme to the oligonucleotides, the amounts of the covalent complex observed for the wild-type and the Arg-136 mutant were identical (Fig. 5). The amount of covalent complex formed by the Asp-111 mutant was also close to that of the wild type while the other mutant enzymes gave lower levels of This experiment also demonstrated that the Arg-136 mutation affected a step in the enzyme relaxation mechanism that took place after DNA cleavage. This could be the strand passage or the DNA religation step. The inter-molecular strand transfer activity of the Arg-136 mutant was compared with the wild type enzyme using the covalent complex formed with this 3Ј-end-labeled oligonucleotide substrate and HindIII-digested DNA as acceptor molecules (26). DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. Aminoacylation was carried out at 25°C at a final concentration of 5, 10, or 50 nM ValRS. As shown in Fig. 4A, yeast ValRS could barely charge E. coli tRNA in vitro compared with the E. coli enzyme under the conditions used. Likewise, E. coli ValRS had a relatively poor charging activity against yeast tRNA in vitro compared with the yeast enzyme (Fig. 4B). This finding is not surprising, considering the fact that the E. coli and yeast enzymes have coevolved with their respective cognate tRNAs and therefore developed certain levels of species specificity or barriers in tRNA recognition. As for the aminoacylation activity of the fusion enzyme (Ad(ScValRS)-EcValRS), our results showed that fusion of the Ad of yeast ValRS to the E. coli enzyme enhanced its aminoacylation activity against yeast tRNA by ϳ3.8-fold (Fig. 4B) but had only a minor effect on its aminoacylation activity against E. coli tRNA (ϳ1.4-fold increase) (Fig. 4A). To illustrate better the difference in aminoacylation activities against yeast tRNA between E. coli ValRS and the fusion enzyme, the protein concentration used for the assay was increased by 5-fold (from 10 to 50 nM). As shown in Fig. 4C, the fusion enzyme showed significantly higher aminoacylation activity against yeast tRNA than did E. coli ValRS, which might explain why the fusion enzyme was able to rescue the growth defect of CW1 on 5-FOA, whereas E. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. subtilis and E. coli valS genes. The orientation of the SpeI-SpeI fragment was subsequently verified by DNA sequencing. Expressions of these constructs were under the control of a constitutive ADH promoter. Fusion of the DNA segment containing the promoter and sequence coding for the MTS and Ad of yeast ValRS to E. coli valS followed a similar protocol, except that the vector used for cloning and expression was pRS425 in this case.
Complementation Assays for Cytoplasmic Function-The yeast VAS1 knock-out strain, CW1, was described previously (31). This strain is maintained by a plasmid containing the wildtype VAS1 gene and a URA3 marker. Complementation assays for cytoplasmic ValRS activity were carried out by introducing a test plasmid carrying the gene of interest and a LEU2 marker into CW1, and the ability of the transformants to grow in the presence of 5-FOA was determined. Starting from a cell density of 4.0 A 600 , cell cultures were 5-fold serially diluted, and 10-l aliquots of each dilution were spotted onto the designated plates containing 5-FOA. Plates were incubated at 30°C for 3-5 days. The transformants evicted the maintenance plasmid with a URA3 marker in the presence of 5-FOA and thus could not grow on the selection medium unless a functional cytoplasmic ValRS was encoded by the test plasmid.
Complementation Assays for Mitochondrial Function-CW1 was cotransformed with a test plasmid (carrying a LEU2 marker) and a second maintenance plasmid (carrying a HIS3 marker) that expresses only the cytoplasmic form of ValRS (due to a mutation in the ATG1 initiator codon). In the presence of 5-FOA, the first maintenance plasmid (carrying a URA3 marker) was evicted from the cotransformants, whereas the second maintenance plasmid was retained. Thus, all cotransformants survived 5-FOA selections due to the presence of the cytoplasmic ValRS derived from the second maintenance plasmid. The mitochondrial phenotypes of the cotransformants were further tested on YPG plates at 30°C, with results documented on day 3 after plating. Because a yeast cell cannot survive on glycerol without functional mitochondria, the cotransformants did not grow on the YPG plates unless a functional mitochondrial ValRS was generated from the test plasmid.
Western Blot Analysis-The protein expression patterns of the constructs used in the complementation assays were determined by a chemiluminescence-based Western blot analysis. INVSc1 (Invitrogen) was first transformed with the constructs of interest, and total protein extracts were prepared from each transformant. Aliquots of the protein extracts (40 g) were loaded onto a mini gel (8 ϫ 10 cm) containing 10% polyacrylamide and electrophoresed at 100 V for 1-2 h. After electrophoresis, the resolved proteins were transferred using a semidry transfer device to a polyvinylidene fluoride membrane in a buffer containing 30 mM glycine, 48 mM Tris base (pH 8.3), 0.037% SDS, and 20% methanol. The membrane was probed with a horseradish peroxidase-conjugated anti-FLAG tag antibody (Sigma) and then exposed to x-ray film after the addition of the appropriate substrates.
Aminoacylation Assay-Aminoacylation reactions were carried out at 25°C in a buffer containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 Purification of the His 6 -tagged proteins was as described previ-ously (32). Determination of active protein concentrations by active-site titration was as described (33). Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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Amino acid substitutions in the herpes simplex virus transactivator VP16 uncouple direct protein-protein interaction and DNA binding from complex assembly and transactivation.
The herpes simplex virus transactivator VP16 directs the assembly of a multicomponent protein-DNA complex that requires the participation of two cellular factors, the POU homeodomain protein Oct-1, which binds independently to response elements, and VCAF-1 (VP16 complex assembly factor; also called HCF, C1), a factor that binds directly to VP16. Equivalent volumes of bound and nonbound material (6 l) were assayed for VCAF-1 activity by mobility shift analysis with GST-Oct-1 and wild type MBP-VP16. Buffer components and protein concentration in the binding assays were normalized as appropriate.
Vhs Binding Assays-Full-length [ 35 S]methionine-labeled HSV-1 vhs protein was prepared by transcription and translation of the in vitro expression plasmid pSPUTKvhs using rabbit reticulocyte lysates (Promega) as described previously (30). For binding assays, amylose beads adsorbed with equivalent amounts of MBP or the various MBP-VP16 fusion proteins were washed sequentially with 10 volumes of CB, CB ϩ 0.2% BSA, and CB ϩ 0.2% BSA ϩ 0.05% Nonidet P-40. 50 l of settled beads were recovered by low speed centrifugation and incubated with radiolabeled Vhs protein (200 l of a 1:100 dilution of programmed reticulocyte lysate). The beads were incubated for 1 h at 4°C with continuous rotation and recovered by centrifugation. Beads were washed once with 10 volumes of CB, three times with 10 volumes of CB ϩ 0.2% BSA, and 3 times with 10 volumes of CB ϩ 0.2% BSA ϩ 0.5% Nonidet P-40. Bound material was eluted by boiling with an equal volume of twice concentrated sodium dodecyl sulfate sample buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
RESULTS
Previous studies have shown that amino acids 140 -250 and 335-390 of VP16 contain determinants that are important for VIC formation and VCAF-1 interaction (19,24,25,27,31). Both regions 1 and 2 are enriched in positively charged residues, in particular arginine residues, relative to the whole protein. Since positively charged amino acids are important in both protein-protein and protein-DNA interactions, we decided to alter selected arginine residues to alanine residues by sitedirected mutagenesis. Alanine residues were chosen, since these would not be expected to alter the conformation of the polypeptide backbone (36). The introduced mutations are shown in Fig. 1A. These include alanine substitutions of arginines at positions 155, 162, 164, 169, 360, 366, and 368. Cys-176 was also mutated to examine the role of this sulfhydryl group. Also, Lys-379 was mutated, since the region surrounding this amino acid has been shown to be surface exposed and to be involved in protein-DNA complex formation (32).
VP16 and the various mutant derivatives were cloned into an MBP expression vector, and the proteins were purified by affinity chromatography on amylose resin ( Fig. 1B; all MBP-VP16 derivatives encoded residues 4 -411 and are thus missing the acidic activation domain). The purified proteins were used to monitor the following properties of VP16: direct DNA binding to the TAATGARAT elements, interaction with DNA-bound Oct-1, direct interaction with VCAF-1, VIC formation, and association with Vhs.
Direct Interaction of VP16 with DNA-Kristie and Sharp (17) first showed that if sufficiently large amounts of VP16 are used in DNA binding assays, VP16 can bind independently and sequence specifically to TAATGARAT elements. Fig. 2A shows the results of DNA binding assays with VP16 carried out in the absence of Oct-1 and VCAF-1 using the labeled ICP0 octa ϩ TAATGARAT element. A 10 -20-fold excess of VP16, compared with the amount sufficient to form VIC in the presence of Oct-1 and VCAF-1 (see below), was used in this case in order to observe direct DNA binding. Elucidation of the basis and physiological relevance for flexibility in the assembly and function of VP16 multicomponent complexes will contribute to our understanding of specificity and selectivity in viral and cellular gene regulation.
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Domain: Biology Chemistry Medicine<|endoftext|>Site-directed Mutagenesis of Conserved Aspartates, Glutamates and Arginines in the Active Site Region of Escherichia coli DNA Topoisomerase I*
DNA topoisomerases (for review, see Refs. Their mechanisms may also share some similarities with topoisomerases. It should be noted that Mg(II) is not absolutely required for the DNA cleavage activity of E. coli DNA topoisomerase I.
To investigate the roles of the carboxylates and arginine residues in the active site region of E. coli topoisomerase I, they were altered by site-directed mutagenesis to alanines, abolishing the acidic or basic functional groups. The mutant enzymes were expressed and purified. Different enzymatic assays were carried out to determine how the mutation affected the interaction of the topoisomerase enzyme with DNA and/or Mg(II).
EXPERIMENTAL PROCEDURES
Materials-All chemical reagents used were ultrapure or Baker analyzed reagent grade. Solutions were prepared with water first deionized with the Barnstead Nanopure system and then passed over a Bio-Rad Chelex 100 resin (100 -200 mesh sodium form) to remove any remaining contaminating metal ions. Tubes, spectrophotometric cells, and glassware for metal ion-sensitive experiments were first washed with 10 mM EDTA and then rinsed extensively with metal-free water * This work was supported by Grant GM54226 from NIGMS, National Institutes of Health, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. Section 1734 solely to indicate this fact. before use. Plasmid DNA was purified by cesium chloride centrifugation.
Enzyme Expression and Purification-Wild-type E. coli DNA topoisomerase I enzyme was expressed from E. coli MV1190 cells transformed with plasmid pJW312. The R136A and R321A mutants were expressed in E. coli AS17 (topA am PLL1(Tc r supD ts ), from R. E. DNA or enzyme-Mg(II) complexes would be needed to arrive at a more detailed mechanism of catalysis by the enzyme.
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Domain: Biology Chemistry Medicine<|endoftext|>Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. The final concentration of ValRS used in the reaction was 5, 10, or 50 nM. Reactions were quenched by spotting 10-l aliquots of the reaction mixture onto Whatman filters soaked in 5% trichloroacetic acid and 1 mM valine. The filters were washed three times, for 15 min each, in ice-cold 5% trichloroacetic acid before liquid scintillation counting. Data were obtained from at least three independent experiments and averaged. Determination of the kinetic parameters, K m and k cat , of the purified enzymes for yeast tRNA Val was as described (15).
Converting E. coli ValRS into a Functional Yeast Cytoplasmic Enzyme-Previous studies showed that a single VAS1
gene specifies both the mitochondrial and cytoplasmic forms of ValRS through alternative use of two in-frame AUG initiator codons, AUG1 and AUG47 (10,31). Hence, the mitochondrial precursor form essentially possesses the same polypeptide sequence as its cytoplasmic counterpart, except for an N-terminal MTS that is subsequently cleaved away by a matrix-processing peptidase upon being imported into mitochondria. Comparison of E. coli and yeast (the cytoplasmic form or the mature mitochondrial form) ValRSs showed that the catalytic cores of these two enzymes are significantly homologous to each another (ϳ42% identity), but the yeast enzyme has an N-terminal polypeptide extension of ϳ97 residues that is absent from its E. coli counterpart (Fig. 1). This Ad has recently been shown to act in cis as a nonspecific TRBD to facilitate tRNA binding and aminoacylation by the enzyme (15).
To test whether the E. coli enzyme can functionally substitute for its yeast homolog in vivo, the wild-type E. coli valS gene (encoding ValRS) was cloned into a yeast shuttle vector, and the ability of the resultant construct to rescue the growth defect of a yeast vas1 Ϫ strain on 5-FOA was tested. As shown in Fig. 2, the construct containing E. coli valS failed to restore the growth phenotype of the knock-out strain CW1 on 5-FOA (Fig. 2B, number 3), suggesting that the cytoplasmic activity of yeast VAS1 cannot be substituted with the bacterial enzyme in vivo. However, fusion of a nonspecific TRBD, such as Arc1p or the Ads of yeast GlnRS and ValRS, to the E. coli enzyme enabled the otherwise nonfunctional bacterial enzyme to act as a yeast enzyme; each of these fusions effectively rescued the growth defect of the knock-out strain on 5-FOA (Fig. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine
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PA28αβ: The Enigmatic Magic Ring of the Proteasome?
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. From this point of view, transit thorough the pore of open gate mutants might not perfectly reflect passage across the PA26/28 central channel. In fact, the crystal structure of PA28Į shows that the aqueous channel through the heptamer has a diameter of 20 Å at its minimum, which is theoretically wide enough for passage of unfolded proteins [34]. However, the homolog-specific inserts present between helices 1 and 2, which are not resolved in the crystal structure, most likely form a ring-like collar on the upper, non-proteasome binding surface of the PA28 heptamer. Although several studies have shown that these loops do not restrict passage of tri-or tetra-peptide fluorogenic substrates [35,67], recent investigations have demonstrated that they can hinder the transit of longer peptides, and conceivably of proteins as well, through the PA28 channel [44]. In fact, we demonstrated that 20S and PA28Įȕ-20S immunoproteasomes hydrolyze proteins at exactly the same slow rates, which implies that denatured substrates transit through the partially or transiently open gate of unligated 20S and the fully open channel of PA28Įȕ-20S particles with comparable efficiency. However, it cannot be excluded that PA28Įȕ might selectively enhance degradation of some specific substrates that are yet to be identified, as has been unambiguously demonstrated for PA28Ȗ [110][111][112][113].
Effects of PA28Įȕ on Peptide Products Generation
Although PA28Įȕ is unable to enhance rates of protein degradation by proteasomes, its association with the 20S particle was found to lead to substantial changes in the patterns of peptides generated, which greatly differ from those produced by 20S and 26S immunoproteasomes [101]. In fact, only ~10% of peptides generated by 20S and 26S immunoproteasomes were found to be 8-10 residues long [101], which is the appropriate length to bind MHC class I heterodimers. Most importantly, association of PA28Įȕ with the ends of 20S immunoproteasomes does not increase the fraction of 8-10 residue peptides generated, but reduces it to 6% of the total. Moreover, the fraction of peptides longer than 10 amino acids, which might serve in MHC class I antigen presentation only after appropriate trimming by aminopeptidases in the cytosol or ER, is larger for 20S than for 26S immunoproteasomes; binding of PA28Įȕ to the 20S particle dramatically reduces the overall efficiency of generation of these longer products. It is thus clear that PA28 does not act simply by expending the fraction of proteasomal products that can be accommodated in the groove of MHC class I molecules directly or after trimming. In this regard, it is worth noting that both 20S and 26S immunoproteasomes display a similar high propensity to release 8-10 residue long products, although 20S has also an increased capacity to generate longer fragments [101]. However, correct evaluation of the overall efficiency of different immunoproteasome species in producing peptides with a size potentially suitable for class I antigen presentation requires normalization of their rates of generation with those of substrate hydrolysis. Therefore, since in vitro 26S immunoproteasomes were found to degrade unfolded proteins at 10-fold higher rates than 20S and PA28Įȕ-20S [84,101,114], it is evident that 26S holoenzymes are potentially the most efficient immunoproteasomal species in terms of generating higher amounts of peptides with the correct size to serve in MHC class I antigen presentation either directly or after aminopeptidase trimming.
PA28 as a Smart Sieve
Surprisingly, PA28Įȕ-20S immunoproteasomes were found to display a reduced ability to generate longer products that, in principle, might depend upon conformational changes in proteasomal active sites. This hypothesis has already been suggested to explain the biochemical properties of PA28 [36,72,75], and specifically its ability to stimulate coordinated dual cleavages of short synthetic peptides (typically 19-25 residues long) by 20S particles [82,83]. This possibility, however, seems unlikely for PA28Įȕ-20S immunoproteasomes since, as already pointed out, association with PA26 does not induce any structural modification of proteasomal catalytic ȕ subunits [71]. Alternatively, PA28Įȕ might primarily act as a molecular sieve that retains longer protein fragments inside the 20S proteolytic chamber until they are cleaved to peptides that are small enough to diffuse to the outside. This later model would be consistent with detailed kinetic analyses showing that PA28 exerts its activating influence by enhancing bi-directional passage of short (3-4 residues) peptides [52] and with an important in vitro/in silico study that identified one of the major factors involved in the enhancement of double cut efficiency induced by PA28 in a reduced efflux of longer peptides out of the 20S particle [115]. Furthermore, it was recently shown that a PA28Įȕ complex lacking the unstructured and highly mobile PA28Į loops surrounding the central pore of the heptameric ring cleaves substrates longer than a nonpeptide more efficiently than wild type PA28. On these bases, it was hypothesized that the flexible loops of PA28 might act as gatekeepers that block the exit of longer peptides from the proteolytic chamber [44]. Further studies will be required to unveil the specific role of these hydrophilic products and to clarify whether they are favoured in some steps of the MHC class I antigen processing pathway.
Acknowledgments
We thank Patrick Moore for assistance in preparation of this manuscript.
Conflicts of Interest
The author declares no conflict of interest.
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Domain: Biology Chemistry Medicine
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Evolutionary Basis of Converting a Bacterial tRNA Synthetase into a Yeast Cytoplasmic or Mitochondrial Enzyme*
Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. By contrast, in higher eukaryotes, nine aminoacyl-tRNA synthetases and three auxiliary proteins (p43, p38, and p18) form a multienzyme complex through interactions of their hydrophobic Ads (25). In addition, ValRS from mammalian cells is exclusively isolated as a high molecular mass complex with the elongation factor EF-1H (26 -28). Recently, several tRNA synthetases from prokaryotes and eukaryotes have been shown to take part in functions beyond aminoacylation, including roles in mitochondrial RNA splicing, transcriptional and translational regulation, cytokine-like activity, and amino acid biosynthesis (29,30).
In this report, we focused on the cross-species and crosscompartmental complementation activities of a bacterial tRNA synthetase in an attempt to understand further the evolutionary pathway that has converted a bacterial tRNA synthetase gene into a dual functional yeast gene possessing both cytoplasmic and mitochondrial activities. Our results show that although Escherichia coli ValRS per se cannot substitute for the cytoplasmic activity of yeast VAS1, fusion of a nonspecific TRBD (such as Arc1p or the Ads of yeast GlnRS and ValRS) to the bacterial enzyme enabled the otherwise nonfunctional enzyme to rescue the growth defect of a VAS1 knock-out strain on 5-fluoroorotic acid (5-FOA). In contrast, the E. coli enzyme, when targeted to mitochondria, could substitute for the mitochondrial activity of VAS1 without the assistance of a cis-acting nonspecific TRBD. These results, together with others, suggest that acquiring a cis-or trans-acting TRBD might be necessary and sufficient for functioning by a yeast cytoplasmic tRNA synthetase, which might explain why so many yeast cytoplasmic aaRSs contain an N-or C-terminal Ad (12). In contrast, obtaining a cis-or trans-acting TRBD does not appear to be necessary for functioning of most yeast mitochondrial aaRSs.
EXPERIMENTAL PROCEDURES
Construction of Plasmids-Cloning of the wild-type yeast VAS1 gene into the high copy number yeast shuttle vector pADH was previously described (31). A short DNA duplex coding for a FLAG (for Western blotting) or His 6 tag (for protein purification) was inserted in-frame at the 3Ј end of the VAS1 gene. To clone the E. coli valS gene, the open reading frame of this gene was first amplified by PCR using bacterial genomic DNA as the template. The forward (with an inserted SpeI site, underlined) and reverse (with an inserted XbaI site, underlined) primers used contained sequences complementary to base pairs Ϫ15 to ϩ18 (5Ј-AACCTGGAAACTAGTATGGAAAA-GACATATAAC-3Ј) and ϩ2842 to ϩ2871 (5Ј-ATCACTGT-GTTTTCTAGACAGCGCGGCGAT-3Ј) of E. coli valS, respectively. After the PCR, the amplified DNA products were digested with the restriction enzymes SpeI and XbaI and then cloned into the appropriate sites of pADH for expression. Cloning of the Bacillus subtilis valS gene into pADH followed a similar protocol.
For fusion of the Ad of yeast ValRS (residues 1-97), the Ad of yeast GlnRS (residues 1-228), or Arc1p to E. coli ValRS, DNA sequence coding for the respective protein domain was PCRamplified as an SpeI-SpeI fragment and inserted in-frame into the SpeI site at the 5Ј end of the open reading frame of E. coli valS, yielding Ad(ScValRS)-EcValRS, Ad(ScGlnRS)-EcValRS, and Arc1p-EcValRS. For fusion of a mitochondrial targeting signal (MTS) to B. subtilis and E. coli ValRSs, DNA sequence coding for the MTS of the mitochondrial precursor form of yeast ValRS (residues 1-46) was PCR-amplified as an SpeI-SpeI fragment and inserted in-frame into the SpeI site at the 5Ј end of the open reading frames of the B. Perhaps this is one of the major reasons why so many yeast cytoplasmic aaRSs possess an N-or C-terminal appendage, whereas most yeast mitochondrial aaRSs do not.
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Domain: Biology Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. In the present study, pMiniHimar RB1 was modified by adding unique restriction sites for insertion of additional genetic elements. In our tests, these elements were promoterless gfpmut3 and mStrawberry, and the resultant vectors can be utilized for random generation of genomic transcriptional fusions. Such vectors can provide a convenient way to conduct genome-wide investigations of gene expression levels under selected conditions (de Lorenzo et al., 1990;Hahn et al., 1991;Boyle-Vavra and Seifert, 1995;Velayudhan et al., 2007).
CONCLUSION
The present report outlined the development of tools needed for genetic manipulation of Delftia sp. Cs1-4. These tools included a new expression cassette (PnpdA-based) that can be used for tagging of chromosomal genes as well as for complementation of knockout mutants, and a pMiniHimar transposon modified to enhance gene recovery and mutant analysis. The effectiveness in Delftia sp. of the Cre-loxP for gene deletion was also demonstrated. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Lastly, for the ΔlasI mutant, the absence of any detectable change in the formation of nanopods suggested that the process was not affected by quorum sensing, at least in the sense that it was regulated by AHL produced by a canonical AHL synthetase. This finding is noteworthy as it helps to narrow the spectrum of possible mechanisms that may control nanopod production.
Efficient targeting for gene inactivation is critical for functional genomic studies and, in bacteria, two widely used systems for generating in-frame, unmarked deletions are those based on sacB counter selection (Jäger et al., 1995;Chen et al., 2010), and Cre-loxP system (Denef et al., 2006;Choi et al., 2008). For strain Cs1-4, the sacB system proved unsuccessful; merodiploids (first recombination) were recovered at a high frequency, but these were not effectively resolved as Delftia sp. Cs1-4 grew in YT agar medium containing 5-15% (wt/vol) sucrose (data not shown). Similar observations have been reported for Streptomyces lividans and some Burkholderia strains, which carry an intrinsic sacBC operon. Alternatively, Cre-loxP system was successfully adapted for gene deletion or insertion, and was an efficient way for recycling antibiotic markers in Delftia sp. Cs1-4. To our knowledge, this is the first report of the Cre-loxP system being used for gene deletion analysis in Delftia spp.
Of the mutants recovered from genome-wide mutagenesis, three were of particular interest as they may encode new functions associated with nanopod production and/or phenanthrene degradation. One of these putatively encoded an Ycf48-like protein. In phototrophs, Ycf48 functions in the assembly and repair of Photosystem II (Komenda et al., 2008;Rengstl et al., 2011). Activities of an Ycf48-like protein that may be related to phenanthrene degradation are unknown, but, given the significant reduction (ca. 64%) in nanopod produced by this mutant, it's interesting to speculate that it may have a role in the assembly of these structures. The putative spoT /relA mutant, had an insertion in a (p)ppGpp synthetase. The alarmone (p)ppGpp primarily governs the stringent response to amino acid starvation (Martinez-Costa et al., 1998;Åberg et al., 2006;Gomez-Escribano et al., 2008;Abranches et al., 2009) and, since growth of the spoT /relA mutant on pyruvate was not impaired, the effect of the mutation appeared related to use of phenanthrene as a carbon source. The third gene of www.frontiersin.org interest, encoding an HlyD-like protein, was clustered with other genes predicted to encode pili formation. But, it remains to be determined how amino acid starvation and pili formation may be connected to phenanthrene degradation. Mutant 7 was not impaired in growth on phenanthrene, but did show decreased expression of npdA, and a depressed level of nanopod production. The protein predicted for the locus bearing the insertion contained a heavy-metal-associated domain that is also found in a number of proteins that transport or detoxify heavy metals; the relation of such a protein to npdA expression and nanopod formation remains to be determined. Minitransposons are widely used for genome-wide mutagenesis in Gram-negative and Gram-positive bacteria (Lampe et al., 1999;Youderian et al., 2003;Maier et al., 2006;Choi et al., 2008) and, compared to other minitransposons, pMiniHimar is advantageous as it does not require host-specific factors for transposition, it lacks site specificity and the transposase is not introduced into the chromosome, thus enhancing insertion stability. The transposition frequency of pMiniHimar was sufficient (>5 × 10 −6 per recipient) for saturation mutagenesis of the strain Cs1-4 genome. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. cepacia (Lefebre and Valvano, 2002). However, in strain Cs1-4, gene expression under Prsp was poor, and not significantly Frontiers in Microbiology | Microbiotechnoloy, Ecotoxicology and Bioremediation different from that of Plac (data not shown). Thus, demonstration of PnpdA as a strong promoter functional in Delftia sp. Cs1-4 has provided a much-needed tool for genetic analyses of this organism, and potentially other related bacteria.
The Δomp32 mutant had an irregular cell shape (Figure 3B), suggesting that Omp32 may have a key role in establishment of cell envelope structure, as shown for other outer membrane proteins (Lazar and Kolter, 1996;Watts and Hunstad, 2008). Analysis of the Δhcp mutant demonstrated that, as opposed to our hypothesis, Hcp did not have a structural function essential for nanopod formation. However, Western blot data indicated that Hcp was associated in some manner with nanopods as the majority of this protein accumulated in the >50-nm diameter fraction along with nanopods (data not shown). It is possible that Hcp was secreted separately from nanopods, and formed extracellular structures that were co-purified with nanopods. If so, such structures were not discernable in samples imaged by transmission electron microscope. Alternatively, Hcp may be associated with nanopods as cargo carried by OMV. In this case, Hcp may function as a virulence factor that may be employed by strain Cs1-4 in interactions with competing bacteria, as has been shown for T6SS in other bacteria (Schwarz et al., 2010;Leung et al., 2011;Records, 2011). Lastly, for the ΔlasI mutant, the absence of any detectable change in the formation of nanopods suggested that the process was not affected by quorum sensing, at least in the sense that it was regulated by AHL produced by a canonical AHL synthetase. This finding is noteworthy as it helps to narrow the spectrum of possible mechanisms that may control nanopod production.
Efficient targeting for gene inactivation is critical for functional genomic studies and, in bacteria, two widely used systems for generating in-frame, unmarked deletions are those based on sacB counter selection (Jäger et al., 1995;Chen et al., 2010), and Cre-loxP system (Denef et al., 2006;Choi et al., 2008). For strain Cs1-4, the sacB system proved unsuccessful; merodiploids (first recombination) were recovered at a high frequency, but these were not effectively resolved as Delftia sp. Cs1-4 grew in YT agar medium containing 5-15% (wt/vol) sucrose (data not shown). Similar observations have been reported for Streptomyces lividans and some Burkholderia strains, which carry an intrinsic sacBC operon. Alternatively, Cre-loxP system was successfully adapted for gene deletion or insertion, and was an efficient way for recycling antibiotic markers in Delftia sp. Cs1-4. To our knowledge, this is the first report of the Cre-loxP system being used for gene deletion analysis in Delftia spp.
Of the mutants recovered from genome-wide mutagenesis, three were of particular interest as they may encode new functions associated with nanopod production and/or phenanthrene degradation. One of these putatively encoded an Ycf48-like protein. In phototrophs, Ycf48 functions in the assembly and repair of Photosystem II (Komenda et al., 2008;Rengstl et al., 2011). Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Cs1-4
The bacterium Delftia sp. Cs1-4 produces novel extracellular structures (nanopods) in conjunction with its growth on phenanthrene. While a full genome sequence is available for strain Cs1-4, genetic tools that could be applied to study phenanthrene degradation/nanopod production have not been reported. Thus, the objectives of this study were to establish such tools, and apply them for molecular analysis of nanopod formation or phenanthrene degradation. Three types of tools were developed or validated. First, we developed a new expression system based on a strong promoter controlling expression of a surface layer protein (NpdA) from Delftia sp. Cs1-4, which was ca. 2,500-fold stronger than the widely used lactose promoter. Second, the Cre-loxP system was validated for generation of markerless, in-frame, gene deletions, and for in-frame gene insertions. The gene deletion function was applied to examine potential roles in nanopod formation of three genes (omp32, lasI, and hcp), while the gene insertion function was used for reporter gene tagging of npdA. Lastly, pMiniHimar was modified to enhance gene recovery and mutant analysis in genome-wide transposon mutagenesis. Application of the latter to strain Cs1-4, revealed several new genes with potential roles in phenanthrene degradation or npdA expression. Collectively, the availability of these tools has opened new avenues of investigation in Delftia sp. Cs1-4 and other related genera/species with importance in environmental toxicology.
INTRODUCTION
Bacteria of the genus Delftia mediate a diversity of processes important in environmental toxicology, including xenobiotic biodegradation and biotransformation of heavy metals (Vacca et al., 2005;De Gusseme et al., 2010;Juarez-Jimenez et al., 2010;Leibeling et al., 2010;Paulin et al., 2010;Zhang et al., 2010;Morel et al., 2011;Yang et al., 2011). Additionally, Delftia spp. have been identified as endobionts in a variety of organisms including humans and, in the latter case, some are emerging as opportunistic pathogens (Hail et al., 2011;Preiswerk et al., 2011). Genome sequence data will be an essential resource for identification of functions in Delftia spp. that are key to these activities, and one recently completed genome is that of the phenanthrene degrader Delftia sp. In addition to its abilities as a phenanthrene degrader, strain Cs1-4 is noteworthy as the organism in which new extracellular structures, termed nanopods, were discovered (Shetty et al., 2011). Nanopods are tubular elements that contain outer membrane vesicles (OMV) within a sheath composed of a surface layer protein (SLP). Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. The reporter vector was then conjugated into strain Cs1-4.
CONSTRUCTION OF STRONG EXPRESSION SYSTEM AND FLUORESCENT PROTEIN REPORTER VECTORS
CONSTRUCTION OF GFP REPORTER VECTOR FOR CHROMOSAOMAL TAGGING OF NPDA
To transcriptionally tag npdA, gfp was inserted immediately downstream of npdA using the Cre-loxP recombination method of Denef et al. (2005). An npdA fragment with the stop codon Frontiers in Microbiology | Microbiotechnoloy, Ecotoxicology and Bioremediation (genome position 5860670-5861289) was amplified using primers Delf22 and Delf23 ( Table 2). The downstream fragment of npdA (genome positions 5860066-5860809) was amplified using primers Delf24 and Delf25 ( Table 2). These fragments were then cloned into pGEM-T easy (pSCH447 and pSCH430). The downstream fragment from pSCH430 was released by digestion with SacII and SacI and inserted into the same sites on pJK100 (pSCH431). The npdA fragment from pSCH447 was released by NdeI and KpnI digestion, and then inserted into the same sites on pSCH431 (pSCH485). The gfpmut3 gene was released from pSCH375 by KpnI and Not I digestion, and assembled into the same sites on pSCH485 (pSCH451). Conjugation of pSCH451 into strain Cs1-4 gave Km r /Tc s colonies, which were recovered for further analysis. The Cre-expressing vector, pCM157, was next introduced into a selected colony (SCH483) in order to remove K m resistance, leading to strain SCH484 (Km s /Tc r ). Curing of pCM157 from SCH484 was done by serial transfers in LB medium. A selected colony (Km s /Tc s ) with green fluorescence was then confirmed for the correct construct by PCR and sequencing (SCH456).
GENOME-WIDE TRANSPOSON MUTAGENESIS
Modification of pHimarEm1 was done to introduce additional unique KpnI-BamHI-SacII restriction sites, to remove the erythromycin resistance gene and to insert genes encoding GFP and RFP. To do so, PCR was done with pHimarEm1 DNA as template, and using forward primer Delf38 and reverse primer Delf39 ( Table 2). The amplicon was digested with BamHI, self-ligated and transformed into E. coli S17 λpir. The gfpmut3 fragment was digested with KpnI and SacII from pSCH375 and inserted into pSC29 at the same restriction sites (pSCH160). The promoterless mStrawberry fragment was then released from pSCH378 by KpnI Frontiers in Microbiology | Microbiotechnoloy, Ecotoxicology and Bioremediation and SacII digestion, inserted into pSCH29 at the same restriction sites (pSCH402), and then introduced into strain SCH456 by conjugation. The Km-resistant colonies were randomly picked and replicated in 96-well plates containing MSM with either pyruvate and or phenanthrene as the carbon source. After incubation with shaking (24 h), the OD 600 and GFP fluorescence were determined (see below).
REPORTER ASSAYS
Renilla luciferase assays were done as described in our prior work (Chen et al., 2009) using a commercially available kit (Promega) according to the manufacturer's protocol. Quantitative analysis of fluorescent protein production was done using a Synergy 2 plate reader with the following conditions (all 0.2-s interval, 22˚C): GFP, excitation at 485 nm, emission 510 nm; RFP, excitation at 574 nm, emission at 596 nm. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Reverse transcription (RT) was done with a 5 -phosphorylated RT primer (Delf1; Table 2). After RT, mRNA was digested with RNaseH, and then cDNA was concatenated using T4 RNA ligase. The region of interest was then amplified via nested PCR using two sets of primers to regions of npdA. In the first PCR, RT products were used as template, and amplified with primers Delf2 and Delf3 ( Table 2). In the second PCR, template was a 10-fold dilution of the round one PCR product, and amplification was done using primers Delf4 and Delf5 ( Table 2). The 5 -RACE products were isolated, purified, ligated into pGEM-T easy and then sequenced.
The npdA fragment including the non-coding and partial structural gene regions was amplified with primers Delf6 and Delf7 (Table 2) using strain Cs1-4 genomic DNA as template. The Renilla luciferase (rluc) gene was amplified from pRL-SV40 using primers Delf8 and Delf9 ( Table 2). These fragments were fused via overlap PCR. To analyze the structure of the putative npdA promoter, deletion derivatives of non-coding fragments upstream of npdA were amplified by employing the same PCR strategy as described above, except using different N-terminal primers, namely Delf10, Delf11, Delf12, Delf13, Delf14, and Delf15 ( Table 2). The above amplicons were inserted in pGEM-T easy, released from this vector by SacI and SacII digestion, and inserted into the same sites of pBBR1MCS-3 to create the deletion series. The reporter vector was then conjugated into strain Cs1-4.
CONSTRUCTION OF STRONG EXPRESSION SYSTEM AND FLUORESCENT PROTEIN REPORTER VECTORS
CONSTRUCTION OF GFP REPORTER VECTOR FOR CHROMOSAOMAL TAGGING OF NPDA
To transcriptionally tag npdA, gfp was inserted immediately downstream of npdA using the Cre-loxP recombination method of Denef et al. (2005). An npdA fragment with the stop codon Frontiers in Microbiology | Microbiotechnoloy, Ecotoxicology and Bioremediation (genome position 5860670-5861289) was amplified using primers Delf22 and Delf23 ( Table 2). The downstream fragment of npdA (genome positions 5860066-5860809) was amplified using primers Delf24 and Delf25 ( Table 2). These fragments were then cloned into pGEM-T easy (pSCH447 and pSCH430). The downstream fragment from pSCH430 was released by digestion with SacII and SacI and inserted into the same sites on pJK100 (pSCH431). The npdA fragment from pSCH447 was released by NdeI and KpnI digestion, and then inserted into the same sites on pSCH431 (pSCH485). The gfpmut3 gene was released from pSCH375 by KpnI and Not I digestion, and assembled into the same sites on pSCH485 (pSCH451). Conjugation of pSCH451 into strain Cs1-4 gave Km r /Tc s colonies, which were recovered for further analysis. The Cre-expressing vector, pCM157, was next introduced into a selected colony (SCH483) in order to remove K m resistance, leading to strain SCH484 (Km s /Tc r ). Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. coli). For solid LB media, Bacto-Agar (Difco, Detroit, MI, USA) was added to a final concentration of 15 g/L. For E. coli, antibiotics were added when required at 100 μg/mL (ampicillin, Ap), 50 μg/mL (kanamycin, Km), or 10 μg/mL (tetracycline, Tc). Kanamycin and tetracycline were used in some Delftia sp. Cs1-4 cultures, and in these cases were added at 300 and 40 μg/mL, respectively.
DNA MANIPULATIONS
Genomic DNA was prepared using a genomic DNA extraction kit (Promega, Madison, WI, USA), and plasmid DNA was purified with the QIAprep spin miniprep kit (QIAGEN, Germantown, MD, USA). Restriction and modification enzymes were purchased from Promega (Madison, WI, USA) or New England Biolabs (Beverly, MA, USA). Klenow fragment or T4 DNA polymerase (Promega) was used to fill in recessed 3 ends and to trim protruding 3 ends of incompatible restriction sites. All PCR amplifications were done with the Failsafe PCR system (Epicenter Technology, Madison, WI, USA). Amplicons were separated in 0.7-1.0% (w/v) agarose gels, and DNA fragments were purified with the QIAquick gel extraction system (QIAGEN). Ligation mixtures were transformed into E. coli JM109 (Promega), and transformants were plated onto LB plates with appropriate antibiotic selection. Resistant colonies were isolated, and then screened for the acquisition of plasmids. All constructs were sequenced to verify structure. For conjugal transfer of plasmids from E. coli to Delftia sp. Cs1-4, LB-grown cultures of both cells were harvested (mid-log phase) by centrifugation, washed with LB and then equal amounts (ca. 10 12 cells of each strain) were mixed, and spotted onto LB plates containing 5 mM CaCl 2 . Following overnight incubation at 22˚C, cells were then scraped off of the plates, diluted, and plated on LB plates containing the appropriate antibiotics.
TRANSCRIPTION START SITE DETERMINATION
Total RNA was isolated from phenanthrene-grown strain Cs1-4 cells, and purified of genomic DNA by DNase I digestion. Analysis by 5 -RACE was done using TaKaRa 5 -full RACE Core set under conditions recommended by the supplier (TaKaRa). Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Thus, we hypothesized that nanopod production may be regulated by quorum sensing.
Testing of the above-described hypotheses has been hindered by a lack of genetic tools that have been developed for use in Delftia spp. The objectives of this study were thus to develop such tools, and apply them for molecular analysis of nanopod formation or phenanthrene degradation. Three types of tools were developed www.frontiersin.org and/or validated. First, a new expression system was developed based on a strong promoter (controlling npdA expression) from Delftia sp. Cs1-4. Second, the Cre-loxP gene deletion system was validated for generation of markerless, in-frame, gene deletions. Third, pMiniHimar was modified to enhance gene recovery and mutant analysis in genome-wide transposon mutagenesis.
BACTERIAL STRAINS, PLASMIDS, AND GROWTH CONDITIONS
Bacterial strains and plasmids used in this work are listed in Table 1. E. coli JM109 was used for cloning. For conjugation, donor strains were either E. coli BW19851 (λ pir) or E. coli S17 (λ pir) and recipient strains were either E. coli TransforMax EC100+ (for propagation of constructs) or Delftia sp. Cs1-4. E. coli strains were routinely grown in Luria-Bertani (LB) broth at 37˚C. Mineral salt medium (MSM; Hickey and Focht, 1990) containing phenanthrene as the sole carbon source (1 mg/mL) was routinely used for Delftia sp. Cs1-4 culture. Liquid cultures were grown with shaking (ca. 200 rpm) at either 25˚C (strain Cs1-4) or 37˚C (E. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>This below document has 2 sentences that end with '(Shetty et al., 2011)', 2 paragraphs that end with 'with importance in environmental toxicology'. It has approximately 490 words, 23 sentences, and 3 paragraph(s).
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Lastly, pMiniHimar was modified to enhance gene recovery and mutant analysis in genome-wide transposon mutagenesis. Application of the latter to strain Cs1-4, revealed several new genes with potential roles in phenanthrene degradation or npdA expression. Collectively, the availability of these tools has opened new avenues of investigation in Delftia sp. Cs1-4 and other related genera/species with importance in environmental toxicology.
INTRODUCTION
Bacteria of the genus Delftia mediate a diversity of processes important in environmental toxicology, including xenobiotic biodegradation and biotransformation of heavy metals (Vacca et al., 2005;De Gusseme et al., 2010;Juarez-Jimenez et al., 2010;Leibeling et al., 2010;Paulin et al., 2010;Zhang et al., 2010;Morel et al., 2011;Yang et al., 2011). Additionally, Delftia spp. have been identified as endobionts in a variety of organisms including humans and, in the latter case, some are emerging as opportunistic pathogens (Hail et al., 2011;Preiswerk et al., 2011). Genome sequence data will be an essential resource for identification of functions in Delftia spp. that are key to these activities, and one recently completed genome is that of the phenanthrene degrader Delftia sp. In addition to its abilities as a phenanthrene degrader, strain Cs1-4 is noteworthy as the organism in which new extracellular structures, termed nanopods, were discovered (Shetty et al., 2011). Nanopods are tubular elements that contain outer membrane vesicles (OMV) within a sheath composed of a surface layer protein (SLP). The latter was termed Nanopod protein A (NpdA), and mutants lacking this protein were unable to form nanopods. Proteomic analyses of nanopods revealed a variety of proteins that were associated with these structures, two being outer membrane protein 32 (Omp32) and hemolysin co-regulated protein (Hcp). These proteins were of interest as we hypothesized that they, along with NpdA, could have key roles in nanopod structure. For Omp32, this hypothesis was based on its occurrence of OMV in nanopods, and Omp32 being the major protein in the outer membrane of strain Cs1-4 (Shetty et al., 2011). The protein Hcp, which is part of the recently discovered type 6 secretion system (T6SS), can self-assemble into ca. 10 nm diameter rings, which subsequently stack into ca. 100 nm tubes (Mougous et al., 2006;Ballister et al., 2008). The functions of such tubes are unknown, but in the case of nanopods, we hypothesized that they could have a structural role in nanopod formation, perhaps forming an inner core. One other gene/protein of interest in nanopod formation was lasI, which is involved in quorum sensing via the acyl homoserine lactone (AHL) synthase it encodes. Its potential connection to nanopod formation was based on two observations: (1) the increased abundance of nanopods in late-growth phase of phenanthrene-grown cultures (Shetty et al., 2011), and (2) the close association of the lone genomic copy of lasI with the phenanthrene degradation gene cluster. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine
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DISCUSSION
Promoters proceeding SLP genes are among the most potent in many bacteria. For example, in Lactobacillus acidophilus, the strength of the SLP gene promoter is roughly twice that controlling the lactate dehydrogenase gene (Boot et al., 1996). Strong promoters may be needed for genes encoding SLP, as SLP are typically among the most abundant cellular proteins, as is the case with NpdA in strain Cs1-4 (Shetty et al., 2011). Thus, to develop a strong expression system, we focused on identification of the npdA promoter.
Collectively, the serial deletion analyses indicated that at least 220 bp upstream of npdA were required for maximal, log phase expression of npdA in strain Cs1-4 growing on phenanthrene. The presence within this region of multiple putative promoters is a feature that appears to be common for genes encoding SLP. For example, the SLP-encoding genes of Lactobacillus brevis ATCC 8287 (Hynönen et al., 2010), Aeromonas salmonicida (Chu et al., www.frontiersin.org 1993), and Bacillus stearothermophilus ATCC 12980 (Jarosch et al., 2000) had at least two promoters, while in Bacillus brevis three promoters were arranged tandemly upstream of the cwp operon (Adachi et al., 1989). The reason(s) why SLP genes have multiple promoters are unknown. Possibly, these could be needed to respond to a variety of stimuli that could affect the expression of SLP genes (Sleytr and Messner, 1983;Adachi et al., 1989;Soual-Hoebeke et al., 1999). As yet, specific functions for the S-layer in Delftia sp. strain Cs1-4 are unknown, however, some involvement in phenanthrene degradation is a possibility as mutants lacking NpdA (and consequently the S-layer) are impaired in their ability to grow on this compound (unpublished data).
Expression systems based on well-characterized promoters such as Plac or Ptac are widely used (Dykxhoorn et al., 1996), but have had limited success in the Burkholderiales (Lefebre and Valvano, 2002). Likewise, for strain Cs1-4, Rluc was weakly expressed under control of Plac, as Rluc activity was ca. 2,500-fold lower than that from PnpdA:rluc. An alternative approach is to use promoters that originate from the Burkholderiales, and one example is the promoter regulating expression of small ribosomal protein S12 (Prsp). The latter promoter has been successfully utilized in Burkholderia xenovorans LB400 (Yu and Tsang, 2006) and in B. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Cs1-4, the transposition frequency of pMiniHimar was ca. 2 × 10 −5 to 5 × 10 −6 per recipient, a frequency comparable to those reported for Shewanella oneidensis, Geobacter sulfurreducens, and B. pseudomallei (Choi et al., 2008;Rollefson et al., 2009). From the 13,000 colonies screened, seven mutants were recovered that were impaired in either growth on phenanthrene (Mutants 1-6; Table 3) or in npdA expression (Mutant 7; Table 3). For the former, three mutants had insertions in the gene cluster encoding the phenanthrene catabolic pathway. Of these, Mutant 3 was intriguing as the gene bearing the insertion was predicted to encode an Ycf48 homolog. For Mutants 5 and 7, insertions were in genes outside of the phenanthene degradation cluster, and were predicted to encode a SpoT/RelAtype (p)ppGpp synthetase, and a HylD Family, type I secretion membrane fusion protein, respectively.
DISCUSSION
Promoters proceeding SLP genes are among the most potent in many bacteria. For example, in Lactobacillus acidophilus, the strength of the SLP gene promoter is roughly twice that controlling the lactate dehydrogenase gene (Boot et al., 1996). Strong promoters may be needed for genes encoding SLP, as SLP are typically among the most abundant cellular proteins, as is the case with NpdA in strain Cs1-4 (Shetty et al., 2011). Thus, to develop a strong expression system, we focused on identification of the npdA promoter.
Collectively, the serial deletion analyses indicated that at least 220 bp upstream of npdA were required for maximal, log phase expression of npdA in strain Cs1-4 growing on phenanthrene. The presence within this region of multiple putative promoters is a feature that appears to be common for genes encoding SLP. For example, the SLP-encoding genes of Lactobacillus brevis ATCC 8287 (Hynönen et al., 2010), Aeromonas salmonicida (Chu et al., www.frontiersin.org 1993), and Bacillus stearothermophilus ATCC 12980 (Jarosch et al., 2000) had at least two promoters, while in Bacillus brevis three promoters were arranged tandemly upstream of the cwp operon (Adachi et al., 1989). The reason(s) why SLP genes have multiple promoters are unknown. Possibly, these could be needed to respond to a variety of stimuli that could affect the expression of SLP genes (Sleytr and Messner, 1983;Adachi et al., 1989;Soual-Hoebeke et al., 1999). As yet, specific functions for the S-layer in Delftia sp. strain Cs1-4 are unknown, however, some involvement in phenanthrene degradation is a possibility as mutants lacking NpdA (and consequently the S-layer) are impaired in their ability to grow on this compound (unpublished data).
Expression systems based on well-characterized promoters such as Plac or Ptac are widely used (Dykxhoorn et al., 1996), but have had limited success in the Burkholderiales (Lefebre and Valvano, 2002). Likewise, for strain Cs1-4, Rluc was weakly expressed under control of Plac, as Rluc activity was ca. 2,500-fold lower than that from PnpdA:rluc. An alternative approach is to use promoters that originate from the Burkholderiales, and one example is the promoter regulating expression of small ribosomal protein S12 (Prsp). The latter promoter has been successfully utilized in Burkholderia xenovorans LB400 (Yu and Tsang, 2006) and in B. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Activities of an Ycf48-like protein that may be related to phenanthrene degradation are unknown, but, given the significant reduction (ca. 64%) in nanopod produced by this mutant, it's interesting to speculate that it may have a role in the assembly of these structures. The putative spoT /relA mutant, had an insertion in a (p)ppGpp synthetase. The alarmone (p)ppGpp primarily governs the stringent response to amino acid starvation (Martinez-Costa et al., 1998;Åberg et al., 2006;Gomez-Escribano et al., 2008;Abranches et al., 2009) and, since growth of the spoT /relA mutant on pyruvate was not impaired, the effect of the mutation appeared related to use of phenanthrene as a carbon source. The third gene of www.frontiersin.org interest, encoding an HlyD-like protein, was clustered with other genes predicted to encode pili formation. But, it remains to be determined how amino acid starvation and pili formation may be connected to phenanthrene degradation. Mutant 7 was not impaired in growth on phenanthrene, but did show decreased expression of npdA, and a depressed level of nanopod production. The protein predicted for the locus bearing the insertion contained a heavy-metal-associated domain that is also found in a number of proteins that transport or detoxify heavy metals; the relation of such a protein to npdA expression and nanopod formation remains to be determined. Minitransposons are widely used for genome-wide mutagenesis in Gram-negative and Gram-positive bacteria (Lampe et al., 1999;Youderian et al., 2003;Maier et al., 2006;Choi et al., 2008) and, compared to other minitransposons, pMiniHimar is advantageous as it does not require host-specific factors for transposition, it lacks site specificity and the transposase is not introduced into the chromosome, thus enhancing insertion stability. The transposition frequency of pMiniHimar was sufficient (>5 × 10 −6 per recipient) for saturation mutagenesis of the strain Cs1-4 genome. In the present study, pMiniHimar RB1 was modified by adding unique restriction sites for insertion of additional genetic elements. In our tests, these elements were promoterless gfpmut3 and mStrawberry, and the resultant vectors can be utilized for random generation of genomic transcriptional fusions. Such vectors can provide a convenient way to conduct genome-wide investigations of gene expression levels under selected conditions (de Lorenzo et al., 1990;Hahn et al., 1991;Boyle-Vavra and Seifert, 1995;Velayudhan et al., 2007).
CONCLUSION
The present report outlined the development of tools needed for genetic manipulation of Delftia sp. Cs1-4. These tools included a new expression cassette (PnpdA-based) that can be used for tagging of chromosomal genes as well as for complementation of knockout mutants, and a pMiniHimar transposon modified to enhance gene recovery and mutant analysis. The effectiveness in Delftia sp. of the Cre-loxP for gene deletion was also demonstrated. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. The presence within this region of multiple putative promoters is a feature that appears to be common for genes encoding SLP. For example, the SLP-encoding genes of Lactobacillus brevis ATCC 8287 (Hynönen et al., 2010), Aeromonas salmonicida (Chu et al., www.frontiersin.org 1993), and Bacillus stearothermophilus ATCC 12980 (Jarosch et al., 2000) had at least two promoters, while in Bacillus brevis three promoters were arranged tandemly upstream of the cwp operon (Adachi et al., 1989). The reason(s) why SLP genes have multiple promoters are unknown. Possibly, these could be needed to respond to a variety of stimuli that could affect the expression of SLP genes (Sleytr and Messner, 1983;Adachi et al., 1989;Soual-Hoebeke et al., 1999). As yet, specific functions for the S-layer in Delftia sp. strain Cs1-4 are unknown, however, some involvement in phenanthrene degradation is a possibility as mutants lacking NpdA (and consequently the S-layer) are impaired in their ability to grow on this compound (unpublished data).
Expression systems based on well-characterized promoters such as Plac or Ptac are widely used (Dykxhoorn et al., 1996), but have had limited success in the Burkholderiales (Lefebre and Valvano, 2002). Likewise, for strain Cs1-4, Rluc was weakly expressed under control of Plac, as Rluc activity was ca. 2,500-fold lower than that from PnpdA:rluc. An alternative approach is to use promoters that originate from the Burkholderiales, and one example is the promoter regulating expression of small ribosomal protein S12 (Prsp). The latter promoter has been successfully utilized in Burkholderia xenovorans LB400 (Yu and Tsang, 2006) and in B. cepacia (Lefebre and Valvano, 2002). However, in strain Cs1-4, gene expression under Prsp was poor, and not significantly Frontiers in Microbiology | Microbiotechnoloy, Ecotoxicology and Bioremediation different from that of Plac (data not shown). Thus, demonstration of PnpdA as a strong promoter functional in Delftia sp. Cs1-4 has provided a much-needed tool for genetic analyses of this organism, and potentially other related bacteria.
The Δomp32 mutant had an irregular cell shape (Figure 3B), suggesting that Omp32 may have a key role in establishment of cell envelope structure, as shown for other outer membrane proteins (Lazar and Kolter, 1996;Watts and Hunstad, 2008). Analysis of the Δhcp mutant demonstrated that, as opposed to our hypothesis, Hcp did not have a structural function essential for nanopod formation. However, Western blot data indicated that Hcp was associated in some manner with nanopods as the majority of this protein accumulated in the >50-nm diameter fraction along with nanopods (data not shown). It is possible that Hcp was secreted separately from nanopods, and formed extracellular structures that were co-purified with nanopods. If so, such structures were not discernable in samples imaged by transmission electron microscope. Alternatively, Hcp may be associated with nanopods as cargo carried by OMV. In this case, Hcp may function as a virulence factor that may be employed by strain Cs1-4 in interactions with competing bacteria, as has been shown for T6SS in other bacteria (Schwarz et al., 2010;Leung et al., 2011;Records, 2011). Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. coli BW19851 (λ pir) or E. coli S17 (λ pir) and recipient strains were either E. coli TransforMax EC100+ (for propagation of constructs) or Delftia sp. Cs1-4. E. coli strains were routinely grown in Luria-Bertani (LB) broth at 37˚C. Mineral salt medium (MSM; Hickey and Focht, 1990) containing phenanthrene as the sole carbon source (1 mg/mL) was routinely used for Delftia sp. Cs1-4 culture. Liquid cultures were grown with shaking (ca. 200 rpm) at either 25˚C (strain Cs1-4) or 37˚C (E. coli). For solid LB media, Bacto-Agar (Difco, Detroit, MI, USA) was added to a final concentration of 15 g/L. For E. coli, antibiotics were added when required at 100 μg/mL (ampicillin, Ap), 50 μg/mL (kanamycin, Km), or 10 μg/mL (tetracycline, Tc). Kanamycin and tetracycline were used in some Delftia sp. Cs1-4 cultures, and in these cases were added at 300 and 40 μg/mL, respectively.
DNA MANIPULATIONS
Genomic DNA was prepared using a genomic DNA extraction kit (Promega, Madison, WI, USA), and plasmid DNA was purified with the QIAprep spin miniprep kit (QIAGEN, Germantown, MD, USA). Restriction and modification enzymes were purchased from Promega (Madison, WI, USA) or New England Biolabs (Beverly, MA, USA). Klenow fragment or T4 DNA polymerase (Promega) was used to fill in recessed 3 ends and to trim protruding 3 ends of incompatible restriction sites. All PCR amplifications were done with the Failsafe PCR system (Epicenter Technology, Madison, WI, USA). Amplicons were separated in 0.7-1.0% (w/v) agarose gels, and DNA fragments were purified with the QIAquick gel extraction system (QIAGEN). Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. The latter was termed Nanopod protein A (NpdA), and mutants lacking this protein were unable to form nanopods. Proteomic analyses of nanopods revealed a variety of proteins that were associated with these structures, two being outer membrane protein 32 (Omp32) and hemolysin co-regulated protein (Hcp). These proteins were of interest as we hypothesized that they, along with NpdA, could have key roles in nanopod structure. For Omp32, this hypothesis was based on its occurrence of OMV in nanopods, and Omp32 being the major protein in the outer membrane of strain Cs1-4 (Shetty et al., 2011). The protein Hcp, which is part of the recently discovered type 6 secretion system (T6SS), can self-assemble into ca. 10 nm diameter rings, which subsequently stack into ca. 100 nm tubes (Mougous et al., 2006;Ballister et al., 2008). The functions of such tubes are unknown, but in the case of nanopods, we hypothesized that they could have a structural role in nanopod formation, perhaps forming an inner core. One other gene/protein of interest in nanopod formation was lasI, which is involved in quorum sensing via the acyl homoserine lactone (AHL) synthase it encodes. Its potential connection to nanopod formation was based on two observations: (1) the increased abundance of nanopods in late-growth phase of phenanthrene-grown cultures (Shetty et al., 2011), and (2) the close association of the lone genomic copy of lasI with the phenanthrene degradation gene cluster. Thus, we hypothesized that nanopod production may be regulated by quorum sensing.
Testing of the above-described hypotheses has been hindered by a lack of genetic tools that have been developed for use in Delftia spp. The objectives of this study were thus to develop such tools, and apply them for molecular analysis of nanopod formation or phenanthrene degradation. Three types of tools were developed www.frontiersin.org and/or validated. First, a new expression system was developed based on a strong promoter (controlling npdA expression) from Delftia sp. Cs1-4. Second, the Cre-loxP gene deletion system was validated for generation of markerless, in-frame, gene deletions. Third, pMiniHimar was modified to enhance gene recovery and mutant analysis in genome-wide transposon mutagenesis.
BACTERIAL STRAINS, PLASMIDS, AND GROWTH CONDITIONS
Bacterial strains and plasmids used in this work are listed in Table 1. E. coli JM109 was used for cloning. For conjugation, donor strains were either E. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Curing of pCM157 from SCH484 was done by serial transfers in LB medium. A selected colony (Km s /Tc s ) with green fluorescence was then confirmed for the correct construct by PCR and sequencing (SCH456).
GENOME-WIDE TRANSPOSON MUTAGENESIS
Modification of pHimarEm1 was done to introduce additional unique KpnI-BamHI-SacII restriction sites, to remove the erythromycin resistance gene and to insert genes encoding GFP and RFP. To do so, PCR was done with pHimarEm1 DNA as template, and using forward primer Delf38 and reverse primer Delf39 ( Table 2). The amplicon was digested with BamHI, self-ligated and transformed into E. coli S17 λpir. The gfpmut3 fragment was digested with KpnI and SacII from pSCH375 and inserted into pSC29 at the same restriction sites (pSCH160). The promoterless mStrawberry fragment was then released from pSCH378 by KpnI Frontiers in Microbiology | Microbiotechnoloy, Ecotoxicology and Bioremediation and SacII digestion, inserted into pSCH29 at the same restriction sites (pSCH402), and then introduced into strain SCH456 by conjugation. The Km-resistant colonies were randomly picked and replicated in 96-well plates containing MSM with either pyruvate and or phenanthrene as the carbon source. After incubation with shaking (24 h), the OD 600 and GFP fluorescence were determined (see below).
REPORTER ASSAYS
Renilla luciferase assays were done as described in our prior work (Chen et al., 2009) using a commercially available kit (Promega) according to the manufacturer's protocol. Quantitative analysis of fluorescent protein production was done using a Synergy 2 plate reader with the following conditions (all 0.2-s interval, 22˚C): GFP, excitation at 485 nm, emission 510 nm; RFP, excitation at 574 nm, emission at 596 nm. All measurements were corrected for background with wild type (WT) Delftia sp. Cs1-4 cells.
DNA SEQUENCE AND SEQUENCE ANALYSIS
The complete genome sequence of Delftia sp. Cs1-4 was deposited in Genbank as accession NC(015563.1. All constructs were sequenced by the dideoxy termination method using an Applied Biosystems (Foster City, CA, USA) 3730 × l DNA Analyzer available at the University of Wisconsin-Madison, Biotechnology Center. GenBank database searches were carried out using the National Center for Biotechnology Information BLAST-N web server.
ANALYSIS OF NPDA PROMOTERS IN DELFTIA SP. CS1-4 AND DEVELOPMENT OF STRONG EXPRESSION SYSTEM
Three TSS were identified for npdA, and were located at (nucleotide) −34-bp (A), −56-bp(G), and −172-bp (A), respectively upstream of the npdA start codon ( Figure 1A). Three putative promoter motifs, PnpdA 1 (TCCTCT-N 15 -TGTCTG), PnpdA 2 (TAGGGG-N 15 -TACGAT), and PnpdA 3 (TACGAT-N 17 -TGGTGG) situated at −38, −61, and −180-bp, respectively were identified ( Figure 1A). Serial deletion of non-coding regions upstream of npdA was done to establish involvement in npdA regulation of one or more of the three putative promoters. There was no significant difference in levels of gene expression between the WT and D1 (npdA −220 bp; Figure 1B). However, further deletion of an 11-bp fragment from D1 (D2, npdA −209 bp) yielded a ca. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. Ligation mixtures were transformed into E. coli JM109 (Promega), and transformants were plated onto LB plates with appropriate antibiotic selection. Resistant colonies were isolated, and then screened for the acquisition of plasmids. All constructs were sequenced to verify structure. For conjugal transfer of plasmids from E. coli to Delftia sp. Cs1-4, LB-grown cultures of both cells were harvested (mid-log phase) by centrifugation, washed with LB and then equal amounts (ca. 10 12 cells of each strain) were mixed, and spotted onto LB plates containing 5 mM CaCl 2 . Following overnight incubation at 22˚C, cells were then scraped off of the plates, diluted, and plated on LB plates containing the appropriate antibiotics.
TRANSCRIPTION START SITE DETERMINATION
Total RNA was isolated from phenanthrene-grown strain Cs1-4 cells, and purified of genomic DNA by DNase I digestion. Analysis by 5 -RACE was done using TaKaRa 5 -full RACE Core set under conditions recommended by the supplier (TaKaRa). Reverse transcription (RT) was done with a 5 -phosphorylated RT primer (Delf1; Table 2). After RT, mRNA was digested with RNaseH, and then cDNA was concatenated using T4 RNA ligase. The region of interest was then amplified via nested PCR using two sets of primers to regions of npdA. In the first PCR, RT products were used as template, and amplified with primers Delf2 and Delf3 ( Table 2). In the second PCR, template was a 10-fold dilution of the round one PCR product, and amplification was done using primers Delf4 and Delf5 ( Table 2). The 5 -RACE products were isolated, purified, ligated into pGEM-T easy and then sequenced.
The npdA fragment including the non-coding and partial structural gene regions was amplified with primers Delf6 and Delf7 (Table 2) using strain Cs1-4 genomic DNA as template. The Renilla luciferase (rluc) gene was amplified from pRL-SV40 using primers Delf8 and Delf9 ( Table 2). These fragments were fused via overlap PCR. To analyze the structure of the putative npdA promoter, deletion derivatives of non-coding fragments upstream of npdA were amplified by employing the same PCR strategy as described above, except using different N-terminal primers, namely Delf10, Delf11, Delf12, Delf13, Delf14, and Delf15 ( Table 2). The above amplicons were inserted in pGEM-T easy, released from this vector by SacI and SacII digestion, and inserted into the same sites of pBBR1MCS-3 to create the deletion series. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. 20% decrease in Rluc activity relative to the WT (Figure 1B) To test the utility of the PnpdA expression system, the genes encoding a GFP and RFP were inserted downstream of the PnpdA cassette, which contained the 220-bp fragment described above.
Transformants appeared green or red under ambient light, indicating strong expression of gfp and mstrawberry, respectively. The apparent high-level expression of these proteins was non-toxic to Delftia sp. Cs1-4, as growth of cultures expressing GFP or RFP was not distinguishable from that of the WT (Figure 2A). Production of GFP and RFP followed similar patterns, with levels increasing with culture growth, achieving stable accumulations upon reaching stationary phase (Figure 2B). In the absence of antibiotic selection, the expression vector was stable in Delftia sp. Cs1-4 for at least 56 generations (Figure 2C).
GENE DELETION AND GENOME-WIDE MUTAGENESIS
For generation of gene knockouts, the vector was used to target omp32, hcp, and lasI. Deletion of all three genes was successful, and confirmed by PCR and/or Southern hybridization. However, none of the gene deletions resulted in a loss of nanopod production, and only the Δomp32 mutant exhibited phenotypes different from that of the WT. In whole cell protein profiles, the latter mutant showed a loss of the predominant band corresponding to Omp32 (Shetty et al., 2011) and appearance of two other proteins, also identified as porins ( Figure 3A). The Δomp32 mutant had an irregular cell shape (Figure 3B), and its growth was impaired on both pyruvate and phenanthrene, but the impact of Omp32 loss appeared to be greater with the latter substrate (Figures 3C,D).
Following conjugal delivery to Delftia sp. Cs1-4, the transposition frequency of pMiniHimar was ca. 2 × 10 −5 to 5 × 10 −6 per recipient, a frequency comparable to those reported for Shewanella oneidensis, Geobacter sulfurreducens, and B. pseudomallei (Choi et al., 2008;Rollefson et al., 2009). From the 13,000 colonies screened, seven mutants were recovered that were impaired in either growth on phenanthrene (Mutants 1-6; Table 3) or in npdA expression (Mutant 7; Table 3). For the former, three mutants had insertions in the gene cluster encoding the phenanthrene catabolic pathway. Of these, Mutant 3 was intriguing as the gene bearing the insertion was predicted to encode an Ycf48 homolog. For Mutants 5 and 7, insertions were in genes outside of the phenanthene degradation cluster, and were predicted to encode a SpoT/RelAtype (p)ppGpp synthetase, and a HylD Family, type I secretion membrane fusion protein, respectively.
DISCUSSION
Promoters proceeding SLP genes are among the most potent in many bacteria. For example, in Lactobacillus acidophilus, the strength of the SLP gene promoter is roughly twice that controlling the lactate dehydrogenase gene (Boot et al., 1996). Strong promoters may be needed for genes encoding SLP, as SLP are typically among the most abundant cellular proteins, as is the case with NpdA in strain Cs1-4 (Shetty et al., 2011). Thus, to develop a strong expression system, we focused on identification of the npdA promoter.
Collectively, the serial deletion analyses indicated that at least 220 bp upstream of npdA were required for maximal, log phase expression of npdA in strain Cs1-4 growing on phenanthrene. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine
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Development of Tools for Genetic Analysis of Phenanthrene Degradation and Nanopod Production by Delftia sp. All measurements were corrected for background with wild type (WT) Delftia sp. Cs1-4 cells.
DNA SEQUENCE AND SEQUENCE ANALYSIS
The complete genome sequence of Delftia sp. Cs1-4 was deposited in Genbank as accession NC(015563.1. All constructs were sequenced by the dideoxy termination method using an Applied Biosystems (Foster City, CA, USA) 3730 × l DNA Analyzer available at the University of Wisconsin-Madison, Biotechnology Center. GenBank database searches were carried out using the National Center for Biotechnology Information BLAST-N web server.
ANALYSIS OF NPDA PROMOTERS IN DELFTIA SP. CS1-4 AND DEVELOPMENT OF STRONG EXPRESSION SYSTEM
Three TSS were identified for npdA, and were located at (nucleotide) −34-bp (A), −56-bp(G), and −172-bp (A), respectively upstream of the npdA start codon ( Figure 1A). Three putative promoter motifs, PnpdA 1 (TCCTCT-N 15 -TGTCTG), PnpdA 2 (TAGGGG-N 15 -TACGAT), and PnpdA 3 (TACGAT-N 17 -TGGTGG) situated at −38, −61, and −180-bp, respectively were identified ( Figure 1A). Serial deletion of non-coding regions upstream of npdA was done to establish involvement in npdA regulation of one or more of the three putative promoters. There was no significant difference in levels of gene expression between the WT and D1 (npdA −220 bp; Figure 1B). However, further deletion of an 11-bp fragment from D1 (D2, npdA −209 bp) yielded a ca. 20% decrease in Rluc activity relative to the WT (Figure 1B) To test the utility of the PnpdA expression system, the genes encoding a GFP and RFP were inserted downstream of the PnpdA cassette, which contained the 220-bp fragment described above.
Transformants appeared green or red under ambient light, indicating strong expression of gfp and mstrawberry, respectively. The apparent high-level expression of these proteins was non-toxic to Delftia sp. Cs1-4, as growth of cultures expressing GFP or RFP was not distinguishable from that of the WT (Figure 2A). Production of GFP and RFP followed similar patterns, with levels increasing with culture growth, achieving stable accumulations upon reaching stationary phase (Figure 2B). In the absence of antibiotic selection, the expression vector was stable in Delftia sp. Cs1-4 for at least 56 generations (Figure 2C).
GENE DELETION AND GENOME-WIDE MUTAGENESIS
For generation of gene knockouts, the vector was used to target omp32, hcp, and lasI. Deletion of all three genes was successful, and confirmed by PCR and/or Southern hybridization. However, none of the gene deletions resulted in a loss of nanopod production, and only the Δomp32 mutant exhibited phenotypes different from that of the WT. In whole cell protein profiles, the latter mutant showed a loss of the predominant band corresponding to Omp32 (Shetty et al., 2011) and appearance of two other proteins, also identified as porins ( Figure 3A). The Δomp32 mutant had an irregular cell shape (Figure 3B), and its growth was impaired on both pyruvate and phenanthrene, but the impact of Omp32 loss appeared to be greater with the latter substrate (Figures 3C,D).
Following conjugal delivery to Delftia sp. Cs1-4, but could also be applied to other related genera and species with importance in environmental toxicology.
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Domain: Biology Environmental Science Chemistry Medicine<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. The soils with communities very different from this central tendency were of two types. The first included soils without a history of disturbance, such as native prairie in Kansas and the forest in Georgia. The second were soils with specific land management practices, such as the conifer plantation in Michigan or tillage with inorganic fertilizers in Georgia. The ability of K-shuff to clearly visualize distinctions between these communities will help describe the role of bacterial communities in soil fertility, crop productivity, sustainability, and other properties of great practical importance.
In conclusion, the power and sensitivity of K-shuff allows the comparison of both structure and composition of microbial communities from gene sequence libraries derived either by high-throughput methods (pyrosequencing and Illumina 1 ) or Sanger methodology. K-shuff offers an ecologically meaningful ordination of structural and compositional differences through the use of multidimensional scaling of its two functions, the intra K-function (IKF) and cross K-function (CKF). Monte Carlo tests of the IKF and CKF can also be used to test for statistically significant differences. K-shuff was also found to be more sensitive than LIB-SHUFF and UniFrac in detecting compositional differences. At the same time, its sensitivity allows an in-depth analysis of communities up to and within an OTU, which at times comprise multiple species, and dissection of small differences that might otherwise remain undetected. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. Thus, K-shuff revealed much more detail about the [20]. CTIF, HPIF and GPIF were inorganic fertilizer-amended soils and CTPL, HPPL and GPPL were poultry litter-amended soils. GF was a nearby forest soil that had not been tilled since 1860. Only libraries from triplicate soil cores collected in summer were combined for these analyses. b N = number of clones in the library. c S = number of OTUs formed at D = 0.03 using DOTUR [4]. compositional differences between the bacterial communities. Previous analyses also noted a progressive decline in the structural diversity during the first thousand years [22]. Similar changes were also seen in the I kf , which declined from 0.204 to 0.176 in the first thousand years and remained relatively constant after that.
Application of K-shuff to studies of the home microbiome
Reanalysis of the home microbiome dataset of Dunn et al. [23] was performed to determine if K-shuff could recapture the major conclusions and provide new insights. Using 100 sequences per sample for all 174 samples, the I kf were calculated for comparison to the OTU richness estimated by Dunn et al. [23]. Within each site, the OTU richness varied 3-10-fold, and the mean OTU richness of different sites varied nearly four-fold. In contrast, the I kf values within each site varied less than 3-fold, and the mean I kf s were relatively constant, varying only from 0.15-0.21 (Fig N in S1 File). The differences in OTU richness and I kf s would be expected if much of the variation was due the presence or absence of rare OTUs, which have little effect on the I kf . Moreover, I kf s of about 0.20 resembled those of seawater, which is consistent with the low diversity expected of the microbiome of surfaces. Thus, the K-shuff analyses suggested that the diversity of the various household surfaces were fairly similar, which was not readily apparent from examination of only OTU richness. [22]. For this analysis, 300 sequences were randomly selected from each replicate and pooled, yielding 1500 sequences for each age in the chronosequence. Similarly, PCoA plot of the variation among sites within the homes using the C kf of K-shuff was compared to the original analysis using the UniFrac distances by Dunn et al. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. First, the relationship between I kf and mixing proportion of the libraries was examined in the seawater and sediment libraries, which were very different. As the mixing proportion increased from 0.5 to 1.0, the I kf of the "seawater" library decreased from 0.227 to 0.181, and the I kf of the "sediment" library increased from 0.227 to 0.305, and there was a clear trend between I kf and ω (Fig F in S1 File). In contrast, as dramatic a trend was not found for the soil and sediment libraries, whose structures were very similar. When the proportion of mixing was 0.5, the I kf was very close to that of the sediment, and the "sediment" library remained close to this value as its proportion increased. The I kf of the "soil' library decreased from 0.304 to 0.277 as its proportion increased to 1.0. Thus, while the expected trends were observed, the changes in I kf were not close to linear with the proportion of each library.
K-shuff performance with Illumina ® dataset
Similar to the Sanger sequences from clone libraries, only small numbers of sequences were needed to calculate the K-shuff functions. For instance, the I kf values calculated for random samples of 100-500 sequences from the cutting board, door trim and pillowcase were very similar (Fig G in S1 File). Additional simulations were then performed using the cutting board (Cb) and pillowcase (Pc) libraries, which appeared to be typical of the diversity within the datasets and a good test of the sensitivity of K-shuff towards structural and composition differences [23]. Similar to the clone libraries, simulations on the reproducibility and effect of sample size on the I kf and C kf found that the mean values were relatively constant but the sampling variation decreased as N increased from 50 to 100. For instance, the mean ± standard deviation of C kf for comparisons between the cutting board (Cb) and pillow case (Pc) libraries were 0.024 ± 0.008, 0.024 ± 0.006 and 0.024 ± 0.005 for N of 50, 75 and 100, respectively (Fig H in S1 File). The small C kf values emphasized that the compositions of these libraries were very similar. Likewise, the mean I kf , ± standard deviation of the Cb and Pc libraries were 0.198 ± 0.006, 0.198 ± 0.005 and 0.198 ± 0.004 and 0.220 ± 0.006, 0.220 ± 0.005 and 0.220 ± 0.005 for N 50, 75, and 100, respectively (Figs I and J in S1 File).
Power analysis for the Illumina 1 dataset was performed using the cutting board (Cb) and pillowcase (Pc) libraries. While the fraction of significantly different I kf and C kf values increased with the mixing proportions, the increases were gradual, consistent with the small differences between the samples. For instance, structural differences between the libraries were only consistently observed at high mixing proportions (Fig K and Table C in S1 File). Similarly, compositional differences between the libraries were only consistently observed for the largest libraries of N = 100 (Fig K in S1 File). These results suggested that it was still possible to detect small differences in I kf and C kf at high mixing proportions even with small sample sizes. Similar to the trends for clone library comparisons, the changes in C kf and I kf were correlated with the mixing proportion of each library (Figs L and M in S1 File).
K-shuff performance with pyrosequencing dataset
For this data, much higher numbers of sequences were required to obtain consistent C kf values. While there was a good correlation, with a r 2 of 0.92, between the C kf values for the 1000 and 1500 sequence libraries, the correlations for the 500 sequence libraries were much lower, r 2 = 0.44 and 0.50, with the 1000 and 1500 sequence libraries, respectively. This result suggested that 500 sequences were not sufficient for reproducible estimates of C kf values with the pyrosequencing libraries. Thus, the libraries composed of 1500 sequences were used for additional analyses (see below). K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. Inspired by Ripley’s K-function for spatial point pattern analysis, the Intra K-function or IKF measures the structural diversity, including both the richness and overall similarity of the sequences, within a library. The Cross K-function or CKF measures the compositional diversity between gene libraries, reflecting both the number of OTUs shared as well as the overall similarity in OTUs. A Monte Carlo testing procedure then enables statistical evaluation of both the structural and compositional diversity between gene libraries. For 16S rRNA gene libraries from complex bacterial communities such as those found in seawater, salt marsh sediments, and soils, K-shuff yields reproducible estimates of structural and compositional diversity with libraries greater than 50 sequences. Similarly, for pyrosequencing libraries generated from a glacial retreat chronosequence and Illumina® libraries generated from US homes, K-shuff required >300 and 100 sequences per sample, respectively. Power analyses demonstrated that K-shuff is sensitive to small differences in Sanger or Illumina® libraries. This extra sensitivity of K-shuff enabled examination of compositional differences at much deeper taxonomic levels, such as within abundant OTUs. This is especially useful when comparing communities that are compositionally very similar but functionally different. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
Introduction
The quantitative comparison of gene sequence libraries has become an essential component of hypothesis-driven ecological research. With the increased affordability of high-throughput sequencing, the analyses of the microbiomes of humans, soils and other ecosystems has focused less on descriptions of microbiomes and more on examining the underlying environmental and biological factors affecting their structures. With the introduction of LIB-SHUFF [1] and other software packages, such as the analysis of molecular variance or AMOVA [2], R -LIBSHUFF [3], DOTUR [4], UniFrac [5], SONS [6], TreeClimber [7], LibraryCompare [8] and Metastats [9], statistical comparisons of communities can now be based on multiple approaches and algorithms. Generally, these comparisons are based on operational taxonomic units (OTUs), which are derived from the sequence datasets using two different approaches: alignment-dependent clustering based on distance cutoffs and an alignment-independent reference based on prior phylotype assignments [10]. While the former is computationally expensive, the latter suffers from the incompleteness of the reference databases, which at times can lead to a major portion of the microbiome left unassigned. Using mock sequence datasets represented in a two-dimensional space of circles and ellipses with known shapes and densities, a systematic evaluation of these tools showed that the current statistical toolbox has the ability to address specific ecological questions concerning the differences among microbial communities [11]. However, these algorithms posed some limitations for complex analyses. For instance, the methods that depend upon assignment of sequences to operational taxonomic units or OTUs require large samples to estimate richness. Similarly, methods that depend upon Monte Carlo testing procedures become computationally expensive when comparing large sequence libraries that are typical of high-throughput technologies. Lastly, there are also limitations in the sensitivity of the algorithms for detecting ecologically relevant differences.
To address these concerns, the utility of a new algorithm for comparison of gene libraries called K-shuff was assessed. K-shuff was designed and implemented in a Fortran program and is motivated by Ripley's K-function [12], which is a powerful statistical tool for spatial point pattern analysis. It uses measures of distances among all pairs of sequences to reflect any aggregation of these sequences as would arise from samples comprised of closely related organisms
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. It is worth noting that even though only six C kf s values were >0.01 (range 0.0033-0.0848), p-values for all comparisons except those between Di and Tv (C kf = 0.0033) were significant (p 0.005). This result indicated that the composition of OTU1 0.14 varied between many of the sites. The implication is that differences between the sites included differences in both the abundance and composition of this OTU.
Within OTU1 0.14 there were 126 OTUs at D = 0.03 with 897 sequences in the largest OTU, namely OTU1 0.03 . MDS analysis of OTU1 0.03 was very different from that of OTU1 0.14 ( Fig 8C). As expected, most of the C kf s calculated for comparisons across the sites were small (range 0.0005-0.0085) except for eight comparisons involving the outer door trim (Do). However, only one OTU1 0.03 sequence was found at Do, and none of the C kf values involving it were significantly different (Table D in S1 File). For that reason, Do was not included in the MDS plot. Nevertheless, these results indicated significant variation in the composition of even this very small group between the samples. For instance, the composition of OTU1 0.03 on the interior door trim (Di) was significantly different from that of the kitchen counter (Kc), refrigerator (Fr) and toilet seat (Ts).
Discussion
Comparison of K-shuff to other methods K-shuff is a sensitive method for detecting small but significant differences in the structural and compositional diversity in gene libraries. Like LIBSHUFF, UniFrac, P-test, F ST and Tree-Climber, it tests the hypothesis that two communities are alike [1,2,3,5,7,27]. All the methods begin with similarity matrices of sequences in gene libraries. However, UniFrac, P-test and TreeClimber generate phylogenetic trees, whose properties are then tested [11]. We specifically compared K-shuff with LIBSHUFF because both methods test the similarity matrices directly, and with UniFrac because it is currently one of the most frequently used tool. The K-shuff IKF and CKF are analogous to the homologous and heterologous coverages as defined in LIB-SHUFF [1]. However, while LIBSHUFF only utilized the evolutionary distances between each sequence and its closest neighbor, the K-functions use information on evolutionary distances among all members of the libraries. Therefore, the K-function uses more of the information contained in the libraries, which may account for its higher sensitivity. Moreover, unlike the heterologous coverage used in LIBSHUFF, the C kf is symmetric or independent of the direction of the comparison, i.e. K ðl 1 ;l 2 Þ ðrÞ ¼ K ðl 2 ;l 1 Þ ðrÞ. Symmetry is a sensible property since both C kf s provide the same information, i.e., the difference in the membership between the two libraries, and should agree with each other. However, unlike UniFrac which detects any differences that tend to assign the total branch length of a tree to a particular community [Schloss 2008], the sensitivity of K-shuff allows for an in-depth analysis of taxa that contribute to differences between communities. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. All the methods begin with similarity matrices of sequences in gene libraries. However, UniFrac, P-test and TreeClimber generate phylogenetic trees, whose properties are then tested [11]. We specifically compared K-shuff with LIBSHUFF because both methods test the similarity matrices directly, and with UniFrac because it is currently one of the most frequently used tool. The K-shuff IKF and CKF are analogous to the homologous and heterologous coverages as defined in LIB-SHUFF [1]. However, while LIBSHUFF only utilized the evolutionary distances between each sequence and its closest neighbor, the K-functions use information on evolutionary distances among all members of the libraries. Therefore, the K-function uses more of the information contained in the libraries, which may account for its higher sensitivity. Moreover, unlike the heterologous coverage used in LIBSHUFF, the C kf is symmetric or independent of the direction of the comparison, i.e. K ðl 1 ;l 2 Þ ðrÞ ¼ K ðl 2 ;l 1 Þ ðrÞ. Symmetry is a sensible property since both C kf s provide the same information, i.e., the difference in the membership between the two libraries, and should agree with each other. However, unlike UniFrac which detects any differences that tend to assign the total branch length of a tree to a particular community [Schloss 2008], the sensitivity of K-shuff allows for an in-depth analysis of taxa that contribute to differences between communities. In our studies, UniFrac proved even less sensitive than LIB-SHUFF, presumably because the process of generating phylogenetic trees greatly reduces the information content of the similarity matrices. Thus, K-shuff gives a more realistic assessment of differences in the communities.
Because of its high sensitivity, K-shuff is especially informative with relatively small libraries, such as those generated by Sanger sequencing. However, even for NexGen libraries, which typically include thousands or millions of sequences, K-shuff may provide valuable insights. Its high sensitivity makes it possible to analyze small subsets of large datasets that might be particularly interesting, such as moderately abundant OTUs. It would also be suitable for examining large numbers of samples generated by multiplexing, which also yield small numbers of sequences for each library [28,29,30]. Because sample size has only a small effect on either I kf or C kf values, it is also practical to test subsamples of very large libraries, which is computationally much less expensive.
Similar to the most frequently used phylogenetic metric UniFrac [5], K-shuff also produces an estimate of distance between the communities that is suitable for ANOVA and clustering analyses. However, C kf , the distance estimate of K-shuff, gives equal weight to both closely related as well as distantly related organisms, thereby allowing a unbiased estimation of the community composition. In contrast, UniFrac gives higher weight to differences arising from distantly related organisms [31]. Another concern with UniFrac is that the distance estimates are dramatically nonlinear. For instance, in mixing simulations of seawater and salt marsh sediment libraries, the UniFrac distances were 0.08, 0.81 and 0.86 at mixing proportions of 0.5, 0.7 and 1, respectively, and were not closely related to the differences in composition of the libraries. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. In essence, this K-function is the empirical cumulative distribution function of the evolutionary distances for a gene library. For every value of evolutionary distance r in the distance matrix, K(r) is the fraction of d ij values less than or equal to r.
The Intra K-function or IKF may be defined as above to describe the genetic diversity among the members of a single library, hereafter referred to its structure, and can be represented as a plot of K(r) as r increases from zero to its maximum value (Fig 1). For some applications, it is also convenient to describe the IKF as a single summary value, I kf , which represents the area above K(r) in the distribution plot (Fig 1). When a library has limited diversity, the IKF will rise rapidly and I kf will be small, as shown in the comparison of Fig 1A and 1E. However, reducing the IKF to a single number necessarily causes loss of information, and multiple IKFs can yield the same I kf value even when the libraries are significantly different, as seen in the comparison of Fig 1A and 1B. Moreover, in contrast to many conventional diversity estimates, the IKF is sensitive to both the number of OTUs and the relatedness between them. This explicit recognition of relatedness, not captured in binning methods, is an important advantage of the method's ability to represent the community diversity, and a community comprised of many closely related sequences would have a lower I kf than a community with same number of OTUs but comprised of distantly related sequences.
While the IKF describes the genetic diversity or structure within each library, the Cross-K-function of CKF provides a natural measure of the dissimilarity in the membership or composition between paired libraries. In the CKF, the K-function is the computed distribution function of the evolutionary distances between pairs of sequences, one from each of the two libraries (Fig 2). When the compositions of the libraries are the same, it is possible to demonstrate that the expected value of the CKF is equal to the IKFs of the two libraries under random allocation or shuffling of members among the two libraries, i.e., the CKF is identical to the IKFs for each library. As the compositions of the libraries become more different, however, the CKF becomes more different from the IKFs. The magnitude of this difference can also be summarized by a single value C kf , which is the sum of the areas between each of the IKFs and the CKF (Fig 2).
Theory behind K-shuff
Suppose that we have a collection of gene sequences and that the evolutionary distances d ij have been computed for all pairs of sequences i and j in the collection. Separate empirical Kfunctions were designed to provide nonparametric summary measures of the richness or evenness of genetic diversity within a library and the dissimilarity of the composition between libraries. The first empirical K-function, the Intra K-function or IKF describes the genetic diversity of a library, S l . It may be regarded as the cumulative distribution function of evolutionary distances among members of the library S l . It is: Here, n l is the number of sequences in the library S l and N is the total number of sequences in all libraries being studied. I(E) is an indicator function that takes the value of 1 if E is true and 0 if otherwise. The quantity d ij is the evolutionary distance between sequences i and j.
For describing the dissimilarity in the membership between a pair of libraries S l 1 and S l 2 , we consider the following bivariate empirical K-function, the Cross K-function or CKF. It is: Iðd ij rÞIði 2 S l 1 ÞIðj 2 S l 2 Þ; see also Diggle [16]. Here, n l 1 and n l 2 are the number of sequences in libraries S l 1 and S l 2 , respectively. The quantity d ij is now the distance between a sequence i of library S l 1 and a sequence j of library S l 2 .
Plotting K ðl 1 ;l 2 Þ ðrÞ against evolutionary distance r yields a description of the compositional similarity of the two libraries. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. These sites were chosen because they varied in diversity and showed low, intermediate, and high OTU richness, respectively (see When ω = 1.0, the libraries had their maximum difference because there was no mixing. For each combination of N and ω, the T I and T c test statistics for the I kf and C kf , respectively, were calculated on 1000 pairs of mixed libraries, and the fraction of comparisons that were significantly different or the power was calculated.
Results
General application of K-shuff K-shuff was first evaluated using 80-member clone libraries of Sanger sequences generated from seawater (SW), marsh sediment (MS), cropped soil (CS) and forest soil (FS) bacterial communities. Because of their longer reads, Sanger libraries better represent the taxonomic diversity of bacterial communities than many NexGen libraries [26]. These libraries were chosen to represent both simple and complex communities and communities from different environments. The SW community was composed of groups of closely related OTUs drawn from just a few phyla and had a relatively low diversity (Fig 3A). This was reflected in the IKF by the rapid increase in K(r) at low levels of evolutionary distance, followed by a plateau and then increase at high levels of evolutionary distance of about 0.3. In contrast, the diversity of the MS community was much greater, and the IKF remained low at low levels of evolutionary distance. The IKFs for the soil communities were very similar to those of the MS. These general observations were also reflected in the I kf values, which were 0.214, 0.302, 0.290, and 0.283 for the SW, MS, CS, and FS communities, respectively. While the differences between the SW I kf and the other values were all significant at p <0.001, the I kf of the two soil communities were not significantly different from each other (p = 0.820) or that of the MS (p = 0.18 and 0.09, respectively). These analyses confirm those of Lasher et al. [18], who had shown that the structural diversity of the bacterial communities of MS from Sapelo Island and soils were similar.
Although the structures of the MS and soil communities were not significantly different, their compositions differed significantly from each other as well as the SW community with p 0.003. For the comparison of the SW and MS communities, this difference was reflected in the CKF, whose value remained low at low evolutionary distances or r values before rapidly increasing at high levels of r (Fig 3A). Although the compositions of all the communities were significantly different, the magnitude of the differences varied greatly. For instance, the C kf value between the soil communities was 0.020, compared to 0.06-0.09 and >0.16 for comparisons between the soil and MS and SW communities, respectively ( Fig 3B). Consistent with the low C kf value, the soil communities possessed many OTUs in common and had a similar overall composition of their major phylogenetic groups [20]. UniFrac also indicated a similar hierarchical clustering of these communities ( Fig 3B). However, the p-values were only marginally significant (p 0.06). Moreover, the magnitude of the difference between the soil communities was nearly equal to their differences from the MS community for either the unweighted or weighted UniFrac distances, which was not consistent with comparisons of either the major phylogenetic groups or OTUs present [18,20]. Thus, K-shuff appeared to provide a better representation of the compositional diversity among these communities.
Power analysis and simulations of K-shuff performance with clone libraries
To determine the sensitivity of K-shuff to differences in the composition of the libraries, power analyses were performed by mixing the libraries from different communities. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. Because technical replicates were not performed, it was not possible to determine if the noise was due to variation in the PCR, cloning, and other aspects of the library preparation and sequencing or the inherent variability of the bacterial communities.
Although K-shuff indicated that, with one exception, the composition of the bacterial communities from all the treatments at all three sites were significantly different, for many ). This cluster of similar communities was well separated from those in the native prairie (BNP) in Kansas, cropland and pastures amended with inorganic fertilizer (CTIF, HPIF, and GPIF) in Georgia, forest which had not been disturbed other than for logging for over 135 years (GF) from Georgia, (Table 2). For instance, all the indices tested agreed that the forest and one of the croplands (CTIF) had the lowest diversity. However, only the I kf consistently indicated that soils amended with poultry litter had a higher diversity than soils amended with inorganic fertilizer. This conclusion was supported by analysis of the compositional diversity, which found that a number of bacterial groups were more abundant in the litter-amended soils [20]. Moreover, the absence of complete agreement between the indices was not surprising. The I kf was the only index that also considered the relatedness of the OTUs in addition to their number.
To determine if K-shuff would provide more insight into pyrosequencing datasets, libraries from a soil chronosequence dating from 60 y to 120 ky at the Franz Josef glacier on New Zealand were analyzed [22]. Previously, Bray-Curtis analysis of the 250 most abundant OTUs suggested that the soil communities were largely described by a monotonic function of change with age and failed to detect significant differences between the soil ages of 130 and 280 years; 530, 1k, and 12k years; and 5k, 12k, 60k, and 120k years [22]. In contrast, the CKF comparisons between the soil ages were all significantly different with p 0.001, and the C kf values varied within the range of 0.0028 and 0.0317. The MDS plot of the C kf values demonstrated complicated changes in the composition of the soil bacterial community with increasing age across the chronosequence (Fig 6). Thus, K-shuff revealed much more detail about the [20]. CTIF, HPIF and GPIF were inorganic fertilizer-amended soils and CTPL, HPPL and GPPL were poultry litter-amended soils. GF was a nearby forest soil that had not been tilled since 1860. Only libraries from triplicate soil cores collected in summer were combined for these analyses. b N = number of clones in the library. c S = number of OTUs formed at D = 0.03 using DOTUR [4]. compositional differences between the bacterial communities. Previous analyses also noted a progressive decline in the structural diversity during the first thousand years [22]. Similar changes were also seen in the I kf , which declined from 0.204 to 0.176 in the first thousand years and remained relatively constant after that.
Application of K-shuff to studies of the home microbiome
Reanalysis of the home microbiome dataset of Dunn et al. [23] was performed to determine if K-shuff could recapture the major conclusions and provide new insights. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>This below document has
* 2 sentences that start with 'The quantity d ij is',
* 2 sentences that end with 'S l '. It has approximately 619 words, 27 sentences, and 8 paragraph(s).
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. The magnitude of this difference can also be summarized by a single value C kf , which is the sum of the areas between each of the IKFs and the CKF (Fig 2).
Theory behind K-shuff
Suppose that we have a collection of gene sequences and that the evolutionary distances d ij have been computed for all pairs of sequences i and j in the collection. Separate empirical Kfunctions were designed to provide nonparametric summary measures of the richness or evenness of genetic diversity within a library and the dissimilarity of the composition between libraries. The first empirical K-function, the Intra K-function or IKF describes the genetic diversity of a library, S l . It may be regarded as the cumulative distribution function of evolutionary distances among members of the library S l . It is: Here, n l is the number of sequences in the library S l and N is the total number of sequences in all libraries being studied. I(E) is an indicator function that takes the value of 1 if E is true and 0 if otherwise. The quantity d ij is the evolutionary distance between sequences i and j.
For describing the dissimilarity in the membership between a pair of libraries S l 1 and S l 2 , we consider the following bivariate empirical K-function, the Cross K-function or CKF. It is: Iðd ij rÞIði 2 S l 1 ÞIðj 2 S l 2 Þ; see also Diggle [16]. Here, n l 1 and n l 2 are the number of sequences in libraries S l 1 and S l 2 , respectively. The quantity d ij is now the distance between a sequence i of library S l 1 and a sequence j of library S l 2 .
Plotting K ðl 1 ;l 2 Þ ðrÞ against evolutionary distance r yields a description of the compositional similarity of the two libraries. For pairs of libraries that are compositionally similar, K ðl 1 ;l 2 Þ ðrÞ will increase rapidly with increasing r. For dissimilar libraries, K ðl 1 ;l 2 Þ ðrÞ will increase slowly. This may be reduced to the index which is the sum of the areas between each of the IKFs and the CKF.
To determine the homogeneity of the genetic diversity or structure among libraries S 1 , S 2 ,Á Á Á,S L , the following test statistic was used in analogy with an analysis of variance (ANOVA) test proposed by Cuevas et al. [17] for functional data: where " K ðrÞ ¼ X L l¼1 n l K ðlÞ ðrÞ N is the weighted average of the IKFs. When all libraries are sampled from the same population, the statistical expectations of all IKFs are the same as their average, resulting in a small T l . Otherwise, when the structures are very different, the T l -value will be large.
Following Singleton et al. [1], a Monte Carlo procedure was used to approximate the distributions of T l and T c and obtain their corresponding p-values. Specifically, sequences are randomly shuffled across libraries for a large number of times, say 999 times. After each shuffling, T l as well as T c -values are generated. The distribution of each test statistic is approximated from the frequency distribution of the (e.g., 999) generated T j -values. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. In addition, p-value can be approximated by the frequency of the generated T j -values that are greater than the observed one.
Validation of K-shuff
Using K-shuff. K-shuff is available for download as Linux source code and Windows executable (current version 1.1) from [URL] along with test datasets and the user manual. All analyses were performed on a personal computer with 2.8 GHz processor and 4 GB RAM. To run K-shuff, a PHYLIP-formatted distance matrix and a control file are required. Because K-shuff is capable of performing multiple comparisons in a single run, one should include all libraries in a single distance matrix. The control file is a simple text file containing parameter settings for the run, such as the path to the distance matrix file, number of libraries in the matrix, number of sequences in each library, etc.
16S rRNA gene datasets. To demonstrate its general applicability and sensitivity, K-shuff was used to examine 16S rRNA gene libraries constructed using the Sanger method and pyrosequencing and Illumina 1 platforms. These data sets were chosen to be representative of interesting bacterial communities that varied in composition as well as structural diversity. Libraries of 16S rRNA gene clone sequences for sea water and salt marsh sediments were from Lasher et al. [18]. These libraries comprised Sanger sequences of about 400 bp. Libraries for cropland, forest and grassland soils from Georgia, Kansas and Michigan were from Jangid et al. [19,20,21], respectively. Prepared by Sanger sequencing, the sequences were typically about 800 bp. Because all of these clone libraries were essentially prepared under an identical protocol, the influence of experimental variations on the observed results were expected to be minimal. For the high-throughput methods, microbial communities from soils formed by the retreating Franz Josef glacier on the South Island of New Zealand were sampled by pyrosequencing as described by Jangid et al. [22]. rRNA gene pyrosequences from this chronosequence had an average read length of 260 bp and are deposited in SRP006445.2. For the Illumina 1 platform, sequences from the home life study by Dunn et al. [23] were used. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
==
Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. The small C kf values emphasized that the compositions of these libraries were very similar. Likewise, the mean I kf , ± standard deviation of the Cb and Pc libraries were 0.198 ± 0.006, 0.198 ± 0.005 and 0.198 ± 0.004 and 0.220 ± 0.006, 0.220 ± 0.005 and 0.220 ± 0.005 for N 50, 75, and 100, respectively (Figs I and J in S1 File).
Power analysis for the Illumina 1 dataset was performed using the cutting board (Cb) and pillowcase (Pc) libraries. While the fraction of significantly different I kf and C kf values increased with the mixing proportions, the increases were gradual, consistent with the small differences between the samples. For instance, structural differences between the libraries were only consistently observed at high mixing proportions (Fig K and Table C in S1 File). Similarly, compositional differences between the libraries were only consistently observed for the largest libraries of N = 100 (Fig K in S1 File). These results suggested that it was still possible to detect small differences in I kf and C kf at high mixing proportions even with small sample sizes. Similar to the trends for clone library comparisons, the changes in C kf and I kf were correlated with the mixing proportion of each library (Figs L and M in S1 File).
K-shuff performance with pyrosequencing dataset
For this data, much higher numbers of sequences were required to obtain consistent C kf values. While there was a good correlation, with a r 2 of 0.92, between the C kf values for the 1000 and 1500 sequence libraries, the correlations for the 500 sequence libraries were much lower, r 2 = 0.44 and 0.50, with the 1000 and 1500 sequence libraries, respectively. This result suggested that 500 sequences were not sufficient for reproducible estimates of C kf values with the pyrosequencing libraries. Thus, the libraries composed of 1500 sequences were used for additional analyses (see below). Because fewer sequences were required for reproducible estimates of C kf values for both Sanger libraries and Illumina sequencing, the requirement of large numbers of sequences seems to inherent to either pyrosequencing per se or the associated methods of sample preparation.
Application of K-shuff to studies of soil bacterial communities
A total of 126 16S rRNA gene libraries prepared by Sanger sequencing from soils collected in Georgia, Kansas and Michigan were analyzed by K-shuff [19,20,21]. Each of the three sites was represented by seven treatments, which included different types of vegetation, land management, and soil. For each treatment, six libraries were constructed, three from replicate plots sampled in the summer and three from the same plots sampled the following winter. Each sample comprised five cores, which were pooled prior to DNA extraction and library construction. ANOVA of the C kf confirmed previous observations that the treatments had significant effects on the compositions of the bacterial communities [19,20,21]. However, the magnitude of the treatment effects depended on the site, with sites with the largest differences in land use having the largest effects (Table 1). Thus, treatment effects in Georgia, where the sites included cropland, pasture and forest, were larger than those in Kansas, where the treatments corresponded to just cropland and prairie. In contrast, the effect of season all three sites was modest. Notably, the variation among the replicates was only somewhat lower than the variation between treatments even though only 56 out of the 126 CKFs between replicates were significantly different at p<0.05 (Table 1). This result suggested that 'noise' limited the ability to observe small differences in these communities. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. In contrast, the C kf values of K-shuff were 0.015, 0.049 and 0.121 at the same mixing proportions. Thus, K-shuff is more reflective of the differences in community composition. I kf as a novel estimate of structural diversity K-shuff provides I kf , which is a novel estimate of the structural diversity that reflects both the relatedness among OTUs or phylogenetic diversity as well as their relative abundances. Thus, a community comprised of many closely related sequences would have a smaller I kf than a community with same numbers of OTUs but comprised of distantly related sequences. Essentially this would not lead to change in memberships for an OTU thereby maintaining both the evenness and richness of the two communities. Further, because many commonly used indices, such as Shannon, Simpson, and Chao 1 are not a measure of phylogenetic diversity, their values would only change with changing richness, evenness or a larger sample size [32]. In addition, these estimates are biased towards richness (Shannon), evenness or dominance (Simpson), or lose information by reducing the data through clustering to obtain OTU abundances for determining singletons and doubletons (Chao 1) [32]. Because abundant OTUs dominate the IKF, the I kf is not very sensitive to the presence of rare OTUs or the 'long tails' frequently observed in microbial communities. Thus, while it is intuitively satisfying to capture multiple sources of diversity in a single value, the complex calculation of I kf makes it difficult to attribute differences to a single source, such as OTU richness or phylogenetic diversity.
As a result, the value of the I kf is difficult to assess without examining more applications. In the cases examined here, K-shuff analyses appeared to provide useful insights into the diversity of the microbial communities of cropland, pasture and forest soils as well as household surfaces. Moreover, it allowed comparisons of very different communities from seawater, marsh sediments and soils. In the long run, because of its high sensitivity and novel approach to structural diversity, it may provide new insights into the nature and structure of microbial communities. Although tested with 16S rRNA gene libraries, the methodology could be easily extended to other genes of interest and should be useful for other ecological and microbiological applications.
Compared to some methods, K-shuff is computationally expensive. However, the run times can be greatly reduced by subsampling large data sets. In our analysis of the subsampled dataset comprising 17366 sequences from Dunn et al. [23], the I kf and C kf were calculated in less than five minutes. However, calculating the p-values took about 4.5 h for the same dataset with 1000 permutations. With new programming languages and algorithms being developed, we believe that this limitation will be overcome soon.
As a test of the applicability of K-shuff, bacterial communities collected from a variety of soil types, land-use, vegetation, seasons and climates across the United States were analyzed. Although K-shuff detected significant differences between most of these communities, many of these differences were very small. Of particular interest was the similarity of many soil communities regardless of the differences in soil type, land uses, and regional climate. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
==
Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. These libraries comprised Sanger sequences of about 400 bp. Libraries for cropland, forest and grassland soils from Georgia, Kansas and Michigan were from Jangid et al. [19,20,21], respectively. Prepared by Sanger sequencing, the sequences were typically about 800 bp. Because all of these clone libraries were essentially prepared under an identical protocol, the influence of experimental variations on the observed results were expected to be minimal. For the high-throughput methods, microbial communities from soils formed by the retreating Franz Josef glacier on the South Island of New Zealand were sampled by pyrosequencing as described by Jangid et al. [22]. rRNA gene pyrosequences from this chronosequence had an average read length of 260 bp and are deposited in SRP006445.2. For the Illumina 1 platform, sequences from the home life study by Dunn et al. [23] were used. First, from a total of 1,719,177 quality-filtered reads a random subset of 1000 reads per sample were extracted as used by the authors in their analysis, which resulted in 174,000 reads from 174 samples representing nine standardized locations within 18-20 different homes. For subsequent analyses of the effect of sample size, sequence subsets were then selected from the 1000 sequence data per sample. For the PCoA of C kf , 100 sequences were randomly subsampled from each library. Randomization from each of the 174 samples yielded between 95-100 sequences for a total of 17366 sequences, which were then used for the final analysis.
All sequences were aligned against the SILVA reference alignment within MOTHUR [24], and a PHYLIP-formatted squared distance matrix was prepared. Conventional diversity estimates were calculated using OTUs clustered at D = 0.03 using the average neighbor algorithm in MOTHUR.
General application of K-shuff. To test a common potential application of K-shuff, the composition of four relatively small libraries of bacterial 16S rRNA genes were compared. These 80 member libraries comprised sequences randomly selected from larger libraries constructed from estuarine seawater (SW) and salt marsh sediment (MS) from the Sapelo Island Microbial Observatory [18] and forest (FS) and cropland soil (CS) from the J. Phil Campbell Sr. Natural Resource Conservation Center in Georgia [20].
The differences between the communities were determined using non-metric multi-dimensional scaling (MDS) analysis in Statistica v10 (www.statsoft.org). K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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|
K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. Because fewer sequences were required for reproducible estimates of C kf values for both Sanger libraries and Illumina sequencing, the requirement of large numbers of sequences seems to inherent to either pyrosequencing per se or the associated methods of sample preparation.
Application of K-shuff to studies of soil bacterial communities
A total of 126 16S rRNA gene libraries prepared by Sanger sequencing from soils collected in Georgia, Kansas and Michigan were analyzed by K-shuff [19,20,21]. Each of the three sites was represented by seven treatments, which included different types of vegetation, land management, and soil. For each treatment, six libraries were constructed, three from replicate plots sampled in the summer and three from the same plots sampled the following winter. Each sample comprised five cores, which were pooled prior to DNA extraction and library construction. ANOVA of the C kf confirmed previous observations that the treatments had significant effects on the compositions of the bacterial communities [19,20,21]. However, the magnitude of the treatment effects depended on the site, with sites with the largest differences in land use having the largest effects (Table 1). Thus, treatment effects in Georgia, where the sites included cropland, pasture and forest, were larger than those in Kansas, where the treatments corresponded to just cropland and prairie. In contrast, the effect of season all three sites was modest. Notably, the variation among the replicates was only somewhat lower than the variation between treatments even though only 56 out of the 126 CKFs between replicates were significantly different at p<0.05 (Table 1). This result suggested that 'noise' limited the ability to observe small differences in these communities. Because technical replicates were not performed, it was not possible to determine if the noise was due to variation in the PCR, cloning, and other aspects of the library preparation and sequencing or the inherent variability of the bacterial communities.
Although K-shuff indicated that, with one exception, the composition of the bacterial communities from all the treatments at all three sites were significantly different, for many ). This cluster of similar communities was well separated from those in the native prairie (BNP) in Kansas, cropland and pastures amended with inorganic fertilizer (CTIF, HPIF, and GPIF) in Georgia, forest which had not been disturbed other than for logging for over 135 years (GF) from Georgia, (Table 2). For instance, all the indices tested agreed that the forest and one of the croplands (CTIF) had the lowest diversity. However, only the I kf consistently indicated that soils amended with poultry litter had a higher diversity than soils amended with inorganic fertilizer. This conclusion was supported by analysis of the compositional diversity, which found that a number of bacterial groups were more abundant in the litter-amended soils [20]. Moreover, the absence of complete agreement between the indices was not surprising. The I kf was the only index that also considered the relatedness of the OTUs in addition to their number.
To determine if K-shuff would provide more insight into pyrosequencing datasets, libraries from a soil chronosequence dating from 60 y to 120 ky at the Franz Josef glacier on New Zealand were analyzed [22]. Previously, Bray-Curtis analysis of the 250 most abundant OTUs suggested that the soil communities were largely described by a monotonic function of change with age and failed to detect significant differences between the soil ages of 130 and 280 years; 530, 1k, and 12k years; and 5k, 12k, 60k, and 120k years [22]. In contrast, the CKF comparisons between the soil ages were all significantly different with p 0.001, and the C kf values varied within the range of 0.0028 and 0.0317. The MDS plot of the C kf values demonstrated complicated changes in the composition of the soil bacterial community with increasing age across the chronosequence (Fig 6). K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
==
Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. First, from a total of 1,719,177 quality-filtered reads a random subset of 1000 reads per sample were extracted as used by the authors in their analysis, which resulted in 174,000 reads from 174 samples representing nine standardized locations within 18-20 different homes. For subsequent analyses of the effect of sample size, sequence subsets were then selected from the 1000 sequence data per sample. For the PCoA of C kf , 100 sequences were randomly subsampled from each library. Randomization from each of the 174 samples yielded between 95-100 sequences for a total of 17366 sequences, which were then used for the final analysis.
All sequences were aligned against the SILVA reference alignment within MOTHUR [24], and a PHYLIP-formatted squared distance matrix was prepared. Conventional diversity estimates were calculated using OTUs clustered at D = 0.03 using the average neighbor algorithm in MOTHUR.
General application of K-shuff. To test a common potential application of K-shuff, the composition of four relatively small libraries of bacterial 16S rRNA genes were compared. These 80 member libraries comprised sequences randomly selected from larger libraries constructed from estuarine seawater (SW) and salt marsh sediment (MS) from the Sapelo Island Microbial Observatory [18] and forest (FS) and cropland soil (CS) from the J. Phil Campbell Sr. Natural Resource Conservation Center in Georgia [20].
The differences between the communities were determined using non-metric multi-dimensional scaling (MDS) analysis in Statistica v10 (www.statsoft.org). For this, the values of the C kf were used as an input matrix for the plots. In addition, PCoA of the compositional parameter (C kf ) matrix using Sorenson distance was used to identify clustering of groups along ordination axes for the household microbiome (see below). A test that household bacterial communities were significantly different (α<0.01) from one another was determined using the multiresponse permutation procedure (MRPP) in PC-ORD Version 6 [25]. A measure of the effect size, based on a chance corrected within group-agreement (A-value) was used to assess heterogeneity within and between groups.
Power analysis and simulations of K-shuff performance. To evaluate the utility of Kshuff more systematically, larger 16S rRNA gene libraries were compiled from both Sanger and high-throughput datasets. Clone library simulations were carried out by combining 1000 sequences from each of SW, MS and soils (FS+CS) that were representative of communities that varied in composition as well as structural diversity (Fig A in S1 File). Pyrosequences were obtained from a soil chronosequence dating from 60 y to 120 ky at the Franz Josef glacier on New Zealand [22]. For each of the nine ages in the chronosequence, libraries of 2000 sequences were prepared from each of five replicate samples or 10000 for each age. To evaluate the number of sequences required for K-shuff, 100, 200 or 300 sequences were randomly selected from each replicate, and the sequences from replicates were pooled for K-shuff analyses of 500, 1000, and 1500 sequences for each age.
Similar simulations were also carried out on the Illumina 1 datasets. Thus, the least number of sequences required for a reproducible measure of IKF and CKF was determined from random samples of 100, 200, 300, 400 and 500 sequences per sample for Cutting board (19 samples), Pillowcase (20 samples), and Door Exterior (18 samples) communities for a total of 57 samples. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. The expectation was that a sensitive statistic would detect differences in the libraries at a low mixing ratio. The power curves for both T I and T c , the test statistics for the I kf and C kf respectively, increased rapidly with the mixing proportion (or ω) for the MS and SW libraries, indicating that K-shuff readily distinguished libraries whose structures and compositions differed (Fig 4). For instance, at a mixing proportion of 0.70, the power of T I to distinguish structural differences between the libraries was already 0.782 for N = 50, 0.945 for N = 75, and 0.994 for N = 100, where N is the size of the libraries (Fig 4 and Table A in S1 File). Similarly, the power of T c to distinguish compositional differences was above 0.90 even at N = 50. Compositional differences between the MS and soil bacterial communities were also readily detected. At a mixing proportion of 0.70, the power was 0.698 for N = 50, 0.901 for N = 75, and 0.998 for N = 100 (Table B in S1 File). Previously, the sediment libraries had been found to possess a structural diversity comparable to soil [18]. Consistent with this result, the power of T I remained low under all the conditions tested (Table B in S1 File). In conclusion, the test statistics had a high power to detect small differences in the libraries, and compositional differences were detected in the absence of large structural differences.
K-shuff was also more sensitive to small changes in the library composition than widely used alternative methods. Based upon 50 replicate libraries, the power of K-shuff to detect significant differences between SW and MS libraries with ω = 0.70 was 0.92, or essentially the same as with 1000 replicates. In contrast, the powers of LIBSHUFF and UniFrac under the same conditions were 0.32 and 0.16, respectively. Thus, K-shuff was much more sensitive that either of these two commonly used methods.
It can be demonstrated that the expected values of both C kf and I kf do not depend on sample size. However, variation in the means is expected as a consequence of higher sampling variances in the smaller samples. Simulations were performed to determine the effect of the sample size N on the sampling variation in I kf and C kf . Sampling variation in K-shuff decreased with increasing N. For instance, the mean ± standard errors of C kf for comparisons of soil and MS were 0.065 ± 0.015, 0.064 ± 0.010, 0.063 ± 0.008, and 0.063 ± 0.007 for N of 25, 50, 75, and 100, respectively (Fig B in S1 File). This effect was also observed for the I kf , of the soil and marine sediments libraries (Figs C and D in S1 File). For instance, the mean ± standard error of the I kf for soil libraries were 0.268 ± 0.019, 0.275 ± 0.012, 0.277 ± 0.009, and 0.277 ± 0.008 for N of 25, 50, 75, and 100, respectively. An important implication of this result is that the libraries do not have to be the same size to compare these parameters.
If the I kf reflected the structures of libraries, it would also be expected to become more like the highly represented library as the mixing proportion increased. Like the C kf , the relationship between I kf and ω was expected to be complex. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. [23] (Fig 7). The MRPP of the compositional parameter (C kf ) matrix from the household bacterial communities was significantly different (α<0.01), with clusters represented by PCoA. Each of the bacterial communities from different household habitats were patterned along two axes explaining 75.9 (Axis 1) and 13.2% (Axis 2) of the variance. A measure of the effect size based on a chance corrected within group-agreement test (A = 0.2) indicated that heterogeneity between groups was greater than that within groups. These results are independent of sample size and thus help to support the veracity of the p-values. Likewise, the MDS plot of the C kf showed similar clustering as the PCoA (Fig O in S1 File). Overall, these results indicated that ordination using the C kf matrix explained a much greater proportion of the variance than did analysis using UniFrac.
To utilize the extra sensitivity afforded by K-shuff, analyses were performed on some of the abundant OTUs to explore differences within the members of an OTU across the nine sites. At D = 0.14, a total of 4664 OTUs were found for the entire subsampled dataset (N = 17366), with the largest OTU (OTU1 0.14 ) comprising 4271 sequences affiliated with the Firmicutes. Based on MDS analysis of the C kf s, OTU1 0.14 showed a similar clustering pattern as the entire subsampled dataset (Fig 8A and 8B). Communities from sites Dh, Di and Tv were tightly clustered whereas Fr, Kc, Cb and Ts, Pc were spread across the plot in two loose clusters. It is worth noting that even though only six C kf s values were >0.01 (range 0.0033-0.0848), p-values for all comparisons except those between Di and Tv (C kf = 0.0033) were significant (p 0.005). This result indicated that the composition of OTU1 0.14 varied between many of the sites. The implication is that differences between the sites included differences in both the abundance and composition of this OTU.
Within OTU1 0.14 there were 126 OTUs at D = 0.03 with 897 sequences in the largest OTU, namely OTU1 0.03 . MDS analysis of OTU1 0.03 was very different from that of OTU1 0.14 ( Fig 8C). As expected, most of the C kf s calculated for comparisons across the sites were small (range 0.0005-0.0085) except for eight comparisons involving the outer door trim (Do). However, only one OTU1 0.03 sequence was found at Do, and none of the C kf values involving it were significantly different (Table D in S1 File). For that reason, Do was not included in the MDS plot. Nevertheless, these results indicated significant variation in the composition of even this very small group between the samples. For instance, the composition of OTU1 0.03 on the interior door trim (Di) was significantly different from that of the kitchen counter (Kc), refrigerator (Fr) and toilet seat (Ts).
Discussion
Comparison of K-shuff to other methods K-shuff is a sensitive method for detecting small but significant differences in the structural and compositional diversity in gene libraries. Like LIBSHUFF, UniFrac, P-test, F ST and Tree-Climber, it tests the hypothesis that two communities are alike [1,2,3,5,7,27]. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. So, more information is retained when compared to LIBSHUFF which only uses the distance between each sequence and its nearest neighbor. In contrast to spatial point processes, the distances among gene sequences typically encompass hundreds of dimensions, where each sequence position comprises a separate dimension. Thus, some ideal properties, such as stationarity and isotropy, which are typically assumed in developing K-function theories, are not relevant. However, this limit does not hinder the usefulness of K-functions for this application. As indicated by Diggle et al. [13] and the results of our case studies detailed below, empirical K-functions still render meaningful scientific interpretations.
In this subject, we perform a systematic evaluation of the reproducibility, sensitivity and power of K-shuff, using previously reported 16S rRNA gene sequence datasets obtained using the Sanger method and pyrosequencing and Illumina 1 platforms. In addition, we compare its performance with LIBSHUFF, the first statistical tool for this purpose, as well as the most frequently used tool today, UniFrac. We show that when correctly employed, K-shuff allows testing of multiple hypotheses in a single process and has the potential to significantly improve our understanding of ecologically meaningful aspects of microbial communities.
Materials and Methods
The K-shuff Metric K-shuff identifies spatial clustering based on the reduced second moment measure, or K-function [14,15]. In general, the K-function is defined based on a distance measure, which in this context is set to be the evolutionary distance between the gene sequences. Denoting the evolutionary distance between sequences i and j by d ij , the empirical K-function for a library of sequences of size N is:
Iðd ij rÞ;
where I(E) is an indicator function (= 1, when E is true; = 0, if otherwise), and r is any positive number on the real line of evolutionary distances. In essence, this K-function is the empirical cumulative distribution function of the evolutionary distances for a gene library. For every value of evolutionary distance r in the distance matrix, K(r) is the fraction of d ij values less than or equal to r.
The Intra K-function or IKF may be defined as above to describe the genetic diversity among the members of a single library, hereafter referred to its structure, and can be represented as a plot of K(r) as r increases from zero to its maximum value (Fig 1). For some applications, it is also convenient to describe the IKF as a single summary value, I kf , which represents the area above K(r) in the distribution plot (Fig 1). When a library has limited diversity, the IKF will rise rapidly and I kf will be small, as shown in the comparison of Fig 1A and 1E. However, reducing the IKF to a single number necessarily causes loss of information, and multiple IKFs can yield the same I kf value even when the libraries are significantly different, as seen in the comparison of Fig 1A and 1B. Moreover, in contrast to many conventional diversity estimates, the IKF is sensitive to both the number of OTUs and the relatedness between them. This explicit recognition of relatedness, not captured in binning methods, is an important advantage of the method's ability to represent the community diversity, and a community comprised of many closely related sequences would have a lower I kf than a community with same number of OTUs but comprised of distantly related sequences.
While the IKF describes the genetic diversity or structure within each library, the Cross-K-function of CKF provides a natural measure of the dissimilarity in the membership or composition between paired libraries. In the CKF, the K-function is the computed distribution function of the evolutionary distances between pairs of sequences, one from each of the two libraries (Fig 2). When the compositions of the libraries are the same, it is possible to demonstrate that the expected value of the CKF is equal to the IKFs of the two libraries under random allocation or shuffling of members among the two libraries, i.e., the CKF is identical to the IKFs for each library. As the compositions of the libraries become more different, however, the CKF becomes more different from the IKFs. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. Moreover, it allowed comparisons of very different communities from seawater, marsh sediments and soils. In the long run, because of its high sensitivity and novel approach to structural diversity, it may provide new insights into the nature and structure of microbial communities. Although tested with 16S rRNA gene libraries, the methodology could be easily extended to other genes of interest and should be useful for other ecological and microbiological applications.
Compared to some methods, K-shuff is computationally expensive. However, the run times can be greatly reduced by subsampling large data sets. In our analysis of the subsampled dataset comprising 17366 sequences from Dunn et al. [23], the I kf and C kf were calculated in less than five minutes. However, calculating the p-values took about 4.5 h for the same dataset with 1000 permutations. With new programming languages and algorithms being developed, we believe that this limitation will be overcome soon.
As a test of the applicability of K-shuff, bacterial communities collected from a variety of soil types, land-use, vegetation, seasons and climates across the United States were analyzed. Although K-shuff detected significant differences between most of these communities, many of these differences were very small. Of particular interest was the similarity of many soil communities regardless of the differences in soil type, land uses, and regional climate. The soils with communities very different from this central tendency were of two types. The first included soils without a history of disturbance, such as native prairie in Kansas and the forest in Georgia. The second were soils with specific land management practices, such as the conifer plantation in Michigan or tillage with inorganic fertilizers in Georgia. The ability of K-shuff to clearly visualize distinctions between these communities will help describe the role of bacterial communities in soil fertility, crop productivity, sustainability, and other properties of great practical importance.
In conclusion, the power and sensitivity of K-shuff allows the comparison of both structure and composition of microbial communities from gene sequence libraries derived either by high-throughput methods (pyrosequencing and Illumina 1 ) or Sanger methodology. K-shuff offers an ecologically meaningful ordination of structural and compositional differences through the use of multidimensional scaling of its two functions, the intra K-function (IKF) and cross K-function (CKF). Monte Carlo tests of the IKF and CKF can also be used to test for statistically significant differences. K-shuff was also found to be more sensitive than LIB-SHUFF and UniFrac in detecting compositional differences. At the same time, its sensitivity allows an in-depth analysis of communities up to and within an OTU, which at times comprise multiple species, and dissection of small differences that might otherwise remain undetected. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. For this, the values of the C kf were used as an input matrix for the plots. In addition, PCoA of the compositional parameter (C kf ) matrix using Sorenson distance was used to identify clustering of groups along ordination axes for the household microbiome (see below). A test that household bacterial communities were significantly different (α<0.01) from one another was determined using the multiresponse permutation procedure (MRPP) in PC-ORD Version 6 [25]. A measure of the effect size, based on a chance corrected within group-agreement (A-value) was used to assess heterogeneity within and between groups.
Power analysis and simulations of K-shuff performance. To evaluate the utility of Kshuff more systematically, larger 16S rRNA gene libraries were compiled from both Sanger and high-throughput datasets. Clone library simulations were carried out by combining 1000 sequences from each of SW, MS and soils (FS+CS) that were representative of communities that varied in composition as well as structural diversity (Fig A in S1 File). Pyrosequences were obtained from a soil chronosequence dating from 60 y to 120 ky at the Franz Josef glacier on New Zealand [22]. For each of the nine ages in the chronosequence, libraries of 2000 sequences were prepared from each of five replicate samples or 10000 for each age. To evaluate the number of sequences required for K-shuff, 100, 200 or 300 sequences were randomly selected from each replicate, and the sequences from replicates were pooled for K-shuff analyses of 500, 1000, and 1500 sequences for each age.
Similar simulations were also carried out on the Illumina 1 datasets. Thus, the least number of sequences required for a reproducible measure of IKF and CKF was determined from random samples of 100, 200, 300, 400 and 500 sequences per sample for Cutting board (19 samples), Pillowcase (20 samples), and Door Exterior (18 samples) communities for a total of 57 samples. These sites were chosen because they varied in diversity and showed low, intermediate, and high OTU richness, respectively (see When ω = 1.0, the libraries had their maximum difference because there was no mixing. For each combination of N and ω, the T I and T c test statistics for the I kf and C kf , respectively, were calculated on 1000 pairs of mixed libraries, and the fraction of comparisons that were significantly different or the power was calculated.
Results
General application of K-shuff K-shuff was first evaluated using 80-member clone libraries of Sanger sequences generated from seawater (SW), marsh sediment (MS), cropped soil (CS) and forest soil (FS) bacterial communities. Because of their longer reads, Sanger libraries better represent the taxonomic diversity of bacterial communities than many NexGen libraries [26]. These libraries were chosen to represent both simple and complex communities and communities from different environments. The SW community was composed of groups of closely related OTUs drawn from just a few phyla and had a relatively low diversity (Fig 3A). This was reflected in the IKF by the rapid increase in K(r) at low levels of evolutionary distance, followed by a plateau and then increase at high levels of evolutionary distance of about 0.3. In contrast, the diversity of the MS community was much greater, and the IKF remained low at low levels of evolutionary distance. The IKFs for the soil communities were very similar to those of the MS. These general observations were also reflected in the I kf values, which were 0.214, 0.302, 0.290, and 0.283 for the SW, MS, CS, and FS communities, respectively. While the differences between the SW I kf and the other values were all significant at p <0.001, the I kf of the two soil communities were not significantly different from each other (p = 0.820) or that of the MS (p = 0.18 and 0.09, respectively). K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. For pairs of libraries that are compositionally similar, K ðl 1 ;l 2 Þ ðrÞ will increase rapidly with increasing r. For dissimilar libraries, K ðl 1 ;l 2 Þ ðrÞ will increase slowly. This may be reduced to the index which is the sum of the areas between each of the IKFs and the CKF.
To determine the homogeneity of the genetic diversity or structure among libraries S 1 , S 2 ,Á Á Á,S L , the following test statistic was used in analogy with an analysis of variance (ANOVA) test proposed by Cuevas et al. [17] for functional data: where " K ðrÞ ¼ X L l¼1 n l K ðlÞ ðrÞ N is the weighted average of the IKFs. When all libraries are sampled from the same population, the statistical expectations of all IKFs are the same as their average, resulting in a small T l . Otherwise, when the structures are very different, the T l -value will be large.
Following Singleton et al. [1], a Monte Carlo procedure was used to approximate the distributions of T l and T c and obtain their corresponding p-values. Specifically, sequences are randomly shuffled across libraries for a large number of times, say 999 times. After each shuffling, T l as well as T c -values are generated. The distribution of each test statistic is approximated from the frequency distribution of the (e.g., 999) generated T j -values. In addition, p-value can be approximated by the frequency of the generated T j -values that are greater than the observed one.
Validation of K-shuff
Using K-shuff. K-shuff is available for download as Linux source code and Windows executable (current version 1.1) from [URL] along with test datasets and the user manual. All analyses were performed on a personal computer with 2.8 GHz processor and 4 GB RAM. To run K-shuff, a PHYLIP-formatted distance matrix and a control file are required. Because K-shuff is capable of performing multiple comparisons in a single run, one should include all libraries in a single distance matrix. The control file is a simple text file containing parameter settings for the run, such as the path to the distance matrix file, number of libraries in the matrix, number of sequences in each library, etc.
16S rRNA gene datasets. To demonstrate its general applicability and sensitivity, K-shuff was used to examine 16S rRNA gene libraries constructed using the Sanger method and pyrosequencing and Illumina 1 platforms. These data sets were chosen to be representative of interesting bacterial communities that varied in composition as well as structural diversity. Libraries of 16S rRNA gene clone sequences for sea water and salt marsh sediments were from Lasher et al. [18]. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. These analyses confirm those of Lasher et al. [18], who had shown that the structural diversity of the bacterial communities of MS from Sapelo Island and soils were similar.
Although the structures of the MS and soil communities were not significantly different, their compositions differed significantly from each other as well as the SW community with p 0.003. For the comparison of the SW and MS communities, this difference was reflected in the CKF, whose value remained low at low evolutionary distances or r values before rapidly increasing at high levels of r (Fig 3A). Although the compositions of all the communities were significantly different, the magnitude of the differences varied greatly. For instance, the C kf value between the soil communities was 0.020, compared to 0.06-0.09 and >0.16 for comparisons between the soil and MS and SW communities, respectively ( Fig 3B). Consistent with the low C kf value, the soil communities possessed many OTUs in common and had a similar overall composition of their major phylogenetic groups [20]. UniFrac also indicated a similar hierarchical clustering of these communities ( Fig 3B). However, the p-values were only marginally significant (p 0.06). Moreover, the magnitude of the difference between the soil communities was nearly equal to their differences from the MS community for either the unweighted or weighted UniFrac distances, which was not consistent with comparisons of either the major phylogenetic groups or OTUs present [18,20]. Thus, K-shuff appeared to provide a better representation of the compositional diversity among these communities.
Power analysis and simulations of K-shuff performance with clone libraries
To determine the sensitivity of K-shuff to differences in the composition of the libraries, power analyses were performed by mixing the libraries from different communities. The expectation was that a sensitive statistic would detect differences in the libraries at a low mixing ratio. The power curves for both T I and T c , the test statistics for the I kf and C kf respectively, increased rapidly with the mixing proportion (or ω) for the MS and SW libraries, indicating that K-shuff readily distinguished libraries whose structures and compositions differed (Fig 4). For instance, at a mixing proportion of 0.70, the power of T I to distinguish structural differences between the libraries was already 0.782 for N = 50, 0.945 for N = 75, and 0.994 for N = 100, where N is the size of the libraries (Fig 4 and Table A in S1 File). Similarly, the power of T c to distinguish compositional differences was above 0.90 even at N = 50. Compositional differences between the MS and soil bacterial communities were also readily detected. At a mixing proportion of 0.70, the power was 0.698 for N = 50, 0.901 for N = 75, and 0.998 for N = 100 (Table B in S1 File). Previously, the sediment libraries had been found to possess a structural diversity comparable to soil [18]. Consistent with this result, the power of T I remained low under all the conditions tested (Table B in S1 File). In conclusion, the test statistics had a high power to detect small differences in the libraries, and compositional differences were detected in the absence of large structural differences.
K-shuff was also more sensitive to small changes in the library composition than widely used alternative methods. Based upon 50 replicate libraries, the power of K-shuff to detect significant differences between SW and MS libraries with ω = 0.70 was 0.92, or essentially the same as with 1000 replicates. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. In contrast, the powers of LIBSHUFF and UniFrac under the same conditions were 0.32 and 0.16, respectively. Thus, K-shuff was much more sensitive that either of these two commonly used methods.
It can be demonstrated that the expected values of both C kf and I kf do not depend on sample size. However, variation in the means is expected as a consequence of higher sampling variances in the smaller samples. Simulations were performed to determine the effect of the sample size N on the sampling variation in I kf and C kf . Sampling variation in K-shuff decreased with increasing N. For instance, the mean ± standard errors of C kf for comparisons of soil and MS were 0.065 ± 0.015, 0.064 ± 0.010, 0.063 ± 0.008, and 0.063 ± 0.007 for N of 25, 50, 75, and 100, respectively (Fig B in S1 File). This effect was also observed for the I kf , of the soil and marine sediments libraries (Figs C and D in S1 File). For instance, the mean ± standard error of the I kf for soil libraries were 0.268 ± 0.019, 0.275 ± 0.012, 0.277 ± 0.009, and 0.277 ± 0.008 for N of 25, 50, 75, and 100, respectively. An important implication of this result is that the libraries do not have to be the same size to compare these parameters.
If the I kf reflected the structures of libraries, it would also be expected to become more like the highly represented library as the mixing proportion increased. Like the C kf , the relationship between I kf and ω was expected to be complex. First, the relationship between I kf and mixing proportion of the libraries was examined in the seawater and sediment libraries, which were very different. As the mixing proportion increased from 0.5 to 1.0, the I kf of the "seawater" library decreased from 0.227 to 0.181, and the I kf of the "sediment" library increased from 0.227 to 0.305, and there was a clear trend between I kf and ω (Fig F in S1 File). In contrast, as dramatic a trend was not found for the soil and sediment libraries, whose structures were very similar. When the proportion of mixing was 0.5, the I kf was very close to that of the sediment, and the "sediment" library remained close to this value as its proportion increased. The I kf of the "soil' library decreased from 0.304 to 0.277 as its proportion increased to 1.0. Thus, while the expected trends were observed, the changes in I kf were not close to linear with the proportion of each library.
K-shuff performance with Illumina ® dataset
Similar to the Sanger sequences from clone libraries, only small numbers of sequences were needed to calculate the K-shuff functions. For instance, the I kf values calculated for random samples of 100-500 sequences from the cutting board, door trim and pillowcase were very similar (Fig G in S1 File). Additional simulations were then performed using the cutting board (Cb) and pillowcase (Pc) libraries, which appeared to be typical of the diversity within the datasets and a good test of the sensitivity of K-shuff towards structural and composition differences [23]. Similar to the clone libraries, simulations on the reproducibility and effect of sample size on the I kf and C kf found that the mean values were relatively constant but the sampling variation decreased as N increased from 50 to 100. For instance, the mean ± standard deviation of C kf for comparisons between the cutting board (Cb) and pillow case (Pc) libraries were 0.024 ± 0.008, 0.024 ± 0.006 and 0.024 ± 0.005 for N of 50, 75 and 100, respectively (Fig H in S1 File). K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. With the introduction of LIB-SHUFF [1] and other software packages, such as the analysis of molecular variance or AMOVA [2], R -LIBSHUFF [3], DOTUR [4], UniFrac [5], SONS [6], TreeClimber [7], LibraryCompare [8] and Metastats [9], statistical comparisons of communities can now be based on multiple approaches and algorithms. Generally, these comparisons are based on operational taxonomic units (OTUs), which are derived from the sequence datasets using two different approaches: alignment-dependent clustering based on distance cutoffs and an alignment-independent reference based on prior phylotype assignments [10]. While the former is computationally expensive, the latter suffers from the incompleteness of the reference databases, which at times can lead to a major portion of the microbiome left unassigned. Using mock sequence datasets represented in a two-dimensional space of circles and ellipses with known shapes and densities, a systematic evaluation of these tools showed that the current statistical toolbox has the ability to address specific ecological questions concerning the differences among microbial communities [11]. However, these algorithms posed some limitations for complex analyses. For instance, the methods that depend upon assignment of sequences to operational taxonomic units or OTUs require large samples to estimate richness. Similarly, methods that depend upon Monte Carlo testing procedures become computationally expensive when comparing large sequence libraries that are typical of high-throughput technologies. Lastly, there are also limitations in the sensitivity of the algorithms for detecting ecologically relevant differences.
To address these concerns, the utility of a new algorithm for comparison of gene libraries called K-shuff was assessed. K-shuff was designed and implemented in a Fortran program and is motivated by Ripley's K-function [12], which is a powerful statistical tool for spatial point pattern analysis. It uses measures of distances among all pairs of sequences to reflect any aggregation of these sequences as would arise from samples comprised of closely related organisms. So, more information is retained when compared to LIBSHUFF which only uses the distance between each sequence and its nearest neighbor. In contrast to spatial point processes, the distances among gene sequences typically encompass hundreds of dimensions, where each sequence position comprises a separate dimension. Thus, some ideal properties, such as stationarity and isotropy, which are typically assumed in developing K-function theories, are not relevant. However, this limit does not hinder the usefulness of K-functions for this application. As indicated by Diggle et al. [13] and the results of our case studies detailed below, empirical K-functions still render meaningful scientific interpretations.
In this subject, we perform a systematic evaluation of the reproducibility, sensitivity and power of K-shuff, using previously reported 16S rRNA gene sequence datasets obtained using the Sanger method and pyrosequencing and Illumina 1 platforms. In addition, we compare its performance with LIBSHUFF, the first statistical tool for this purpose, as well as the most frequently used tool today, UniFrac. We show that when correctly employed, K-shuff allows testing of multiple hypotheses in a single process and has the potential to significantly improve our understanding of ecologically meaningful aspects of microbial communities.
Materials and Methods
The K-shuff Metric K-shuff identifies spatial clustering based on the reduced second moment measure, or K-function [14,15]. In general, the K-function is defined based on a distance measure, which in this context is set to be the evolutionary distance between the gene sequences. Denoting the evolutionary distance between sequences i and j by d ij , the empirical K-function for a library of sequences of size N is:
Iðd ij rÞ;
where I(E) is an indicator function (= 1, when E is true; = 0, if otherwise), and r is any positive number on the real line of evolutionary distances. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. In our studies, UniFrac proved even less sensitive than LIB-SHUFF, presumably because the process of generating phylogenetic trees greatly reduces the information content of the similarity matrices. Thus, K-shuff gives a more realistic assessment of differences in the communities.
Because of its high sensitivity, K-shuff is especially informative with relatively small libraries, such as those generated by Sanger sequencing. However, even for NexGen libraries, which typically include thousands or millions of sequences, K-shuff may provide valuable insights. Its high sensitivity makes it possible to analyze small subsets of large datasets that might be particularly interesting, such as moderately abundant OTUs. It would also be suitable for examining large numbers of samples generated by multiplexing, which also yield small numbers of sequences for each library [28,29,30]. Because sample size has only a small effect on either I kf or C kf values, it is also practical to test subsamples of very large libraries, which is computationally much less expensive.
Similar to the most frequently used phylogenetic metric UniFrac [5], K-shuff also produces an estimate of distance between the communities that is suitable for ANOVA and clustering analyses. However, C kf , the distance estimate of K-shuff, gives equal weight to both closely related as well as distantly related organisms, thereby allowing a unbiased estimation of the community composition. In contrast, UniFrac gives higher weight to differences arising from distantly related organisms [31]. Another concern with UniFrac is that the distance estimates are dramatically nonlinear. For instance, in mixing simulations of seawater and salt marsh sediment libraries, the UniFrac distances were 0.08, 0.81 and 0.86 at mixing proportions of 0.5, 0.7 and 1, respectively, and were not closely related to the differences in composition of the libraries. In contrast, the C kf values of K-shuff were 0.015, 0.049 and 0.121 at the same mixing proportions. Thus, K-shuff is more reflective of the differences in community composition. I kf as a novel estimate of structural diversity K-shuff provides I kf , which is a novel estimate of the structural diversity that reflects both the relatedness among OTUs or phylogenetic diversity as well as their relative abundances. Thus, a community comprised of many closely related sequences would have a smaller I kf than a community with same numbers of OTUs but comprised of distantly related sequences. Essentially this would not lead to change in memberships for an OTU thereby maintaining both the evenness and richness of the two communities. Further, because many commonly used indices, such as Shannon, Simpson, and Chao 1 are not a measure of phylogenetic diversity, their values would only change with changing richness, evenness or a larger sample size [32]. In addition, these estimates are biased towards richness (Shannon), evenness or dominance (Simpson), or lose information by reducing the data through clustering to obtain OTU abundances for determining singletons and doubletons (Chao 1) [32]. Because abundant OTUs dominate the IKF, the I kf is not very sensitive to the presence of rare OTUs or the 'long tails' frequently observed in microbial communities. Thus, while it is intuitively satisfying to capture multiple sources of diversity in a single value, the complex calculation of I kf makes it difficult to attribute differences to a single source, such as OTU richness or phylogenetic diversity.
As a result, the value of the I kf is difficult to assess without examining more applications. In the cases examined here, K-shuff analyses appeared to provide useful insights into the diversity of the microbial communities of cropland, pasture and forest soils as well as household surfaces. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science
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K-shuff: A Novel Algorithm for Characterizing Structural and Compositional Diversity in Gene Libraries
K-shuff is a new algorithm for comparing the similarity of gene sequence libraries, providing measures of the structural and compositional diversity as well as the significance of the differences between these measures. Using 100 sequences per sample for all 174 samples, the I kf were calculated for comparison to the OTU richness estimated by Dunn et al. [23]. Within each site, the OTU richness varied 3-10-fold, and the mean OTU richness of different sites varied nearly four-fold. In contrast, the I kf values within each site varied less than 3-fold, and the mean I kf s were relatively constant, varying only from 0.15-0.21 (Fig N in S1 File). The differences in OTU richness and I kf s would be expected if much of the variation was due the presence or absence of rare OTUs, which have little effect on the I kf . Moreover, I kf s of about 0.20 resembled those of seawater, which is consistent with the low diversity expected of the microbiome of surfaces. Thus, the K-shuff analyses suggested that the diversity of the various household surfaces were fairly similar, which was not readily apparent from examination of only OTU richness. [22]. For this analysis, 300 sequences were randomly selected from each replicate and pooled, yielding 1500 sequences for each age in the chronosequence. Similarly, PCoA plot of the variation among sites within the homes using the C kf of K-shuff was compared to the original analysis using the UniFrac distances by Dunn et al. [23] (Fig 7). The MRPP of the compositional parameter (C kf ) matrix from the household bacterial communities was significantly different (α<0.01), with clusters represented by PCoA. Each of the bacterial communities from different household habitats were patterned along two axes explaining 75.9 (Axis 1) and 13.2% (Axis 2) of the variance. A measure of the effect size based on a chance corrected within group-agreement test (A = 0.2) indicated that heterogeneity between groups was greater than that within groups. These results are independent of sample size and thus help to support the veracity of the p-values. Likewise, the MDS plot of the C kf showed similar clustering as the PCoA (Fig O in S1 File). Overall, these results indicated that ordination using the C kf matrix explained a much greater proportion of the variance than did analysis using UniFrac.
To utilize the extra sensitivity afforded by K-shuff, analyses were performed on some of the abundant OTUs to explore differences within the members of an OTU across the nine sites. At D = 0.14, a total of 4664 OTUs were found for the entire subsampled dataset (N = 17366), with the largest OTU (OTU1 0.14 ) comprising 4271 sequences affiliated with the Firmicutes. Based on MDS analysis of the C kf s, OTU1 0.14 showed a similar clustering pattern as the entire subsampled dataset (Fig 8A and 8B). Communities from sites Dh, Di and Tv were tightly clustered whereas Fr, Kc, Cb and Ts, Pc were spread across the plot in two loose clusters. K-shuff will therefore prove beneficial for conventional microbiome analysis as well as specific hypothesis testing.
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Domain: Biology Medicine Computer Science<|endoftext|>Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Ice worms were collected from two distinct glaciers in Alaska, Harding Icefield and Byron Glacier, and glacier surfaces were also sampled for comparison. Marked differences were observed in bacterial community structures between the ice worm and glacier surface samples. Several bacterial phylotypes were detected almost exclusively in the ice worms, and these bacteria were phylogenetically affiliated with either animal-associated lineages or, interestingly, clades mostly consisting of glacier-indigenous species. The former included bacteria that belong to Mollicutes, Chlamydiae, Rickettsiales, and Lachnospiraceae, while the latter included Arcicella and Herminiimonas phylotypes. Among these bacteria enriched in ice worm samples, Mollicutes, Arcicella, and Herminiimonas phylotypes were abundantly and consistently detected in the ice worm samples; these phylotypes constituted the core microbiota associated with the ice worm. A fluorescence in situ hybridization analysis showed that Arcicella cells specifically colonized the epidermis of the ice worms. Other bacterial phylotypes detected in the ice worm samples were also abundantly recovered from the respective habitat glaciers; these bacteria may be food for ice worms to digest or temporary residents. Nevertheless, some were overrepresented in the ice worm RNA samples; they may also function as facultative gut bacteria. Our results indicate that the community structure of bacteria associated with ice worms is distinct from that in the associated glacier and includes worm-specific and facultative, glacier-indigenous lineages.
Glaciers, which are harsh environments that are permanently covered with ice and snow, have unique ecosystems composed of psychrophilic/psychrotolerant organisms (3,33). Recent studies revealed that microorganisms thriving on glacial surfaces have a marked impact on global biogeochemical cycles and surface ice melting (2,34,37,40). Although the importance of glacier ecosystems has been recognized and many glaciers have been examined, most studies have focused on microorganisms on the glacier surface, and knowledge on glacier invertebrates is limited.
The glacier ice worm, Mesenchytraeus solifugus (phylum Annelida; family Enchytraeidae), is the largest metazoan and regarded as the predominant consumer in North American maritime glaciers (17,35). Ice worms are nocturnal in summer seasons; they reside deep beneath the glacier surface in the daytime to avoid sunlight, and emerge at dusk on the surface to forage food, which mainly consists of unicellular green algae (17,35). Their behaviors in winter seasons are unknown. Their physiology and phylogeny have also been investigated (9,14,18,38); however, limited information is available on their ecological roles in glacial environments. In our previous study, the bacterial community structures physically associ-ated with the ice worm were analyzed by cloning bacterial 16S rRNA genes (27). The findings obtained revealed that the ice worm harbors glacier-derived and animal gut-specific bacterial lineages, and that the novel Mollicutes bacterium, "Candidatus Vermiplasma glacialis," colonizes the gut wall of ice worms as a dominant bacterial member. However, the number of analyzed specimens and sequences were limited; therefore, more comprehensive surveys are needed in order to obtain a deeper understanding of the community structure of bacteria associated with ice worms.
In the exhibit survey, we aimed to clarify the taxonomic composition of the bacterial microbiota associated with the ice worm in more detail by deep sequencing 16S rRNA gene amplicons. In order to identify the core symbiotic microbiota, if one exists, ice worms were collected from two distinct glaciers in Alaska, Harding Icefield and Byron Glacier, and their associated bacterial communities were compared. The 16S rRNA gene and its transcripts were simultaneously analyzed to assess the activity of each bacterial species. Furthermore, bacterial community structures from the glacier surfaces were examined, and the specificity of the bacterial species associated with ice worms was evaluated. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine<|endoftext|>Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Due to the harsh survival conditions of the ice worm, its populations have been geographically isolated among habitat glaciers (9, 10). Thus, bacterial lineages that specifically inhabit the ice worm body might also be isolated and exhibit different taxonomic compositions in the respective glaciers.
Conclusion
The results of the present study strongly suggest that not only animal-associated bacterial lineages, such as Mollicutes, Rickettsiales, Parachlamydiaceae, and Lachnospiraceae, but also glacier-indigenous bacteria, including Arcicella and Herminiimonas, benefit from the presence of the ice worm. The body surface and intestinal tract of the ice worm appear to provide a unique habitat, which is potentially rich with nutrients, to microbes in glacier ecosystems with limited resources, thereby driving the formation of a worm-associated bacterial microbiota that is distinct from that in glaciers. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine
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Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Arcicella and Herminiimonas are both aerobic and heterotrophic bacteria; the former utilize various carbohydrates (7) and the latter utilize organic acids as carbon sources (15,20). Arcicella species potentially feed on mucus and excrement secreted by the ice worm, and Herminiimonas species, although their localization remains unknown, may incorporate metabolites produced by the host or other bacterial species.
Marked differences in OTU compositions between the Harding and Byron ice worm samples were largely attributable to differences in bacterial community structures between the Harding and Byron glacier surfaces ( Fig. 2 and 3), which may be caused by environmental differences between these two glaciers. For example, the sampling sites in Harding were around the center of the large icefield, in which any influence from the shore should be minimal, whereas those in Byron were located in a small avalanche cone, which was contiguous with soil and covered with plant fragments possibly derived from shore vegetation. These locational differences may lead to different physicochemical and nutritional conditions, which have a marked impact on the bacterial microbiota, as reported in previous studies (25,36). This is analogous to findings obtained in terrestrial earthworms; the bacterial microbiota in the earthworm gut changes based on the soil used as food (41). In addition, several OTUs specific to the ice worm, such as Mollicutes OTU23265 and OTU11203, Lachnospiraceae OTU742, Parachlamydiaceae OTU9924, and Brevinema OTU21396, also exhibited differential compositional patterns between the Harding and Byron worms (Fig. 1, 3, and Fig. S3). This might reflect the isolation of ice worm populations. Due to the harsh survival conditions of the ice worm, its populations have been geographically isolated among habitat glaciers (9, 10). Thus, bacterial lineages that specifically inhabit the ice worm body might also be isolated and exhibit different taxonomic compositions in the respective glaciers.
Conclusion
The results of the present study strongly suggest that not only animal-associated bacterial lineages, such as Mollicutes, Rickettsiales, Parachlamydiaceae, and Lachnospiraceae, but also glacier-indigenous bacteria, including Arcicella and Herminiimonas, benefit from the presence of the ice worm. The body surface and intestinal tract of the ice worm appear to provide a unique habitat, which is potentially rich with nutrients, to microbes in glacier ecosystems with limited resources, thereby driving the formation of a worm-associated bacterial microbiota that is distinct from that in glaciers. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine<|endoftext|>Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. In addition, two and three sets of the glacier surface samples from Harding and Byron (designated as Harding surface/Byron surface), respectively, were similarly analyzed. In total, 4,208 OTUs were identified from 1,965,105 paired-end reads. The mean number (with SD) of the observed OTUs in each sample category was as follows; 79±16 (Harding worm DNA), 546±350 (Harding worm RNA), 101±44 (Byron worm DNA), 1,105±583 (Byron worm RNA), 230±160 (Harding surface DNA), 757±297 (Harding surface RNA), 116±30 (Byron surface DNA), and 810±274 (Byron surface RNA). Thus, the observed number of OTUs as well as the Chao1 species richness estimate and reciprocal Simpson's diversity index were higher in RNA samples than in DNA samples in any of the worm or glacier surface samples (Table S1). The rank abundance curves of the OTUs showed that RNA samples contained much rare OTUs than DNA samples (Fig. S1).
The taxonomic composition of bacteria was basically consistent among the biological replicates for each sample category (Fig. 1). In contrast, marked differences were observed among the different sample categories. For example, Entomoplasmatales and Mycoplasmataceae sequences were abundantly detected in ice worm samples, whereas only a few were recovered from the glacier surface samples. Most of the Mycoplasmataceae OTUs in the ice worm samples were identical or closely related to the "Ca. Vermiplasma" sequences obtained in our previous study (27), and differed from OTU13756, which accounted for 1.6% of sequences in the "Harding surface DNA 2" sample ( Fig. 1). The OTU13756 sequence was identical to the sequences of two Mycoplasmataceae bacteria in the NCBI nr/nt database: the hemotrophic cattle pathogen "Candidatus Mycoplasma haemobos" (KJ883514) (19) and uncultured bacterium clone GYs3-84 (JX493242) obtained from soil in China (44). Entomoplasmatales OTUs were also identical or closely related to the phylotypes obtained in our previous study (27). Lachnospiraceae OTUs were exclusively recovered from the Harding worm samples. The closest relative of the dominant Lachnospiraceae OTU742 was a sequence obtained from a termite gut sample (AB089001) with 93.3% sequence similarity. Cytophagaceae, predominated by the genus Arcicella (25 out of 85 Cytophagaceae OTUs and 91.8% of the total Cytophagaceae reads), were frequently detected in both worm and glacier surface samples, but were more abundant in the former, particularly in Byron worms (Fig. 1).
Core microbiota associated with ice worms
In order to identify dominant bacterial OTUs that were consistently associated with ice worms, we first listed OTUs for which the frequency in a sample was >0.1%. These OTUs were then sorted into four groups: (i) OTUs dominant in both Harding and Byron worm samples (designated as "core OTUs"); (ii) OTUs dominant in Harding worm samples only; (iii) OTUs dominant in Byron worm samples only; (iv) other dominant OTUs, which did not fit into groups (i) to (iii), but were included in the 10 most abundant OTUs (see the legend to Fig. 2) (Fig. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine
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This below document has 2 sentences that end with 'glacier surface samples',
2 sentences that end with 'RNA samples (Fig',
2 sentences that end with 'in RNA samples'. It has approximately 465 words, 26 sentences, and 7 paragraph(s).
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Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. 3).
The majority of OTUs that were only dominant in either the Harding or Byron worm samples were also abundantly recovered from their corresponding habitat glacier surface samples, but were less abundantly detected in the other glacier surface samples (Fig. 3). However, several OTUs, such as OTU742 (Lachnospiraceae), OTU9924 (Parachlamydiaceae), OTU3927 (Entomoplasmatales), and OTU18119 (Rickettsiales), were specifically detected in either the Harding or Byron worm samples, but were almost never found in any of the glacier surface samples. Parachlamydiaceae OTU9924 showed the highest sequence similarity (92.5%) to "Candidatus Metachlamydia lacustris" (GQ221847), which is a parasite of the aquatic amoeba, Saccamoeba lacustris (8). OTU18199 belonged to the uncultured Rickettsiales clade LWSR-14 in the SILVA v111 database, but shared only 84.3% sequence identity with its closest relative in the database, Sphingorhabdus marina (KT899836) isolated from Ny-Alesund, Svalbard. OTU3927 was closest to the Entomoplasmatales core OTU, OTU23265.
Differences in taxonomic compositions between DNA and RNA samples
An analysis using DESeq2 indicated that 62 out of 1,114 OTUs and 217 out of 2,878 OTUs in Harding and Byron worm samples, respectively, showed significant differences in their abundance between DNA and RNA samples (Fig. 3). For example, three Mollicutes OTUs (OTU23265, OTU11203, and OTU3927) were significantly more abundant in DNA samples than in RNA samples. Conversely, several glacierindigenous OTUs of Alphaproteobacteria, Betaproteobacteria, and Planctomycetacia, including OTU11712 and OTU20203 (Acetobacteraceae), OTU8825 (Comamonadaceae), OTU15614 (Methylophilaceae), and OTU25211 (Planctomycetaceae), were more abundantly detected in RNA samples. The dominant Lachnospiraceae OTU742 was also significantly more abundant in RNA samples (Fig. 3).
We examined the in situ localization of Arcicella OTU cells, which were phylogenetically affiliated with a glacierindigenous lineage, but greatly enriched in the ice worm samples (Fig. 3). A FISH analysis revealed that Arcicella cells specifically colonized in clusters on the outer surface of the epidermis of Harding and Byron worms (Fig. 4).
The deep sequencing of 16S rRNA genes and their transcripts clearly revealed specific and facultative relationships between bacteria and glacier ice worms. Significant differences were observed in bacterial community structures between ice worms and their associated glacier surface samples. In particular, several OTUs were almost exclusively detected in the ice worm samples. Among them, the "Ca. Vermiplasma" and Lachnospiraceae members belong to lineages specific to animal intestinal tracts (27). Although the physiology of "Ca. Vermiplasma" currently remains unknown, Lachnospiraceae bacteria reportedly participate in polysaccharide fermentation in the gut of terrestrial earthworms (30,32). Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine<|endoftext|>Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Comparative genomics of Clostridiales families (Lachnospiraceae, Ruminococcaceae, and Clostridiaceae) also indicated that Lachnospiraceae and Ruminococcaceae bacteria in the mammalian gut are more specialized for the degradation of plant-derived recalcitrant substrates, such as cellulose, than Clostridiaceae bacteria (4). Since algal cells are one of the main food sources of ice worms (17,27), Lachnospiraceae members may play an important role in the digestion of algal polysaccharides in the ice worm gut.
Several Rickettsiales and Parachlamydiaceae OTUs were also almost exclusively detected in the ice worm samples. These OTUs are phylogenetically related to parasites or endosymbionts of eukaryotes, including algae, ciliates, and amoebas. These OTUs may have been derived from intracellular bacteria that were hosted by ingested eukaryotic organisms, such as green algae and ciliates. However, the frequency of the chloroplast 16S rRNA gene or transcript sequences, which should represent the abundance of food-derived sequences, did not markedly differ between the ice worm and glacier surface samples (Table S2). Therefore, it is more likely that Rickettsiales and Parachlamydiaceae members specifically inhabit the ice worm body, although the localization of these bacteria remains unknown. Alternatively, these bacteria might proliferate in the ice worm body by infection from ingested eukaryotic organisms; it has been reported that certain members of the Rickettsiales or Parachlamydiaceae can be horizontally transferred from their original hosts to phylogenetically distinct host organisms by ingestion or mucosal infection (26,39,43).
It is notable that Arcicella OTU19485 and Herminiimonas OTU17386, which belong to glacier-indigenous lineages, were greatly enriched in the ice worm samples. Indeed, the colonization of Arcicella cells was observed on the surface of the ice worm body (Fig. 4). These upshots argue that glacier-indigenous bacteria can form tight associations with ice worms, and also suggest that these bacteria have been specialized to associate with ice worms. Arcicella and Herminiimonas are both aerobic and heterotrophic bacteria; the former utilize various carbohydrates (7) and the latter utilize organic acids as carbon sources (15,20). Arcicella species potentially feed on mucus and excrement secreted by the ice worm, and Herminiimonas species, although their localization remains unknown, may incorporate metabolites produced by the host or other bacterial species.
Marked differences in OTU compositions between the Harding and Byron ice worm samples were largely attributable to differences in bacterial community structures between the Harding and Byron glacier surfaces ( Fig. 2 and 3), which may be caused by environmental differences between these two glaciers. For example, the sampling sites in Harding were around the center of the large icefield, in which any influence from the shore should be minimal, whereas those in Byron were located in a small avalanche cone, which was contiguous with soil and covered with plant fragments possibly derived from shore vegetation. These locational differences may lead to different physicochemical and nutritional conditions, which have a marked impact on the bacterial microbiota, as reported in previous studies (25,36). This is analogous to findings obtained in terrestrial earthworms; the bacterial microbiota in the earthworm gut changes based on the soil used as food (41). In addition, several OTUs specific to the ice worm, such as Mollicutes OTU23265 and OTU11203, Lachnospiraceae OTU742, Parachlamydiaceae OTU9924, and Brevinema OTU21396, also exhibited differential compositional patterns between the Harding and Byron worms (Fig. 1, 3, and Fig. S3). This might reflect the isolation of ice worm populations. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine
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Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Our results provide an insight into the unique niche occupied by ice worms with their associated bacterial consortium in glacial ecosystems.
Sample collection
Sampling expeditions were carried out in the upper area of Exit Glacier located in Harding Icefield (August 2014) and in Byron Glacier (August 2015). Both glaciers are located in the Kenai Peninsula, Alaska, and are approximately 90 km apart. In Harding Icefield, sampling was conducted at two sites; an ice surface site (60°09.622'N, 149°47.482'W) and snowpack site (60°09.167'N, 149°46.795'W) (Table S1). In Byron Glacier, samples were collected from the ice surfaces of an avalanche cone (60°45.672'N, 148°50.89'W) below the terminus of the glacier. Ice worms and glacier surface samples were collected with sterile stainless steel scoops. All ice worm samples were collected at dusk when ice worms started to appear on the surface of the glaciers. Specimens were preserved on site in RNAlater ® solution (Ambion, Austin, TX, USA). Some collected ice worms were kept alive in melted glacier surface ice. All fixed samples were kept frozen with ice during transportation to a laboratory in Japan. Specimens preserved in RNAlater ® were stored at -80°C, and live ice worms were reared at 4°C.
Nucleic acid extraction
PCR, RT-PCR, and amplicon sequencing
RNA samples were reverse-transcribed into cDNA using the SuperScript ® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) with random hexamers. PCR was conducted using Phusion ® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) with the Bacteria-specific 341F and 785R primer set, which targets the V3-V4 region (approx. 400 bp) of 16S rRNA. Primer sequences contained the Illumina overhang adapter sequence ( [URL]_ sequencing_library_preparation.html). The PCR program was as follows: initial denaturation at 98°C for 30 s, 25 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and a final extension at 72°C for 7 min. Nextera ® XT indices (Illumina, San Diego, CA, USA) were attached to the amplicons by eight additional PCR cycles. After purification with the Agencourt ® AMPure ® XP (Beckman Coulter, Brea, CA, USA), DNA was quantified with the Qubit ® dsDNA High Sensitivity Assay Kit (Invitrogen). Sequencing was conducted on the Illumina MiSeq TM platform with MiSeq reagent kit v3.
Quality check of sequence reads and assignment to operational taxonomic units (OTUs)
Paired-end sequence reads were merged, primer trimmed, and quality filtered using the program package USEARCH v8.0 (12). Quality filtering was performed using the fastq filter command with the following parameters: minimum length=350 bp, no ambiguous bases allowed, and maximum expected errors for all bases in a read were less than 1. The quality of reads was evaluated using FastQC ( [URL] rRNA sequences were sorted into OTUs with a criterion of 97% sequence identity using the program UCLUST implemented in the QIIME v1.8.0 package with the furthest-neighbor clustering algorithm (6,12). Chimeric sequences were identified using UCHIME (13), and singletons were also eliminated from subsequent analyses. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine<|endoftext|>Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. The taxonomic assignment of OTUs was performed with the taxonomic classification service implemented in the SINA Alignment Service ( [URL]/) using the SILVA 126 database with default parameter settings, except for the minimum identity with the query sequence, set to 0.80. OTUs assigned to Eukarya, Archaea, mitochondria, chloroplasts, and "unclassified" were removed. We also removed OTUs affiliated with the genera Halomonas and Shewanella; we concluded that these two bacterial groups were reagent contaminants because they commonly appeared in distinct samples, including those not related to this study (data not shown). We further eliminated OTUs that could not be aligned with other 16S rRNA sequences.
Statistical analysis
The similarity of bacterial community structures was evaluated with QIIME using nonmetric multidimensional scaling (NMDS) based on the Bray-Curtis similarity index. Differences among clusters were statistically tested by an analysis of similarity (ANOSIM) with 999 permutations. The separation of clusters in the NMDS plot was correlated to OTU abundance by calculating Spearman's correlation coefficient with mothur v1.37.0 (29).
In each of the 16S rRNA OTUs recovered from the ice worm samples, differences in abundance between DNA and RNA samples were statistically tested in R v3.2.2 using the DESeq2 package with the negative binomial Wald test (23). If the adjusted P value was less than 0.05, we considered the OTU abundance to be significantly different between DNA and RNA samples.
Phylogenetic analysis
Nearly full-length 16S rRNA gene sequences obtained in our previous study (27) were aligned using ARB (24) with manual corrections. In total, 1,312 (for Arcicella) and 1,218 (for Herminiimonas) unambiguously aligned nucleotide sites, which corresponded to positions 69-1440 and 94-1390 in Escherichia coli (J01695), respectively, were used for a phylogenetic analysis. Maximum likelihood (ML) and neighbor-joining (NJ) trees were constructed in MEGA7 (22) with 500 bootstrap resamplings. The GTR+Γ+I nucleotide substitution model and maximum composite likelihood model were used for ML and NJ tree reconstructions, respectively.
Nucleotide sequence accession numbers
Datasets of 16S rRNA reads sequenced in this study have been deposited in DDBJ/EMBL/GenBank under the accession number DRA005157.
Taxonomic composition and species richness of bacteria
Three and six sets of the ice worm samples, each set containing five individual worms, from Harding and Byron (designated as Harding worm/Byron worm), respectively, were subjected to 16S rRNA amplicon sequencing. In addition, two and three sets of the glacier surface samples from Harding and Byron (designated as Harding surface/Byron surface), respectively, were similarly analyzed. In total, 4,208 OTUs were identified from 1,965,105 paired-end reads. The mean number (with SD) of the observed OTUs in each sample category was as follows; 79±16 (Harding worm DNA), 546±350 (Harding worm RNA), 101±44 (Byron worm DNA), 1,105±583 (Byron worm RNA), 230±160 (Harding surface DNA), 757±297 (Harding surface RNA), 116±30 (Byron surface DNA), and 810±274 (Byron surface RNA). Thus, the observed number of OTUs as well as the Chao1 species richness estimate and reciprocal Simpson's diversity index were higher in RNA samples than in DNA samples in any of the worm or glacier surface samples (Table S1). The rank abundance curves of the OTUs showed that RNA samples contained much rare OTUs than DNA samples (Fig. S1).
The taxonomic composition of bacteria was basically consistent among the biological replicates for each sample category (Fig. 1). In contrast, marked differences were observed among the different sample categories. For example, Entomoplasmatales and Mycoplasmataceae sequences were abundantly detected in ice worm samples, whereas only a few were recovered from the glacier surface samples. Most of the Mycoplasmataceae OTUs in the ice worm samples were identical or closely related to the "Ca. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine
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Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Although the importance of glacier ecosystems has been recognized and many glaciers have been examined, most studies have focused on microorganisms on the glacier surface, and knowledge on glacier invertebrates is limited.
The glacier ice worm, Mesenchytraeus solifugus (phylum Annelida; family Enchytraeidae), is the largest metazoan and regarded as the predominant consumer in North American maritime glaciers (17,35). Ice worms are nocturnal in summer seasons; they reside deep beneath the glacier surface in the daytime to avoid sunlight, and emerge at dusk on the surface to forage food, which mainly consists of unicellular green algae (17,35). Their behaviors in winter seasons are unknown. Their physiology and phylogeny have also been investigated (9,14,18,38); however, limited information is available on their ecological roles in glacial environments. In our previous study, the bacterial community structures physically associ-ated with the ice worm were analyzed by cloning bacterial 16S rRNA genes (27). The findings obtained revealed that the ice worm harbors glacier-derived and animal gut-specific bacterial lineages, and that the novel Mollicutes bacterium, "Candidatus Vermiplasma glacialis," colonizes the gut wall of ice worms as a dominant bacterial member. However, the number of analyzed specimens and sequences were limited; therefore, more comprehensive surveys are needed in order to obtain a deeper understanding of the community structure of bacteria associated with ice worms.
In the exhibit survey, we aimed to clarify the taxonomic composition of the bacterial microbiota associated with the ice worm in more detail by deep sequencing 16S rRNA gene amplicons. In order to identify the core symbiotic microbiota, if one exists, ice worms were collected from two distinct glaciers in Alaska, Harding Icefield and Byron Glacier, and their associated bacterial communities were compared. The 16S rRNA gene and its transcripts were simultaneously analyzed to assess the activity of each bacterial species. Furthermore, bacterial community structures from the glacier surfaces were examined, and the specificity of the bacterial species associated with ice worms was evaluated. Our results provide an insight into the unique niche occupied by ice worms with their associated bacterial consortium in glacial ecosystems.
Sample collection
Sampling expeditions were carried out in the upper area of Exit Glacier located in Harding Icefield (August 2014) and in Byron Glacier (August 2015). Both glaciers are located in the Kenai Peninsula, Alaska, and are approximately 90 km apart. In Harding Icefield, sampling was conducted at two sites; an ice surface site (60°09.622'N, 149°47.482'W) and snowpack site (60°09.167'N, 149°46.795'W) (Table S1). In Byron Glacier, samples were collected from the ice surfaces of an avalanche cone (60°45.672'N, 148°50.89'W) below the terminus of the glacier. Ice worms and glacier surface samples were collected with sterile stainless steel scoops. All ice worm samples were collected at dusk when ice worms started to appear on the surface of the glaciers. Specimens were preserved on site in RNAlater ® solution (Ambion, Austin, TX, USA). Some collected ice worms were kept alive in melted glacier surface ice. All fixed samples were kept frozen with ice during transportation to a laboratory in Japan. Specimens preserved in RNAlater ® were stored at -80°C, and live ice worms were reared at 4°C.
Nucleic acid extraction
PCR, RT-PCR, and amplicon sequencing
RNA samples were reverse-transcribed into cDNA using the SuperScript ® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) with random hexamers. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine<|endoftext|>Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. Vermiplasma" sequences obtained in our previous study (27), and differed from OTU13756, which accounted for 1.6% of sequences in the "Harding surface DNA 2" sample ( Fig. 1). The OTU13756 sequence was identical to the sequences of two Mycoplasmataceae bacteria in the NCBI nr/nt database: the hemotrophic cattle pathogen "Candidatus Mycoplasma haemobos" (KJ883514) (19) and uncultured bacterium clone GYs3-84 (JX493242) obtained from soil in China (44). Entomoplasmatales OTUs were also identical or closely related to the phylotypes obtained in our previous study (27). Lachnospiraceae OTUs were exclusively recovered from the Harding worm samples. The closest relative of the dominant Lachnospiraceae OTU742 was a sequence obtained from a termite gut sample (AB089001) with 93.3% sequence similarity. Cytophagaceae, predominated by the genus Arcicella (25 out of 85 Cytophagaceae OTUs and 91.8% of the total Cytophagaceae reads), were frequently detected in both worm and glacier surface samples, but were more abundant in the former, particularly in Byron worms (Fig. 1).
Core microbiota associated with ice worms
In order to identify dominant bacterial OTUs that were consistently associated with ice worms, we first listed OTUs for which the frequency in a sample was >0.1%. These OTUs were then sorted into four groups: (i) OTUs dominant in both Harding and Byron worm samples (designated as "core OTUs"); (ii) OTUs dominant in Harding worm samples only; (iii) OTUs dominant in Byron worm samples only; (iv) other dominant OTUs, which did not fit into groups (i) to (iii), but were included in the 10 most abundant OTUs (see the legend to Fig. 2) (Fig. 3).
The majority of OTUs that were only dominant in either the Harding or Byron worm samples were also abundantly recovered from their corresponding habitat glacier surface samples, but were less abundantly detected in the other glacier surface samples (Fig. 3). However, several OTUs, such as OTU742 (Lachnospiraceae), OTU9924 (Parachlamydiaceae), OTU3927 (Entomoplasmatales), and OTU18119 (Rickettsiales), were specifically detected in either the Harding or Byron worm samples, but were almost never found in any of the glacier surface samples. Parachlamydiaceae OTU9924 showed the highest sequence similarity (92.5%) to "Candidatus Metachlamydia lacustris" (GQ221847), which is a parasite of the aquatic amoeba, Saccamoeba lacustris (8). OTU18199 belonged to the uncultured Rickettsiales clade LWSR-14 in the SILVA v111 database, but shared only 84.3% sequence identity with its closest relative in the database, Sphingorhabdus marina (KT899836) isolated from Ny-Alesund, Svalbard. OTU3927 was closest to the Entomoplasmatales core OTU, OTU23265.
Differences in taxonomic compositions between DNA and RNA samples
An analysis using DESeq2 indicated that 62 out of 1,114 OTUs and 217 out of 2,878 OTUs in Harding and Byron worm samples, respectively, showed significant differences in their abundance between DNA and RNA samples (Fig. 3). For example, three Mollicutes OTUs (OTU23265, OTU11203, and OTU3927) were significantly more abundant in DNA samples than in RNA samples. Conversely, several glacierindigenous OTUs of Alphaproteobacteria, Betaproteobacteria, and Planctomycetacia, including OTU11712 and OTU20203 (Acetobacteraceae), OTU8825 (Comamonadaceae), OTU15614 (Methylophilaceae), and OTU25211 (Planctomycetaceae), were more abundantly detected in RNA samples. The dominant Lachnospiraceae OTU742 was also significantly more abundant in RNA samples (Fig. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
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Domain: Biology Environmental Science Medicine
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Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. 3).
We examined the in situ localization of Arcicella OTU cells, which were phylogenetically affiliated with a glacierindigenous lineage, but greatly enriched in the ice worm samples (Fig. 3). A FISH analysis revealed that Arcicella cells specifically colonized in clusters on the outer surface of the epidermis of Harding and Byron worms (Fig. 4).
The deep sequencing of 16S rRNA genes and their transcripts clearly revealed specific and facultative relationships between bacteria and glacier ice worms. Significant differences were observed in bacterial community structures between ice worms and their associated glacier surface samples. In particular, several OTUs were almost exclusively detected in the ice worm samples. Among them, the "Ca. Vermiplasma" and Lachnospiraceae members belong to lineages specific to animal intestinal tracts (27). Although the physiology of "Ca. Vermiplasma" currently remains unknown, Lachnospiraceae bacteria reportedly participate in polysaccharide fermentation in the gut of terrestrial earthworms (30,32). Comparative genomics of Clostridiales families (Lachnospiraceae, Ruminococcaceae, and Clostridiaceae) also indicated that Lachnospiraceae and Ruminococcaceae bacteria in the mammalian gut are more specialized for the degradation of plant-derived recalcitrant substrates, such as cellulose, than Clostridiaceae bacteria (4). Since algal cells are one of the main food sources of ice worms (17,27), Lachnospiraceae members may play an important role in the digestion of algal polysaccharides in the ice worm gut.
Several Rickettsiales and Parachlamydiaceae OTUs were also almost exclusively detected in the ice worm samples. These OTUs are phylogenetically related to parasites or endosymbionts of eukaryotes, including algae, ciliates, and amoebas. These OTUs may have been derived from intracellular bacteria that were hosted by ingested eukaryotic organisms, such as green algae and ciliates. However, the frequency of the chloroplast 16S rRNA gene or transcript sequences, which should represent the abundance of food-derived sequences, did not markedly differ between the ice worm and glacier surface samples (Table S2). Therefore, it is more likely that Rickettsiales and Parachlamydiaceae members specifically inhabit the ice worm body, although the localization of these bacteria remains unknown. Alternatively, these bacteria might proliferate in the ice worm body by infection from ingested eukaryotic organisms; it has been reported that certain members of the Rickettsiales or Parachlamydiaceae can be horizontally transferred from their original hosts to phylogenetically distinct host organisms by ingestion or mucosal infection (26,39,43).
It is notable that Arcicella OTU19485 and Herminiimonas OTU17386, which belong to glacier-indigenous lineages, were greatly enriched in the ice worm samples. Indeed, the colonization of Arcicella cells was observed on the surface of the ice worm body (Fig. 4). These upshots argue that glacier-indigenous bacteria can form tight associations with ice worms, and also suggest that these bacteria have been specialized to associate with ice worms. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
==
Domain: Biology Environmental Science Medicine<|endoftext|>Bacterial Microbiota Associated with the Glacier Ice Worm Is Dominated by Both Worm-Specific and Glacier-Derived Facultative Lineages
The community structure of bacteria associated with the glacier ice worm Mesenchytraeus solifugus was analyzed by amplicon sequencing of 16S rRNA genes and their transcripts. PCR was conducted using Phusion ® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) with the Bacteria-specific 341F and 785R primer set, which targets the V3-V4 region (approx. 400 bp) of 16S rRNA. Primer sequences contained the Illumina overhang adapter sequence ( [URL]_ sequencing_library_preparation.html). The PCR program was as follows: initial denaturation at 98°C for 30 s, 25 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, and a final extension at 72°C for 7 min. Nextera ® XT indices (Illumina, San Diego, CA, USA) were attached to the amplicons by eight additional PCR cycles. After purification with the Agencourt ® AMPure ® XP (Beckman Coulter, Brea, CA, USA), DNA was quantified with the Qubit ® dsDNA High Sensitivity Assay Kit (Invitrogen). Sequencing was conducted on the Illumina MiSeq TM platform with MiSeq reagent kit v3.
Quality check of sequence reads and assignment to operational taxonomic units (OTUs)
Paired-end sequence reads were merged, primer trimmed, and quality filtered using the program package USEARCH v8.0 (12). Quality filtering was performed using the fastq filter command with the following parameters: minimum length=350 bp, no ambiguous bases allowed, and maximum expected errors for all bases in a read were less than 1. The quality of reads was evaluated using FastQC ( [URL] rRNA sequences were sorted into OTUs with a criterion of 97% sequence identity using the program UCLUST implemented in the QIIME v1.8.0 package with the furthest-neighbor clustering algorithm (6,12). Chimeric sequences were identified using UCHIME (13), and singletons were also eliminated from subsequent analyses. The taxonomic assignment of OTUs was performed with the taxonomic classification service implemented in the SINA Alignment Service ( [URL]/) using the SILVA 126 database with default parameter settings, except for the minimum identity with the query sequence, set to 0.80. OTUs assigned to Eukarya, Archaea, mitochondria, chloroplasts, and "unclassified" were removed. We also removed OTUs affiliated with the genera Halomonas and Shewanella; we concluded that these two bacterial groups were reagent contaminants because they commonly appeared in distinct samples, including those not related to this study (data not shown). We further eliminated OTUs that could not be aligned with other 16S rRNA sequences.
Statistical analysis
The similarity of bacterial community structures was evaluated with QIIME using nonmetric multidimensional scaling (NMDS) based on the Bray-Curtis similarity index. Differences among clusters were statistically tested by an analysis of similarity (ANOSIM) with 999 permutations. The separation of clusters in the NMDS plot was correlated to OTU abundance by calculating Spearman's correlation coefficient with mothur v1.37.0 (29).
In each of the 16S rRNA OTUs recovered from the ice worm samples, differences in abundance between DNA and RNA samples were statistically tested in R v3.2.2 using the DESeq2 package with the negative binomial Wald test (23). If the adjusted P value was less than 0.05, we considered the OTU abundance to be significantly different between DNA and RNA samples.
Phylogenetic analysis
Nearly full-length 16S rRNA gene sequences obtained in our previous study (27) were aligned using ARB (24) with manual corrections. In total, 1,312 (for Arcicella) and 1,218 (for Herminiimonas) unambiguously aligned nucleotide sites, which corresponded to positions 69-1440 and 94-1390 in Escherichia coli (J01695), respectively, were used for a phylogenetic analysis. Maximum likelihood (ML) and neighbor-joining (NJ) trees were constructed in MEGA7 (22) with 500 bootstrap resamplings. The GTR+Γ+I nucleotide substitution model and maximum composite likelihood model were used for ML and NJ tree reconstructions, respectively.
Nucleotide sequence accession numbers
Datasets of 16S rRNA reads sequenced in this study have been deposited in DDBJ/EMBL/GenBank under the accession number DRA005157.
Taxonomic composition and species richness of bacteria
Three and six sets of the ice worm samples, each set containing five individual worms, from Harding and Byron (designated as Harding worm/Byron worm), respectively, were subjected to 16S rRNA amplicon sequencing. Future analyses on the metabolism and genomes of ice worms and their associated microbiota will provide a deeper understanding of their ecological functions in glacier ecosystems.
==
Domain: Biology Environmental Science Medicine
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Influenza A Virus Induces an Immediate Cytotoxic Activity in All Major Subsets of Peripheral Blood Mononuclear Cells
Background A replication defective influenza A vaccine virus (delNS1 virus) was developed. In conclusion our data highlight the pleiotropic functions of immune cells and provide further evidence of a close inter-relation of the adaptive and the innate immune system.
Ethics Statement
The use of human peripheral blood has been approved by the ethical committee of the Medical University of Vienna, Austria. The use of animals has been approved by the Ministry of Science of Austria.
Cells and Viruses
Infection of PBMCs and monocultures with influenza virus
After written informed consent, peripheral blood mononuclear cells (PBMCs) from healthy donors and cancer patients were isolated using Ficoll-Hypaque (Amersham Pharmacia Biotech AB, Sweden) density gradient centrifugation. To deplete CD56+, CD3+, CD14+ and CD19+ cells PBMCs were separated with the corresponding microbeads (Miltenyi Biotec, Germany). All depletions were carried out according to the manufacturer's instructions. Cell depletions were confirmed by flow cytometry.
Monocultures of human CD56+, CD3+, CD14+ and CD19+ cells were obtained by magnetic separation using cell specific isolation kits (Milteny Biotec, Germany) following the manufacturer's instructions. As determined by flow cytometry, the procedure resulted in .90% pure cell isolates. Immediately after isolation PBMCs were infected with delNS1 or PR8 virus at an m.o.i. of 0.02 in serum-free OPTIPRO medium containing 4 mM glutamine. After incubation for 30 min. at room temperature, the inoculums were removed and cells were washed. Uninfected control cells were treated in the same manner except that no virus was added (mock infection). Virus-activated and nonactivated PBMCs were cultured at a density of 5610 6 cells/ml in RPMI-1640 medium supplemented with 2 mM glutamine and 10% heat inactivated FCS for 24 h prior to co-culture with tumor cells.
To determine the amount of the infectious viral particles in the supernatant of the virus-activated PBMCs, 1ml of supernatant was centrifuged at 25 000 r.p.m. for 18h, at +4uC. The pellet was resuspended in 500 ml of serum-free OPTIPRO medium containing 4 mM glutamine and 100 ml of that suspension was used to determine the viral concentration via plaque assay on Vero cells.
For the stimulation of PBMCs with the CpG reagent ODN2216, cells were cultured for 24 h (4610 6 /ml) in the presence of CpG ODN 2216 (12.5 mg/ml; VBC-Biotech, Austria) in RPMI-1640 medium supplemented with 2 mM glutamine and 10% heat inactivated FCS.
To activate PBMCs with INFa, the recombinant form (Roche, Switzerland) was added at the final concentration of 3000 IU/ml 16 h before cells were co-cultured with tumor cells.
Animals
Six week old BALB/c female mice were purchased from Himberg (Austria). All animals were maintained at standard conditions and fed a standard diet and water ad libitum.
Spleens were removed from mice under aseptic conditions and homogenized. After the lysis of erythrocytes, cells were washed and resuspended in serum-free OPTIPRO medium and infected with virus as described above.
Cytotoxicity assay and inhibition experiments
Tumor cells were seeded in 96-well flat-bottom plates at a density of 5610 3 /well and left for several hours to adhere. The cytotoxicity of influenza virus activated or non-activated PBMCs was tested in duplicates against tumor cells at different effector-totarget cell ratios after 24 h of co-culture. The tumor cell viability was determined by colorimetric Easy for You Assay Kit (EZ4U Kit, Biomedica, Austria) according to the manufacturers instructions and absorbance at 450 nm was measured spectrophotometrically using a Dynatech Microplate Reader 5000 (Dynatech Laboratories Inc., Chantilly, VA). Results were calculated as the percentage of viability equaling the (OD of co-culture-OD of PBMCs)/OD tumor cells6100.
To determine the sensitivity of cancer cells to the infection with delNS1 virus, the tumor cells A375, Sk-Mel 28, CaCo-2 and MCF-7 were seeded as described above and infected with a m.o.i. of 0.2 and 0.02. 10,000 events were acquired for each sample and the percentage of positive cells was reported.
==
Domain: Biology Medicine<|endoftext|>Influenza A Virus Induces an Immediate Cytotoxic Activity in All Major Subsets of Peripheral Blood Mononuclear Cells
Background A replication defective influenza A vaccine virus (delNS1 virus) was developed. Stimulation of the adaptive immune response has been regarded as substantial for viral clearance for a long time. In contrast, the relevance of the stimulation of the innate immune system for viral clearance became recently evident when Gazit et al. demonstrated that mice lacking the natural killer (NK) cell receptor Ncr-1 die from otherwise non-lethal influenza A virus infection [1]. NK-cells which are stimulated by the viral hemagglutinin are regarded as the main protagonists of virus induced cytotoxicity of the innate immune system. However, innate cytotoxicity does not seem to be restricted to NK-cells. A plasmacytoid dendritic cell line is also able to exert a cytotoxic effect after contact with influenza A virus [2]. Thus, different cell types might contribute to virus induced innate cytotoxicity.
Apart from the relevance for viral infections, the innate immune system also provides multiple ways of tumor cell destruction due to its potency of effective cytotoxicity towards degenerated cells. It was possible to treat murine cancer by adoptive transfer of splenocytes, bone marrow cells or enriched peripheral macrophages from cancer resistant CR mice [3]. The tumor ablative effect of leukocytes is not restricted to a single cell type but transfer of multiple subsets of leukocytes [4] can promote cytotoxic effects. Tumor ablation by induction of the innate immune system has already led to clinical success. Stimulating toll like receptor (TLR) 7/8 with imiquimod leads to therapeutic effects on basaliomas. This appears to be mediated by cytotoxic activity of dendritic cells present in the tumor tissue [5]. Thus, efficient stimulation of cells of the innate immune system might offer a therapeutic window for cancer therapy in humans [6].
Therapeutic stimulation of the innate immune system may also be induced by viruses. Specifically, oncolytic viruses-viruses which have been generated to conditionally replicate in the tumor tissue [7]-might exert such effects [8]. We have previously developed the first prototype of an oncolytic influenza A virus, based on a deletion of the viral non-structural NS1 gene. This genotype restricts the virus to replication in protein kinase R (PKR)-and IFN-defective tumor cells but not in normal cells [9,10]. In general, influenza A viruses might be attractive immune stimulants since they effectively activate the endosomal TLR 3 and 7 [11,12]. In addition to this property, NS1 deletion viruses have a specific immune stimulatory phenotype, since the virus lacks its natural viral antagonist of the IFN pathway. Consequently, the NS1 deletion virus effectively induces cytokines of the innate immune system such as TNF, IL-1b and type I IFN [13][14][15]. 10,000 events were acquired for each sample and the percentage of positive cells was reported.
==
Domain: Biology Medicine
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Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study
Background Epigenetic regulation is an important mechanism leading to cancer initiation and promotion. Follow-up data of at least six months were available for 74 of the 106 patients with VIN and 25 of the 59 patients with VSCC. Mean follow-up time was 67.8 months (SD ± 41.8, range 8 -169 months) and 50.6 months (SD ± 42.7, range 6 -149 months) in patients with VIN and VSCCs, respectively. Table 1 shows the patient's age and p16 status in VIN and VSCC. Table 2 shows the clinicopathological data of the patients with VSCC included in the study.
Tissue Microarray construction
Two tissue microarrays (TMA), one for the VIN and one for the VSCC cases, were constructed using a semiautomatic tissue arrayer (Beecher Instruments, Woodland, USA) as previously described [17]. Areas involving vulvar cancer or VIN were marked on hematoxylin/ eosin-stained sections. Cylindrical cores 0.6 mm in diameter were punched out of the corresponding paraffin embedded block and inserted into a recipient block. Two different spots from each tumor were punched out.
Additionally, immunohistochemical staining with p16 (dilution 1:200, Santa Cruz Biotechnology, clone: sc-56330, CA-95060 Santa Cruz, United States of America) Mean age, standard deviation (SD) and range are given in years.
Immunohistochemical staining of HDAC isoforms was scored by applying a semiquantitative immunoreactivity scoring system (IRS). Therefore the percentage of positive cells was categorized as none (0), less then 10% of the cells (1), 10-50% of the cells (2), 51-80% of the cells (3), and more then 80% of the cells (4). The intensity was graded as absent (0), weakly positive (1+), moderately positive (2 +) or strongly positive (3+). The IRS score results from multiplying the area-score with the intensity of immunoreactivity, as described elsewhere [25]. It ranges from 0 to 12. Nuclear staining of HDACs was considered positive, whereas cytoplasmic staining was regarded as nonspecific. Both TMAs were scored by two observers (N. S. & R. C.) who were blinded to the clinicopathological information of each sample. The two cores of each individual tumor were scored separately, and the mean score of the two twin tissue cores was attributed to a single patient.
To assess correlations and associations between expression of HDACs and clinicopathological parameters, Spearman's rho (bivariate correlation analysis), Fisher's exact test and χ-square tests were applied, where appropriate. p-values of < 0.05 were considered significant. SPSS 18.0 package software (SPSS Inc., Chicago/Illinois, USA) was used.
HDAC expression in VIN and VSCC
Nuclear HDAC 1, 2 and 3 staining could be evaluated in 163 of 165 cases (98.8%). In 9 cases out of 163 (5.5%), only one of both tissue cores could be analyzed. Investigations of HDAC-i for the topical or systemic treatment of VIN and VSCC are warranted.
==
Domain: Biology Medicine<|endoftext|>This below document has
* 2 sentences that end with 'the survival of M',
* 2 sentences that end with 'immune response elicited against M'. It has approximately 382 words, 24 sentences, and 4 paragraph(s).
<<<<>>>>
Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. TF is a FK506 binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (PPIase) that is considered a highly conserved ribosome-associated chaperone found in a vast number of bacteria (Hoffmann et al., 2010). This protein has been reported to be essential for protein synthesis and survival under conditions of stress (Wu et al., 2011). The present study indicated that overexpression of Rv2629 induced the expression of TF and FFP and might improve bacterial survival in the persistence state.
The success of the pathogenicity of M. tuberculosis is largely attributed to its ability to manipulate the host immune responses. The genome of M. tuberculosis encodes multiple immune-modulatory proteins that have been implicated in the pathogenicity of this strain. In agreement with the main concept of the current study, it has been shown that the immune-modulatory protein PE_PGRS41 boosted the survival of M. smegmatis within macrophages and was accompanied by enhanced cytotoxic cell death via inhibition of apoptosis and autophagy. This indicated that specific bacterial proteins can act as virulence factors that enhance the pathogenic properties of M. smegmatis (Deng et al., 2017). To the best of our knowledge the implication of the antigenic protein Rv2629 in the virulence of M. smegmatis has not been reported to date. The number of studies that have examined the contribution of this antigen in the immune response elicited against M. tuberculosis is very limited (eight in total). The majority of the studies on Rv2629 have focused on the contribution of this protein to the immune response elicited against M. tuberculosis as well as in the resistance developed against agents such as Rifampicin (Chakravorty et al., 2008;Louw et al., 2009;Niki et al., 2012). In addition, polymorphisms of Rv2629 have been used as potential markers for the identification process of certain M. tuberculosis genotypes (Alonso et al., 2011;Zhang et al., 2014). Consequently, the current study offers novel evidence on the potential interplay of the Rv2629 protein regarding the survival of M. tuberculosis.
Based on KEGG database, the overlap value between MSMEG_1130 and Rv2629 was 366. Data are representative of two experiments.
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Domain: Biology Medicine<|endoftext|>This below document has
* 2 sentences that start with 'Typhi-specific modulation of the expression',
* 2 sentences that start with 'Typhi-specific up-regulation of PD-1',
* 2 sentences that start with 'Typhi-specific PD-1 expression',
* 2 sentences that end with 'Fig 3B and 3E)',
* 2 sentences that end with '(Fig 3B and 3E)'. It has approximately 319 words, 24 sentences, and 3 paragraph(s).
<<<<>>>>
Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. As for the measurement of homing potential, PBMC from volunteers challenged with wild-type S. Typhi were stimulated with S. Typhi-infected autologous B-LCL or non-infected B-LCL. Relative (net) S. Typhi-specific modulation of the expression of the activation molecules listed above was determined by subtracting the values obtained following stimulation with non-infected B-LCL from stimulation with S. Typhi-infected B-LCL.
There was considerable variability among volunteers in the S. Typhi-specific modulation of the expression of all activation molecules. We observed that S. Typhi-specific up-regulation of PD-1, CD27, LFA-1, NRP-1, and Tim-3 was present before challenge in many volunteers ( At early time-points (days 1-4) following challenge, however, we observed a notable increase in S. Typhi-specific expression of HLA-DR resulting in significantly higher expression in TD than in No TD volunteers (p = 0.015-mixed effects regression model) (Fig 3A and 3D). In contrast, S. Typhispecific HLA-DR expression on circulating T reg in No TD volunteers decreased slightly after challenge (days 1-4), returning to baseline levels by 21-28 days post-challenge (Fig 3A and 3D).
We also identified marked up-regulation of the expression of PD-1 by S. Typhi-infected targets in circulating T reg isolated from TD compared to No TD volunteers post-challenge ( Fig 3B and 3E). S. Typhi-specific up-regulation of PD-1 expression increased gradually in TD volunteers following challenge with the highest levels identified on days 21-28 post-challenge ( Fig 3B and 3E). Distinctly, however, among those volunteers who did not develop disease we noted a general down-regulation of S. Typhi-specific PD-1 expression in circulating T reg 1-9 days post-challenge with a significant increase between the days 5-9 and days 10-14 post-challenge time groups (Fig 3B and 3E). S. Typhi-specific PD-1 expression returned to baseline levels by days 21-28 post-challenge (Fig 3B and 3E). These opposite trends resulted in significantly higher up-regulation of S. All volunteers provided written informed consent.
==
Domain: Biology Medicine
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Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. UKs, also known as UMP kinases, are key enzymes in the synthesis of nucleoside triphosphates. The members of the UMP kinase family catalyze the conversion of UMP to UDP, an essential step in the pyrimidine metabolic pathway in a variety of bacteria (Yoshida et al., 2012). In many bacterial genomes, the gene tends to be located immediately downstream of elongation factor T and upstream of ribosome recycling factor. This enzyme has been shew to be essential in bacteria. The product of pyrH (Rv2883) gene, a member of the UMP kinase family, has been shew to be essential for M. tuberculosis growth by the rapid screening method (Lee et al., 2007;Rostirolla et al., 2011). Genetic studies have provided evidence that UMP kinases are essential for the growth of both Gram-negative (e.g., E. coli) and Gram-positive bacteria (e.g., Streptococcus pneumoniae) (Yamanaka et al., 1992;Fassy et al., 2004). Therefore, it can be speculated that Rv2629 delays entry in the log-phase by inhibiting protein biosynthesis. Delayed growth under conditions of dormancy is likely to be beneficial for the survival of bacteria in the host, and therefore may contribute to the increased pathogenicity of the strain. Although Rv2629 has been characterized as a dormancy protein that is induced under anaerobic conditions of M. tuberculosis, the main focus of research has been toward the ability of this protein to confer resistance to bacterial strains (Florczyk et al., 2003;Wang et al., 2007). The current study investigates the novel hypothesis that the reduced growth and dormancy-type characteristics of M. smegmatis and M. tuberculosis are not entirely dependent on antibiotic resistance. The drug tolerance observed in the present study may be due to alternative mechanisms, such as the survival of the bacilli in a non-replicating and/or low metabolic state. Similarly, it has been reported that the phenotypic tolerance acquired in M. tuberculosis to isoniazid treatment is via the activation of mycobacterial DNA activating protein (MDP1) and the regulation of kat G transcription (Niki et al., 2012). The aforementioned results supported the functional overlap of Rv2629 between M. smegmatis and M. Data are representative of two experiments.
==
Domain: Biology Medicine<|endoftext|>Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. The M. smegmatis cultures were diluted when an OD 600 value of 0.05 was obtained. Aliquots of 50 mL were dispensed in conical flasks and grown under continuous shaking at 220 rpm and aerobic conditions. The growth and survival were assayed by optical density readings (OD 600 ) every 3 h following inoculation. The experiments were performed in triplicate.
The growth curve of recombinant M. tuberculosis H37Ra was detected using the BACTEC MGIT 960 Mycobacterial Detection System (Becton, Dickinson and Company, United States), as determined by the manufacturer's instructions.
Infection of THP-1 Cells with M. tuberculosis Strains and M. smegmatis Strains THP-1 cells (5 × 10 5 ) were stimulated with 5 ng/mL PMA (Sigma Chemical Co, St. Louis, MO, United States) for 24 h prior to infection. The infection was conducted using H37Rv, clinical M. tuberculosis strain 934, and recombinant M. smegmatis in the log-phase at a multiplicity of infection (MOI) value of 1:10 (37 • C, 5% CO 2 , 4 h). Extracellular bacilli were removed by washing cells three times with PBS and the cells were cultured for 1 h in complete medium in the presence of 100 µg/mL gentamycin. The medium was replaced every 12 h during incubation, and the cells were lysed using 1% Triton X-100 solution at specific time points (0, 24, 48, 72, and 96 h). The precipitates were collected by centrifugation (2000 g, 20 min) for total RNA extraction, as described above. The number of CFU of intracellular mycobacteria and the viable bacteria present in the supernatant were determined on Middlebrook 7H10 agar with 10 % ADC.
Mouse Infection
The culture of MSP and MSW was conducted in 5 mL of Middlebrook7H9 medium supplemented with 10% ADC and 500 mg sterile 2-mm glass beads at 37 • C under continuous shaking. The cultures isolated during the log phase were centrifuged at 500 g in order to remove clumps, and the cells were washed twice with sterile saline. The cell suspension was adjusted to an OD 600 of 0.8, corresponding to a concentration of 5 × 10 7 CFU/mL. Female Balb/c mice (6-8-week-old) were infected intravenously with 0.1 mL of the bacterial suspension. The size of inoculum was confirmed by CFU counts.
The ability of survival and dissemination of each strain in vivo was assessed in six mice per group that were sacrificed by cervical dislocation on 0, 3, 6, 9, 12, 15, 20, and 35 days after infection. Data are representative of two experiments.
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Domain: Biology Medicine<|endoftext|>Influenza A Virus Induces an Immediate Cytotoxic Activity in All Major Subsets of Peripheral Blood Mononuclear Cells
Background A replication defective influenza A vaccine virus (delNS1 virus) was developed. In contrast, Draghi et al. [29] and He et al. [31] demonstrated that CD56+CD3-cells by themselves are unable to generate IFNc production upon stimulation with influenza virus. However, IFNc production by NK cells was possible by co-cultivation with dendritic cells producing IL-12 or with CD3+ cells producing IL-2, respectively. None of these cytokines were induced in our CD56+ cell cultures. One difference to the above mentioned studies is the fact that the CD56+ population we isolated still contained CD56+CD3+. Thus, the interaction of CD56+CD3-and CD56+CD3+ cells might lead to IFNc production via cell-cell interaction. However, CD56+CD3+ are not likely to produce IFNc on their own, since the CD3+ population containing this subset did not produced IFNc. An important aspect of our work is the fact that we have used a highly immunogenic, replication defective mutant delNS1 virus, which has been shown to induce a stronger innate immune response than the wild type virus [13][14][15], whereas Draghi et al. [29] and He et al. [31] used an influenza wild type virus. Moreover, we observed a cytokine response when virus was used at low m.o.i. of 0.02, whereas other authors only observed a cytokine response at an m.o.i. of 10 or higher [29]. It is known that cytokine patterns of immune cells following influenza A virus stimulation are critically dependent on the m.o.i. [32]. At a high m.o.i. all cytokines expressing cells might be directly stimulated and possibly lysed by the virus. Being able to use the virus at a low m.o.i. we allowed a second wave of cytokine stimulation. 10,000 events were acquired for each sample and the percentage of positive cells was reported.
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Domain: Biology Medicine<|endoftext|>Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. Homing of T reg to sites of specific inflammation has been previously shown [16,20,21,22,23]. Integrin α4β7 is an important molecule associated with homing of lymphocytes to the gut, the site of initial encounter with S. Typhi [28]. Here we identified, for the first time, significantly higher pre-challenge gut homing potential of circulating T reg (up-regulation of S. Typhi-specific integrin α4β7 expression) in volunteers who were subsequently diagnosed with typhoid disease compared to those who were not. Following challenge, however, there was a significant decrease in S. Typhi-specific integrin α4β7 expression on circulating T reg , suggesting that these T reg left the peripheral blood, presumably as a result of homing to the gut microenvironment. It is currently unclear why S. Typhi-specific T reg expressing differential levels of integrin α4β7 were observed among volunteers before challenge. Participants were recruited in a non-endemic area and are, therefore, unlikely to have previously encountered S. Typhi. However, the S. Typhi genome has a high degree of homology with other Enterobacteriaceae. Thus, differences in baseline T reg responses could be the result of previous encounters with other enteric Gram negative bacilli, including those present in the normal gut microbiota. We have previously reported that oral immunization of volunteers with attenuated oral S. Typhi vaccines elicits S. Typhi-specific T EM which expressed, or not, the gut homing molecule integrin α4β7 [11,29]. It has been shown that T cells activated in the gut preferentially express high levels of integrin α4β7 compared to T cells primed in peripheral lymph nodes [30]. Therefore, it is possible that T reg initially primed in the gut would express higher levels of integrin α4β7 upon re-stimulation resulting in recirculation to the site of initial antigen encounter. It is thus reasonable to speculate that higher levels of T reg homing to the gut may suppress local T eff responses resulting in ineffectual control of the infection ultimately leading to typhoid diagnosis. All volunteers provided written informed consent.
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Domain: Biology Medicine
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Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. Typhi-specific expression of PD-1 in circulating T reg in TD compared to No TD volunteers at days 5-9 (p = 0.0097-mixed effects regression model) and 21-28 post-challenge (p = 0.0092-mixed effects regression model) (Fig 3B and 3E).
Interestingly, we observed increased up-regulation of the expression of CD27 on S. Typhispecific T reg in No TD compared to TD volunteers (Fig 3C and 3F). While the trend was present at most time-points, this difference was statistically significant only in the day 10-14 time frame (after typhoid diagnosis and initiation of antibiotics), (p = 0.031-mixed effects regression model). In a subset of volunteers, S. Typhi-specific CD39 expression was also measured. Although there were only a small number of samples tested, we identified up-regulation of S. Typhi-specific CD39 expression on circulating T reg following challenge in TD volunteers. This increase peaked at days 10-14 post-challenge and was significantly higher than pre-challenge Differential kinetics of activation of circulating S. Typhi-specific T reg between TD and No TD volunteers To further explore changes in S. Typhi-specific modulation of the expression of activation molecules over time, we examined kinetic curves of individual volunteers. While significant differences were not detected in the mean expression of LFA-1 between TD and No TD volunteers, the kinetic patterns were remarkably different in volunteers diagnosed, or not, with typhoid following challenge. Despite considerable variation among volunteers, a pattern of increased expression of LFA-1 around the time of disease was identified in a majority of TD volunteers (4/5) while expression remained relatively constant for most No TD volunteers (Fig 4A). We also identified differences in the kinetic patterns of S. Typhi-specific NRP-1 expression. Unlike LFA-1, we observed S. Typhi-specific up-regulation of NRP-1 expression in TD volunteers after diagnosis and initiation of antibiotics (days 14-21 post-challenge) (Fig 4B).
Circulating T reg suppress S. Typhi-specific T eff responses
To further assess the functionality of T reg in the setting of typhoid disease, we performed CD25 depletion assays. PBMC from 4 TD volunteers were either mock depleted (pan anti-mouse IgG) or CD25 depleted (anti-human CD25) using magnetic bead separation. Time-points were selected based on known S. Typhi-specific cytokine responses. All volunteers provided written informed consent.
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Domain: Biology Medicine<|endoftext|>Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. In contrast, S. Typhispecific HLA-DR expression on circulating T reg in No TD volunteers decreased slightly after challenge (days 1-4), returning to baseline levels by 21-28 days post-challenge (Fig 3A and 3D).
We also identified marked up-regulation of the expression of PD-1 by S. Typhi-infected targets in circulating T reg isolated from TD compared to No TD volunteers post-challenge ( Fig 3B and 3E). S. Typhi-specific up-regulation of PD-1 expression increased gradually in TD volunteers following challenge with the highest levels identified on days 21-28 post-challenge ( Fig 3B and 3E). Distinctly, however, among those volunteers who did not develop disease we noted a general down-regulation of S. Typhi-specific PD-1 expression in circulating T reg 1-9 days post-challenge with a significant increase between the days 5-9 and days 10-14 post-challenge time groups (Fig 3B and 3E). S. Typhi-specific PD-1 expression returned to baseline levels by days 21-28 post-challenge (Fig 3B and 3E). These opposite trends resulted in significantly higher up-regulation of S. Typhi-specific expression of PD-1 in circulating T reg in TD compared to No TD volunteers at days 5-9 (p = 0.0097-mixed effects regression model) and 21-28 post-challenge (p = 0.0092-mixed effects regression model) (Fig 3B and 3E).
Interestingly, we observed increased up-regulation of the expression of CD27 on S. Typhispecific T reg in No TD compared to TD volunteers (Fig 3C and 3F). While the trend was present at most time-points, this difference was statistically significant only in the day 10-14 time frame (after typhoid diagnosis and initiation of antibiotics), (p = 0.031-mixed effects regression model). In a subset of volunteers, S. Typhi-specific CD39 expression was also measured. Although there were only a small number of samples tested, we identified up-regulation of S. Typhi-specific CD39 expression on circulating T reg following challenge in TD volunteers. This increase peaked at days 10-14 post-challenge and was significantly higher than pre-challenge Differential kinetics of activation of circulating S. Typhi-specific T reg between TD and No TD volunteers To further explore changes in S. Typhi-specific modulation of the expression of activation molecules over time, we examined kinetic curves of individual volunteers. All volunteers provided written informed consent.
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Domain: Biology Medicine<|endoftext|>Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. Mce4 encodes a cholesterol transporter that is associated with cholesterol metabolism and is essential for mycobacterial persistence. The disruption of mce4 results in failure of M. tuberculosis to maintain chronic infection in mice, while retaining full virulence during the acute phase (Mohn et al., 2008;Pandey and Sassetti, 2008). In contrast to these findings, the MCE family protein and the Mceassociated protein are generally considered as virulence factors, and are associated with lipid transport and host cell invasion (Rodriguez et al., 2015;Perkowski et al., 2016). Ag85B may also contribute to the adherence, invasion, and dissemination of organisms in the host tissue, which can bind to Fn and play a critical role in mycobacterial adherence to the host cells (Kassa et al., 2012). Furthermore, Ag85B serves as an important colonization factor potentially contributing to mycobacterial virulence (Kuo et al., 2012). To date, no proteomic studies have been conducted with regard to the Rv2629 mutant strain of M. smegmatis and/or M. tuberculosis. The 2D-gel electrophoresis indicated that the proteins TF and FFP were expressed in MSW, but not in MSP strains. TF is a FK506 binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (PPIase) that is considered a highly conserved ribosome-associated chaperone found in a vast number of bacteria (Hoffmann et al., 2010). This protein has been reported to be essential for protein synthesis and survival under conditions of stress (Wu et al., 2011). The present study indicated that overexpression of Rv2629 induced the expression of TF and FFP and might improve bacterial survival in the persistence state.
The success of the pathogenicity of M. tuberculosis is largely attributed to its ability to manipulate the host immune responses. The genome of M. tuberculosis encodes multiple immune-modulatory proteins that have been implicated in the pathogenicity of this strain. In agreement with the main concept of the current study, it has been shown that the immune-modulatory protein PE_PGRS41 boosted the survival of M. smegmatis within macrophages and was accompanied by enhanced cytotoxic cell death via inhibition of apoptosis and autophagy. This indicated that specific bacterial proteins can act as virulence factors that enhance the pathogenic properties of M. smegmatis (Deng et al., 2017). Data are representative of two experiments.
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Domain: Biology Medicine
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This below document has
* 2 sentences that end with '(Florczyk et al., 2003)',
* 2 sentences that end with 'al., 2008;Homolka et al., 2009)'. It has approximately 407 words, 23 sentences, and 3 paragraph(s).
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Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. smegmatis) (Homolka et al., 2009). DosR was shown to initiate transcription of an array of genes known as the dosR regulon, which comprises 48 genes containing the DosR-binding site and an ACP family sequence motif (Florczyk et al., 2003). The expression of these genes allows long-term survival of M. tuberculosis under anaerobic conditions in the host (Errey and Blanchard, 2005;Homolka et al., 2009;Alonso et al., 2011).
Rv2629 is a member of the dosR dormancy regulon (Park et al., 2003) due to the presence of an ACP motif predicted by the Gibbs Recursive Sampler and SCAN (Florczyk et al., 2003). The Rv2629 gene is conserved with regard to its expression in several environmental mycobacteria and related strains of M. tuberculosis (Wang et al., 2007). Rv2629 has been reported to be induced by hypoxia and nitric oxide, while it is upregulated under dormancy conditions (Boon and Dick, 2002;Park et al., 2003;Voskuil et al., 2003;Starck et al., 2004), suggesting an association with the dormant state of M. tuberculosis (Starck et al., 2004). Rv2629 was further identified as a drug-resistant protein by 2D-gel electrophoresis and MS analysis (Wang et al., 2007). A previous study determined the Rv2629 sequence variations in 58 MDR strains and in 55 strains from a reference collection that represented the major M. tuberculosis complex pathogens namely, M. tuberculosis Beijing, M. africanum West African 1, M. tuberculosis (H37Rv; ATCC 27294), M. bovis (ATCC 19210), and M. africanum (ATCC 25420) (Homolka et al., 2009). Among these 58 MDR strains, 36 (62%) had the 191A/C mutation (codon 64, Asp to Ala) in Rv2629 that supported an association with rifampicin resistance (Chakravorty et al., 2008;Homolka et al., 2009). The 191C allele was present in the Beijing strains when the MDR strains were stratified according to different phylogenetic lineages that indicated a classical Beijing IS6110 restriction fragment length polymorphism pattern (Chakravorty et al., 2008;Homolka et al., 2009). The IS6110-restriction fragment length polymorphism (IS6110-RFLP) has been applied for the identification of the Beijing strains based on their hybridization patterns (Alonso et al., 2011). The Beijing strains evade the innate immune response and exhibit high capacity to cause and spread TB in humans compared with other M. Data are representative of two experiments.
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Domain: Biology Medicine<|endoftext|>Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. The bacterial pellets were washed twice with 0.02 M PBS, resuspended, and sonicated in 200 µL of 0.02M PBS (supplemented with 1 mM PMSF and 10 mM EDTA) and centrifuged. The supernatant was treated with lysis buffer (8 M urea, 2 M thiourea, 140 mM DTT, 0.5% Biolyte pH 4-7, and 4% CHAPs). The protein concentration was estimated using the Bradford assay. Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel), transferred to a PVDF membrane and detected with anti-Rv2629 rabbit antiserum (in house preparation) and/or the FtsZ monoclonal antibody (4 • C, overnight) followed by a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (Sigma, United States) (37 • C, 1 h). The blots were visualized with BCIP/NBT solution (room temperature, 20 min). The reaction was terminated by addition of deionized water. FtsZ was used as a loading control.
Minimum Inhibitory Concentration Assay
Minimum inhibitory concentrations assay of recombinant M. smegmatis was carried out to detect the relationship between over-expressing Rv2629 proteins and the susceptibility to anti-TB drugs (streptomycin, isoniazid, rifampicin, ethambutol, ofloxacin, levofloxacin, moxifloxacin, amikacin, kanamycin, and capreomycin). Antibiotic dilutions and 96-well plate preparations (Falcon 3072; Becton Dickinson, Lincoln Park, NJ, United States) were carried out as described by Caviedes et al. (2002). Briefly, Rv2629 overexpressing strains were grown in 7H9/ADC at 37 • C until an OD 600 value of 0.5 was obtained. The bacterial suspensions were diluted 1000 times in 7H9 broth and the final diluted broth was dispensed into each well of a 96-well cell culture plate (100 µL/well). The drug storage solutions were diluted to prepare two-fold serial diluted solutions in 7H9 broth and subsequently added to the wells containing the bacterial suspensions. The plate was incubated for 48 h at 37 • C. The wells containing bacterial pellets were denoted as the positive wells, whereas the wells containing clear broth were denoted as the negative wells. The minimum drug concentrations of the negative wells were the MICs of the drugs. All the experiments were carried out in triplicate.
Microarray Analysis
Total RNA from each sample was quantified using the NanoDrop 1000 (Thermo Fisher, United States). RNA integrity was assessed using standard denaturing agarose gel electrophoresis. For microarray analysis, the Agilent Array platform was used according to the manufacturer's standard protocols for sample preparation and microarray hybridization. Briefly, total RNA (1 µg) was amplified and transcribed into fluorescent cRNA, following the manufacturer's Quick Amp Labeling protocol (Version 5.7, Agilent Technologies). Data are representative of two experiments.
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Domain: Biology Medicine<|endoftext|>Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. Additional pathological tests indicated that the pathogenicity of MSW increased and additional damage was caused in the lung tissues compared to the MSP and/or MSM strains. The pulmonary alveolus of mice infected with MSP was damaged slightly and it was the sole cause of bleeding in the bronchial lumen in the absence of granuloma formation during the 15th day following infection. Sporadic granuloma could be found in the small part of the lungs at the 20th day after infection, whereas the inflammatory exudate was absorbed and the main part of the lung recovered to the normal structure 35 days postinfection ( Figure 9A). Apparent changes could be observed in the lung of MSW-infected mice. The normal structure of the major part of the pulmonary alveolus disappeared, which was permeated with inflammatory exudate and associated with the infiltration of a great number of inflammatory cells. The alveolar wall indicated congestion and incrassation, whereas apparent granuloma could be found in the lungs 15 days post-infection. The area and degree of damage increased further 20 days postinfection, and the normal structure of the complete part of the pulmonary alveolus disappeared. The structure of the majority of the pulmonary alveolus recovered, although a certain part of granuloma could be found in the lung (Figure 9B). The pathogenicity that was caused by the Rv2629 expressing strains decreased when a missense mutation (Asp64 to Ala64, A/C) was present at position 191 of the genetic sequence of Rv2629. The pulmonary alveolus of mice infected with MSM was damaged partly and sporadic granuloma could be found in certain parts of the lungs 15 days post-infection. The damaged structure of pulmonary alveolus recovered gradually and the majority of the lung tissue recovered to the normal structure at the 20th and the 35th day week post-infection, respectively, as noted for the MSP strain ( Figure 9C). Consequently, the overexpression of the wild type Rv2629 could increase the survival and pathogenicity of M. smegmatis, whereas the 191 A/C mutation in Rv2629 could weaken the pathogenicity of the Rv2629-expressing strains.
Protein expression maps of M. smegmatis overexpressing Rv2629 were obtained by 2-DE (Figure 10). A total of eight differential proteins were found by comparing the protein differential expressing map on 2-DE gels. A total of five proteins, namely, CHP, UK, TF, AA, and FFP, were identified by MALDI-TOF-MS (Table 3). Nevertheless, the expression pattern of these proteins differed among the strains. TF and FFP were present only on the MSW 2-DE gel, as opposed to the MSP gel. AA was not detected on the MSW 2-DE gel. The expression of UK decreased, while the expression of CHP increased in the MSW compared with the MSP strains.
DISCUSSION
In the present study, an expression analysis of the Rv2629 native protein was conducted in recombinant M. Data are representative of two experiments.
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Domain: Biology Medicine
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Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study
Background Epigenetic regulation is an important mechanism leading to cancer initiation and promotion. For patients with recurrent or metastatic disease, irradiation and chemotherapy offer some benefit; however, response rates are regarded as low [4,5]. Targeted therapies for VSCC have not yet been established in clinical practice [6], but given the low benefit of conventional chemotherapies, novel systemic treatment modalities are urgently needed for these patients.
Epigenetics characterize the hereditary changes in the pattern of gene expression that are not due to changes in DNA sequence. Genetics and epigenetics interact at all stages of cancer development. Epigenetic alterations in mammalian genomes fall into two main categories: DNA methylation and histone modification. Histones are strongly alkaline proteins that are able to package the DNA and condense it into structural units called nucleosomes. Acetylation and deacetylation of histones are performed by histone acetyltransferase (HATs) and histone deacetylases (HDACs), respectively. HDACs increase the affinity of histone complexes to DNA. The chromatin is thereby more condensed and transcriptionally repressed [7][8][9].
Additionally HDACs can modify proteins other than histones, such as transcription factors (e.g. p53, E2F, pRb). Acetylation can also affect protein stability and protein-protein interactions. Therefore, HDACs are emerging as important regulators of cell growth, differentiation, and apoptosis [10,11]. There are at least eighteen deacetylase enzymes known in human cells, categorized into four classes: class I (HDAC 1, 2, 3, 8), class II (HDAC 4,5,6,7,9,10), class III (SIRT 1-7) and class IV (HDAC 11). HDAC1 and HDAC2 are one of the bestcharacterized HDACs. However, the isoenzyme-specific biological functions of HDACs are still mostly unknown [8]. It has been postulated that dysregulated function of HDACs leads to cancer formation and development [11]. Altered HDAC expression is observed in a variety of cancer types, such as prostate adenocarcinoma [12], gastric carcinoma [13], colorectal carcinoma [14], cervical dysplasia and endometrial stromal sarcoma [15]. In vulvar intraepithelial neoplasia and vulvar cancer, no data on HDAC expression has been published.
The aim of this study was to analyze the expression of the class I HDACs 1, 2 and 3 by immunohistochemistry in a series of VIN and VSCC samples using the tissue microarray technique and to correlate the finding with the clinicopathological features of the patients. [16].
Clinical data were available for 158 of the 165 cases. Investigations of HDAC-i for the topical or systemic treatment of VIN and VSCC are warranted.
==
Domain: Biology Medicine<|endoftext|>Influenza A Virus Induces an Immediate Cytotoxic Activity in All Major Subsets of Peripheral Blood Mononuclear Cells
Background A replication defective influenza A vaccine virus (delNS1 virus) was developed. [31] used an influenza wild type virus. Moreover, we observed a cytokine response when virus was used at low m.o.i. of 0.02, whereas other authors only observed a cytokine response at an m.o.i. of 10 or higher [29]. It is known that cytokine patterns of immune cells following influenza A virus stimulation are critically dependent on the m.o.i. [32]. At a high m.o.i. all cytokines expressing cells might be directly stimulated and possibly lysed by the virus. Being able to use the virus at a low m.o.i. we allowed a second wave of cytokine stimulation. This secondary cytokine wave is produced by cells not infected by virus, but activated by cytokines and cell-cell interactions, which have been induced previously by the initial infection. The cytokine composition of this second wave seems to be different from that of the first wave.
With respect to the mechanism of the virus induced cytotoxic effect our transwell analysis indicates that cell-cell interaction mediate around half of the effect, whereas the other half was mediated by soluble factors in the supernatant. Although we assayed for a number of known cytotoxicity-associated soluble factors such as TNF, TRAIL, FASL, NKG2D, perforin or granzyme B, we could not identify single factors responsible for this effect. It should be noted that we have used a type I IFN resistant target cell line, which restricts the latter pathways. In contrast, TRAIL in IFN sensitive target cells lines has been found to play a role in virus stimulated cells, as has been shown for Newcastle Disease virus (NDV) induced tumoricidal effects at high m.o.i. [19]. Our analysis indicates that other factors are able to compensate whenever TRAIL is not functional.
What are the implications of these findings for viral immunology? First, during regular influenza A virus infection all activated immune cells might initially exert cytotoxic functions thus limiting initial viral spread by killing of virus infected cells. Second, we regard the influenza A virus (delNS1) suitable for activation of a cytotoxic immune response to cancer. This might be achieved by ex vivo stimulation of peripheral blood cells with the virus and subsequent adoptive transfer of viral activated cells into the patient. Alternatively, delNS1 virus might be injected as oncolytic viro-immunotherapy directly into the tumor with the intention to induce cytotoxic activity in the anergic immune cells present in the tumor tissue. 10,000 events were acquired for each sample and the percentage of positive cells was reported.
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Domain: Biology Medicine<|endoftext|>Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. RJ and WX provided new tools and reagents; HyW and HhW analyzed data and helped to design experiments; DL, QW and LZ wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: [URL]. 2017.02231/full#supplementary-material FIGURE S1 | Expression levels of Rv2629 modulate the growth of H37Ra strains under aerobic conditions. The growth curve of recombinant H37Ra overexpressing the Rv2629 W (RaW) and/or Rv2629 M (RaM) genes were measured using the BD BACTEC MGIT 960 Mycobacterium growth and detection system. The results are shown as the fluorescence intensity acquired automatically. RaP (transformed with the pMV261 plasmid) was used as a control. Data are representative of two experiments.
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Domain: Biology Medicine
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Activation of Salmonella Typhi-Specific Regulatory T Cells in Typhoid Disease in a Wild-Type S. Typhi will provide much needed information to inform and accelerate the development of novel vaccines for typhoid and other enteric fevers, as well as other enteric infections. For example, strategies to identify vaccines that activate T eff without the concomitant activation of suppressive T reg responses, or that elicit an optimal balance between T eff and T reg responses may result in improved protective efficacy.
Volunteers and isolation of peripheral blood mononuclear cells (PBMC)
Healthy adult volunteers aged 18-60 were recruited by the Centre for Clinical Vaccinology and Tropical Medicine, Oxford, UK, to participate in this study. Volunteers with history of typhoid fever or immunization against typhoid fever were excluded [7]. Volunteers were orally challenged with 1-5x10 4 CFU of wt-S. Typhi (Quailes strain) suspended in sodium bicarbonate at Oxford University in compliance with the National Research Ethic Service (NRES), Oxford Research Ethics Committee A [7]. Close monitoring was performed throughout the study, and at the time of typhoid fever diagnosis (TD, as determined by blood culture-confirmed S. Typhi bacteremia or development of a fever 38°C for 12 hours), volunteers were treated with a 2-week course of antibiotics (Ciprofloxacin, 500mg twice daily). Those volunteers who did not developed typhoid fever (No TD) received a 2-week course of antibiotics at day 14 post-challenge. PBMC collected from 12 randomly selected volunteers (TD n = 6, No TD n = 6) participating in the challenge trial were used in this study. Selection was made based on the number of available PBMC with those volunteers having more PBMC utilized for the studies. PBMC were isolated prior to challenge and at 9-11 time-points following challenge (S1 Fig). Isolation was performed by Lymphoprep gradient centrifugation (Axis-Shield, Oslo, Norway) and PBMC were cryopreserved in liquid nitrogen following standard techniques within four hours of initial blood draw. Viability of cryopreserved PBMC was assessed after thawing of cells and an overnight rest at 37°C with 5% CO 2 (as described in ex vivo stimulation).
Target/stimulator cells B-LCL were generated from autologous PBMC for each volunteer as previously described [45]. Briefly, B-LCL were established using supernatant from the B95.8 cell line (ATCC CRL1612; American Type Culture Collection) as the source of EBV. PBMC from each volunteer were incubated with EBV containing supernatant and cyclosporine (0.5 μg/mL; Sigma, St. Louis, MO) at 37°C with 5% CO 2 for 2-3 weeks. B-LCL were maintained in culture or cryopreserved until use.
Infection of target/stimulator cells
Target cells were infected by incubation with wild-type S. Typhi strain ISP1820 in RPMI 1640 media (Gibco, Carlsbad, CA) without antibiotics for 3 hours at 37°C with 5% CO 2 as previously described [45]. On the day following infection the cells were gamma irradiated (6000 rad). To confirm that targets were infected with S. All volunteers provided written informed consent.
==
Domain: Biology Medicine<|endoftext|>Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study
Background Epigenetic regulation is an important mechanism leading to cancer initiation and promotion. A high proliferation index (Ki-67 area) correlated with high HDAC 1 (p = 0.008, cc = 0.21) and HDAC 2 (p < 0.001, cc = 0.36) expression. Using cut-off levels of 10%, this correlation was confirmed (Table 3). Similar to this findings, applying a cut-off level of 25% results in a significant associations between Ki-67 and HDAC 1 in VIN (p = 0.012) and Ki-67 and HDAC 2 in the VSCC group (p = 0.035).
In the grouped analyses, no significant association between p16 positivity and patient age with HDAC expression was observed (Table 3). In fact, almost equal frequencies were found between p16 and HDAC 1 as well as between patient age and HDAC 2. There was no significant association between tumor size (pT) and high HDAC 2 expression.
Discussion
Based on the expression pattern of histone deacetylating proteins, we hypothesized that epigenetic regulation plays a major role in the pathogenesis of invasive vulvar cancer, as has been demonstrated for several other malignancies [12][13][14][15]. The transformation of non-invasive to invasive vulvar neoplasia may be promoted by epigenetic regulation. To our knowledge, this report is the first on class I HDAC expression in vulvar cancer or vulvar intraepithelial neoplasia. HDAC immunoreactivity score (IRS) has been dichotomized in two groups; HDAC 1 to 3 "high" represent tissue samples with a IRS of 12, HDAC 1 to 3 "low", a IRS less than 12. Clinicopathological parameters investigated in this study are listed in the first column; Vulvar intraepithelial neoplasia (VIN), vulvar squamous cell cancer (VSCC); p-16 as a surrogate marker for human papilloma virus (HPV) infection as described before [21][22][23]; patient younger than 60 years (Age ≤ 60), patient equal or elder than 60 years (Age > 60); related pTNM-stage and tissue differentiation (G1 to G3) in VSCC. Nuclear Ki-67 protein is a marker for cell proliferation. "Ki-67 ≤ 10%" means that 10 percent or less of the cells are proliferating. P16 has been proposed as a surrogate marker for HPV associated neoplasia [21][22][23]. We found no difference between class I HDAC expression in p16-negative or p16-positive tumor tissue. Therefore, the regulation of gene expression by HDACs seems to be independent of HPV infection.
The epigenetic regulation of HDACs has recently been explored as a therapeutic target by the discovery and development of HDAC-inhibitors (HDACi). In-vitro data suggest that HDACi induce cell cycle arrest, differentiation, and apoptosis [15,[29][30][31][32]. The antitumor effects of HDACi emphasize the important role of HDACs in cancer development. However, HDACi affects the activity of several enzymes, and it is difficult to identify the particular functions of different HDAC isoforms involved in cancerogenesis. Several HDACi are currently under clinical investigation involving various hematological malignancies and solid tumors, of which vorinostat has already been approved for the systemic treatment of cutaneous T cell lymphoma [33]. Particularly, in oral squamous cell cancer, there are different phase I and II trials using HDAC-i as a monotherapy or in Figure 2 HDACs immunoreactivity score (IRS) in VIN and vulvar cancer tissue. The mean IRS is represented by a circle, and the 95% confidence interval is represented by an error bar. Investigations of HDAC-i for the topical or systemic treatment of VIN and VSCC are warranted.
==
Domain: Biology Medicine<|endoftext|>Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice
Objective: The aim of the present study was to explore the potential biological role of Rv2629 in Mycobacterium smegmatis and Mycobacterium tuberculosis. Moreover, Blastn data showed that there are 54% identical nucleotide sequences and Blastp data showed that there are 59% similar or identical amino acid sequences between them. The nucleotide and amino acid sequences of two genes are similar, there was certain homology of nucleotide and amino acid sequences between the two genes. Therefore, MSMEG_1130 was the most homologous protein with Rv2629 in M. smegmatis strains, with more than 50% amino acids identical or similar with Rv2629. To further clarify the problem, other low-expression models such as the MRA_2657 low-expression H37Ra strain should be further investigated. Furthermore, there was no experimental data supporting the different function of MSMEG 1130 from Rv2629. On the contrary, MSMEG_1130 might have similar function with Rv2629 related with the growth of M. smegmatis, for our data show MSMEG_1130 low-expression strain exhibited a rapid growth compared with the control. MSMEG_1130 was also considered as a protein related with the growth of M. smegmatis in other study (Lauten et al., 2010).
AUTHOR CONTRIBUTIONS
QW and LZ conceived and supervised the study; DL, KH, WW, CP, and YD performed experiments. RJ and WX provided new tools and reagents; HyW and HhW analyzed data and helped to design experiments; DL, QW and LZ wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: [URL]. 2017.02231/full#supplementary-material FIGURE S1 | Expression levels of Rv2629 modulate the growth of H37Ra strains under aerobic conditions. The growth curve of recombinant H37Ra overexpressing the Rv2629 W (RaW) and/or Rv2629 M (RaM) genes were measured using the BD BACTEC MGIT 960 Mycobacterium growth and detection system. The results are shown as the fluorescence intensity acquired automatically. RaP (transformed with the pMV261 plasmid) was used as a control. Data are representative of two experiments.
==
Domain: Biology Medicine
|
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