1,940 research outputs found
Recommended from our members
Characterization of a Novel Metalloprotease Secreted by the Type II Secretion System in Vibrio cholerae
Vibrio cholerae, a Gram-negative bacterium, is the etiological agent of cholera. V.
cholerae shuttles between the human host and the aquatic reservoir, where it associates
with marine organisms such as copepods and vertebrate fish. The bacteria use the Type II
Secretion System (T2SS) to release proteins that facilitate V. cholerae survival. Here, we
describe a novel T2SS-dependent protein, collagenase (Clg). The analysis of the
predicted amino acid sequence of Clg revealed a signal peptide, pro-peptide, peptidase
M9 domain with a zinc-metalloprotease HEXXH consensus, and two pre-peptidase Cterminal
(PPC) domains. Clg was purified to apparent homogeneity from the culture
supernatant of V. cholerae and its activity was examined using synthetic and putative
natural substrates. The protease showed activity in zymogram assays, against DQ gelatin
and FALGPA, as well as a purified fish collagen but not against tested human host
derived proteins. The enzymatic activity of Clg was blocked in the presence of
metalloprotease inhibitors. Additionally, site-directed mutagenesis of the Clg predicted
catalytic residues followed by enzymatic assays and SDS-PAGE analyses demonstrated
that the zinc-binding motif is crucial for protein activity, but not for protease secretion.
The maturation of Clg, which leads to disassembly of the two PPC domains, was also
demonstrated
Block of NMDA receptor channels by endogenous neurosteroids: implications for the agonist induced conformational states of the channel vestibule
N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, and their dysfunction is implicated in multiple brain disorders. NMDARs can be allosterically modulated by numerous compounds, including endogenous neurosteroid pregnanolone sulfate. Here, we identify the molecular basis of the use-dependent and voltage-independent inhibitory effect of neurosteroids on NMDAR responses. The site of action is located at the extracellular vestibule of the receptor's ion channel pore and is accessible after receptor activation. Mutations in the extracellular vestibule in the SYTANLAAF motif disrupt the inhibitory effect of negatively charged steroids. In contrast, positively charged steroids inhibit mutated NMDAR responses in a voltage-dependent manner. These results, in combination with molecular modeling, characterize structure details of the open configuration of the NMDAR channel. Our results provide a unique opportunity for the development of new therapeutic neurosteroid-based ligands to treat diseases associated with dysfunction of the glutamate system
Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells
Compelling evidence suggests that the epithelial cell–derived cytokine thymic stromal lymphopoietin (TSLP) may initiate asthma or atopic dermatitis through a dendritic cell–mediated T helper (Th)2 response. Here, we describe how TSLP might initiate and aggravate allergic inflammation in the absence of T lymphocytes and immunoglobulin E antibodies via the innate immune system. We show that TSLP, synergistically with interleukin 1 and tumor necrosis factor, stimulates the production of high levels of Th2 cytokines by human mast cells (MCs). We next report that TSLP is released by primary epithelial cells in response to certain microbial products, physical injury, or inflammatory cytokines. Direct epithelial cell–mediated, TSLP-dependent activation of MCs may play a central role in “intrinsic” forms of atopic diseases and explain the aggravating role of infection and scratching in these diseases
Effects of Gyejibongnyeong-hwan on dysmenorrhea caused by blood stagnation: study protocol for a randomized controlled trial
<p>Abstract</p> <p>Background</p> <p>Gyejibongnyeong-hwan (GJBNH) is one of the most popular Korean medicine formulas for menstrual pain of dysmenorrhea. The concept of blood stagnation in Korean medicine is considered the main factor of causing abdominal pain, or cramps, during menstrual periods. To treat the symptoms, GJBNH is used to fluidify the stagnated blood and induce the blood flow to be smooth, reducing pain as the result. The purpose of this trial is to identify the efficacy of GJBNH in dysmenorrhea caused by blood stagnation.</p> <p>Methods</p> <p>This study is a multi-centre, randomised, double-blind, controlled trial with two parallel arms: the group taking GJBNH and the group taking placebo. 100 patients (women from age 18 to 35) will be enrolled to the trial. Through randomization 50 patients will be in experiment arm, and the other 50 patients will be in control arm. At the second visit (baseline), all participants who were already screened that they fulfil both the inclusion and the exclusion criteria will be randomised into two groups. Each group will take the intervention three times per day during two menstrual cycles. After the treatment for two cycles, each patient will be followed up during their 3<sup>rd</sup>, 4<sup>th </sup>and 5<sup>th </sup>menstrual cycles. From the screening (Visit 1) through the second follow-up (Visit 6) the entire process will take 25 weeks.</p> <p>Discussion</p> <p>This trial will provide evidence for the effectiveness of GJBNH in treating periodical pain due to dysmenorrhea that is caused by blood stagnation. The primary outcome between the two groups will be measured by changes in the Visual Analogue Score (VAS) of pain. The secondary outcome will be measured by the Blood Stagnation Scale, the Short-form McGill questionnaire and the COX menstrual symptom scale. Analysis of covariance (ANCOVA) and repeated measured ANOVA will be used to analyze the data analysis.</p> <p>Trial registration</p> <p>Current Controlled Trials: <a href="http://www.controlled-trials.com/ISRCTN30426947">ISRCTN30426947</a></p
Recommended from our members
The Type II Secretion Pathway in Vibrio cholerae Is Characterized by Growth Phase-Dependent Expression of Exoprotein Genes and Is Positively Regulated by σ[superscript E]
Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The
type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera
toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that
under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas
there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V.
cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S.
Subsequent screening of a V. cholerae genomic library suggested that σ[superscript E] stress response, phosphate metabolism, and the second
messenger 3',5'-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σ[superscript E],
we discovered that the upstream region of the T2S operon possesses both the consensus σ[superscript E] and σ⁷⁰ signatures, and deletion of
the σ[superscript E] binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σ[superscript E] stimulated transcription of T2S in wild-type and isogenic ΔrpoE strains of V. cholerae, providing additional support for the idea that the T2S
complex belongs to the σ[superscript E] regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent
expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches
Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity
Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana-exposed to more than a decade of regular ivermectin treatment-have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread.Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations
Recommended from our members
A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen
Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease
cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the
well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the
interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein
constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals,
as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the
characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the
subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of
fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein
enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular
milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose
that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new
lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization
program.This is the publisher’s final pdf. The published article is copyrighted by the American Society for Microbiology and can be found at: http://jb.asm.org/
Current challenges in software solutions for mass spectrometry-based quantitative proteomics
This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.
Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy
ObjectivesWe sought to explore the relationship between a Tcap gene (TCAP)abnormality and cardiomyopathy.BackgroundHypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) cause severe heart failure and sudden death. Recent genetic investigations have revealed that mutations of genes encoding Z-disc components, including titin and muscle LIM protein (MLP), are the primary cause of both HCM and DCM. The Z-disc plays a role in establishing the mechanical coupling of sarcomeric contraction and stretching, with the titin/Tcap/MLP complex serving as a mechanical stretch sensor. Tcap interacts with the calsarcin, which tethers the calcineurin to the Z-disc.MethodsThe TCAPwas analyzed in 346 patients with HCM (236 familial and 110 sporadic cases) and 136 patients with DCM (34 familial and 102 sporadic cases). Two different in vitro qualitative assays—yeast two-hybrid and glutathion S-transferase pull-down competition—were performed in order to investigate functional changes in Tcap's interaction with MLP, titin, and calsarcin-1 caused by the identified mutations and a reported DCM-associated mutation, R87Q.ResultsTwo TCAPmutations, T137I and R153H, were found in patients with HCM, and another TCAPmutation, E132Q, was identified in a patient with DCM. It was demonstrated by the qualitative assays that the HCM-associated mutations augment the ability of Tcap to interact with titin and calsarcin-1, whereas the DCM-associated mutations impair the interaction of Tcap with MLP, titin, and calsarcin-1.ConclusionsThese observations suggest that the difference in clinical phenotype (HCM or DCM) may be correlated with the property of altered binding among the Z-disc components
- …