16 research outputs found

    Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

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    Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

    Soluble guanylyl cyclase activation by HMR-1766 (ataciguat) in cells exposed to oxidative stress

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    Many vascular diseases are characterized by increased levels of ROS that destroy the biological activity of nitric oxide and limit cGMP formation. In the present study, we investigated the cGMP-forming ability of HMR-1766 in cells exposed to oxidative stress. Pretreatment of smooth muscle cells with H 2O2 reduced cGMP production stimulated by sodium nitroprusside (SNP) or BAY 41-2272. However, pretreatment with H 2O2 significantly increased HMR-1766 responses. Similar results were obtained with SIN-1, menadione, and rotenone. In addition, HMR-1766 was more effective in stimulating heme-free sGC compared with the wild-type enzyme. Interestingly, in cells expressing heme-free sGC, H2O 2 inhibited instead of potentiated HMR-1766 responses, suggesting that the ROS-induced enhancement of cGMP formation was heme dependent. Moreover, using truncated forms of sGC, we observed that the NH2-terminus of the β1-subunit is required for the action of HMR-1766. Finally, to study tolerance development to HMR-1766, cells were pretreated with this sGC activator and reexposed to HMR-1766 or SNP. Results from these experiments demonstrated lack of tolerance development to HMR-1766 as well as lack of cross-tolerance with SNP. We conclude that HMR-1766 is an improved sGC activator as it has the ability to activate oxidized/heme-free sGC and is resistant to the development of tolerance; these observations make HMR-1766 a promising agent for treating diseases associated with increased vascular tone combined with enhanced ROS production. Copyright © 2008 the American Physiological Society

    Correction to: Backbone and side chain NMR assignments of the H-NOX domain from Nostoc sp. in complex with BAY58-2667 (cinaciguat) (Biomolecular NMR Assignments, (2021), 15, 1, (53-57), 10.1007/s12104-020-09982-3)

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    In the original publication of the article, the Acknowledgements section was omitted. The section is as follows: Acknowledgments We acknowledge support of this work by the project “INSPIRED-The National Research Infrastructures on Integrated Structural Biology, Drug Screening Efforts and Drug target functional characterization” (MIS 5002550) which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure”, funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund). GD is supported by the EU Horizon 2020 Marie Skłodowska-Curie fellowship “NMRSIGN” (Grant No. 795175). © 2021, Springer Nature B.V

    Synthesis and Characterization of Novel Poly( N

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    Poly(N-vinylcaprolactam-co-itaconic acid), P(VC-co-IA), gels were synthesized in ethanol by using the free radical cross-linking polymerization method at 60 degrees C for 24 h in the presence of azobis(isobutyronitrile) (AIBN) and ally] methacrylate (AMA) as the initiator and the cross-linking agent, respectively. In order to determine the effect of the synthesis medium on the percentage of gelation (PG) and equilibrium swelling value (ESV), an ethanol/distilled water mixture (80:20, v/v) was also used as the synthesis medium for poly(N-vinylcaprolactam) (PVC) and P(VC-co-IA) gels. The swelling behaviors of the gels were investigated in distilled water at various temperatures and in different pH buffer solutions. Structural, morphologic, and thermal characterization studies of the gels were carried out using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermal gravimetric analysis (TGA), respectively. The lowest PG and highest ESV were obtained for the gel including 5 mol % IA, which was the gel synthesized in the ethanol/distilled water mixture. PVC synthesized in ethanol/water mixture had the highest percentage of gelation. All the gels displayed pH- and temperature-sensitive swelling behavior. The swelling kinetics of the copolymer gels synthesized in ethanol was investigated at pH 10.0, and it was determined that gels containing 5 and 10 mol % of IA indicated non-Fickian and case II swelling behavior, respectively
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