The Experts below are selected from a list of 144 Experts worldwide ranked by ideXlab platform
Timothy L. Cover - One of the best experts on this subject based on the ideXlab platform.
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Structural Characterization of the Helicobacter Pylori VacA Toxin by Single Particle Em and X-Ray Crystallography
Biophysical Journal, 2013Co-Authors: Melissa G. Chambers, Tasia M. Pyburn, Christian González-rivera, Scott E. Collier, Yoshimasa Takizawa, D. Borden Lacy, Timothy L. CoverAbstract:Helicobacter pylori is a Gram-negative bacterium that infects the human stomach and contributes to the pathogenesis of peptic ulceration, gastric adenocarcinoma and gastric lymphoma. H. pylori secretes an exotoxin called vacuolating toxin (VacA), known for its ability to induce vacuolation in the cytoplasm of mammalian cells. VacA can cause depolarization of Membrane potential, alteration of mitochondrial Membrane permeability, apoptosis, activation of mitogen-activated protein kinases, inhibition of T cell activation and proliferation, and autophagy. The mechanisms by which these processes occur are not yet fully understood but many of these toxic effects depend on the capacity of VacA to form Anion-Selective Membrane channels. VacA is an 88 kDa protein that contains two distinct domains, p55 and p33. The 88 kDa monomers can assemble into large water-soluble oligomeric “flower”-shaped structures. Using single particle electron microscopy and the random conical tilt approach, we have determined three-dimensional (3D) structures of six distinct VacA oligomeric conformations at ∼15 A resolution. This analysis shows that VacA can organize into a number oligomeric conformations that include both single and double layer hexamers and heptamers. The structures, regardless of oligomeric type, contain two prominent features: extended straight “legs” with a slight kink at the distal end and a central “spoke-like” density that contains two distinct globular domains separated by a thin connecting density. We have also generated structures of three VacA mutant proteins that all form oligomers but differ in activity. Overall, these studies provide the most detailed analysis of p33 structure to date and also provide a more thorough understanding of how VacA forms oligomers.
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Random Mutagenesis of Helicobacter pylori vacA To Identify Amino Acids Essential for Vacuolating Cytotoxic Activity
Infection and Immunity, 2006Co-Authors: Mark S. Mcclain, Gabor Szabo, Zhifeng Shao, Daniel M. Czajkowsky, Victor J. Torres, Timothy L. CoverAbstract:VacA is a secreted toxin that plays a role in Helicobacter pylori colonization of the stomach and may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. In this study, we analyzed a library of plasmids expressing randomly mutated forms of recombinant VacA and identified 10 mutant VacA proteins that lacked vacuolating cytotoxic activity when added to HeLa cells. The mutations included six single amino acid substitutions within an amino-terminal hydrophobic region and four substitutions outside the amino-terminal hydrophobic region. All 10 mutations mapped within the p33 domain of VacA. By introducing mutations into the H. pylori chromosomal vacA gene, we showed that secreted mutant toxins containing V21L, S25L, G121R, or S246L mutations bound to cells and were internalized but had defects in vacuolating activity. In planar lipid bilayer and Membrane depolarization assays, VacA proteins containing V21L and S25L mutations were defective in formation of Anion-Selective Membrane channels, whereas proteins containing G121R or S246L mutations retained channel-forming capacity. These are the first point mutations outside the amino-terminal hydrophobic region that are known to abrogate vacuolating toxin activity. In addition, these are the first examples of mutant VacA proteins that have defects in vacuolating activity despite exhibiting channel activities similar to those of wild-type VacA.
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Functional Properties of the p33 and p55 Domains of the Helicobacter pylori Vacuolating Cytotoxin
Journal of Biological Chemistry, 2005Co-Authors: Victor J. Torres, Mark S. Mcclain, Susan E. Ivie, Timothy L. CoverAbstract:Helicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of Anion-Selective Membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma Membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties.
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Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Mark S. Sundrud, Victor J. Torres, Derya Unutmaz, Timothy L. CoverAbstract:Recent evidence indicates that the secreted Helicobacter pylori vacuolating toxin (VacA) inhibits the activation of T cells. VacA blocks IL-2 secretion in transformed T cell lines by suppressing the activation of nuclear factor of activated T cells (NFAT). In this study, we investigated the effects of VacA on primary human CD4+ T cells. VacA inhibited the proliferation of primary human T cells activated through the T cell receptor (TCR) and CD28. VacA-treated Jurkat T cells secreted markedly diminished levels of IL-2 compared with untreated cells, whereas VacA-treated primary human T cells continued to secrete high levels of IL-2. Further experiments indicated that the VacA-induced inhibition of primary human T cell proliferation was not attributable to VacA effects on NFAT activation or IL-2 secretion. We show here that VacA suppresses IL-2-induced cell-cycle progression and proliferation of primary human T cells without affecting IL-2-dependent survival. Through the analysis of a panel of mutant VacA proteins, we demonstrate that VacA-mediated inhibition of T cell proliferation requires an intact N-terminal hydrophobic region necessary for the formation of Anion-Selective Membrane channels. Remarkably, we demonstrate that one of these mutant VacA proteins [VacA-Δ(6–27)] abrogates the immunosuppressive actions of wild-type VacA in a dominant-negative fashion. We suggest that VacA may inhibit the clonal expansion of T cells that have already been activated by H. pylori antigens, thereby allowing H. pylori to evade the adaptive immune response and establish chronic infection.
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Clustering and Redistribution of Late Endocytic Compartments in Response to Helicobacter pylori Vacuolating Toxin
Molecular Biology of the Cell, 2004Co-Authors: Yi Li, Angela Wandinger-ness, James R. Goldenring, Timothy L. CoverAbstract:Helicobacter pylori VacA is a secreted protein toxin that may contribute to the pathogenesis of peptic ulcer disease and gastric adenocarcinoma. When added to cultured mammalian cells in the presence of weak bases (e.g., ammonium chloride), VacA induces the formation of large cytoplasmic vacuoles. Here, we report a previously unrecognized capacity of VacA to induce clustering and perinuclear redistribution of late endocytic compartments. In contrast to VacA-induced cell vacuolation, VacA-induced clustering and redistribution of late endocytic compartments are not dependent on the presence of weak bases and are not inhibited by bafilomycin A1. VacA mutant toxins defective in the capacity to form Anion-Selective Membrane channels fail to cause clustering and redistribution. VacA-induced clusters of late endocytic compartments undergo transformation into vacuoles after the addition of ammonium chloride. VacA-induced clustering and redistribution of late endocytic compartments occur in cells expressing wild-type or constitutively active Rab7, but not in cells expressing dominant-negative mutant Rab7. In VacA-treated cells containing clustered late endocytic compartments, overexpression of dominant-negative Rab7 causes reversion to a nonclustered distribution. Redistribution of late endocytic compartments to the perinuclear region requires a functional microtubule cytoskeleton, whereas clustering of these compartments and vacuole formation do not. These data provide evidence that clustering of late endocytic compartments is a critical mechanistic step in the process of VacA-induced cell vacuolation. We speculate that VacA-induced alterations in late endocytic Membrane traffic contribute to the capacity of H. pylori to persistently colonize the human gastric mucosa.
Stefan Matile - One of the best experts on this subject based on the ideXlab platform.
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Anion-macrodipole interactions: self-assembling oligourea/amide macrocycles as anion transporters that respond to Membrane polarization.
Journal of the American Chemical Society, 2009Co-Authors: Andreas Hennig, Lucile Fischer, Gilles Guichard, Stefan MatileAbstract:Macrocyclic urea/amide hybrids are introduced as functional, Anion-Selective Membrane transporters in lipid bilayer Membranes. Six derivatives with varying side chains (aliphatic and aromatic) and conformations (parallel and antiparallel carbonyl dipoles) are investigated by fluorescence methods, among which the more active aromatic derivatives were selected for an in-depth study. Strong response of transport activity toward anion exchange and weak response toward cation exchange establish anion selectivity for all macrocycles. “Antiparallel” macrocycles that self-assemble into “antiparallel” nanotubes without macrodipole exhibit Hofmeister selectivity. Parallel macrocycles that self-assemble into parallel nanotubes with strong macrodipole are capable of overcoming the dehydration penalty of the Hofmeister bias. Both systems show additional chloride selectivity. The activity of antiparallel and parallel nanotubes in binary mixtures of bromide/perchlorate and chloride/thiocyanate is over- and underadditive...
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Anion-Macrodipole Interactions: Self-Assembling Oligourea/Amide Macrocycles as Anion Transporters that Respond to Membrane Polarization.
Journal of the American Chemical Society, 2009Co-Authors: Andreas Hennig, Lucile Fischer, Gilles Guichard, Stefan MatileAbstract:Macrocyclic urea/amide hybrids are introduced as functional, Anion-Selective Membrane transporters in lipid bilayer Membranes. Six derivatives with varying side chains (aliphatic and aromatic) and conformations (parallel and antiparallel carbonyl dipoles) are investigated by fluorescence methods, among which the more active aromatic derivatives were selected for an in-depth study. Strong response of transport activity toward anion exchange and weak response toward cation exchange establish anion selectivity for all macrocycles. "Antiparallel" macrocycles that self-assemble into "antiparallel" nanotubes without macrodipole exhibit Hofmeister selectivity. Parallel macrocycles that self-assemble into parallel nanotubes with strong macrodipole are capable of overcoming the dehydration penalty of the Hofmeister bias. Both systems show additional chloride selectivity. The activity of antiparallel and parallel nanotubes in binary mixtures of bromide/perchlorate and chloride/thiocyanate is over- and underadditive, respectively (positive and negative AMFE). The activity of antiparallel nanotubes decreases rapidly with increasing Membrane polarization, whereas parallel nanotubes are inactivated at high and activated by Membrane potentials at low concentration. Hill coefficients of parallel nanotubes decrease significantly with Membrane polarization, whereas those of antiparallel nanotubes increase slightly. The overall unusual characteristics of parallel nanotubes call for a new transport mechanism, where macrodipole-potential interactions account for voltage sensitivity and anion-macrodipole interactions account for anion selectivity.
Andreas Hennig - One of the best experts on this subject based on the ideXlab platform.
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Anion-macrodipole interactions: self-assembling oligourea/amide macrocycles as anion transporters that respond to Membrane polarization.
Journal of the American Chemical Society, 2009Co-Authors: Andreas Hennig, Lucile Fischer, Gilles Guichard, Stefan MatileAbstract:Macrocyclic urea/amide hybrids are introduced as functional, Anion-Selective Membrane transporters in lipid bilayer Membranes. Six derivatives with varying side chains (aliphatic and aromatic) and conformations (parallel and antiparallel carbonyl dipoles) are investigated by fluorescence methods, among which the more active aromatic derivatives were selected for an in-depth study. Strong response of transport activity toward anion exchange and weak response toward cation exchange establish anion selectivity for all macrocycles. “Antiparallel” macrocycles that self-assemble into “antiparallel” nanotubes without macrodipole exhibit Hofmeister selectivity. Parallel macrocycles that self-assemble into parallel nanotubes with strong macrodipole are capable of overcoming the dehydration penalty of the Hofmeister bias. Both systems show additional chloride selectivity. The activity of antiparallel and parallel nanotubes in binary mixtures of bromide/perchlorate and chloride/thiocyanate is over- and underadditive...
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Anion-Macrodipole Interactions: Self-Assembling Oligourea/Amide Macrocycles as Anion Transporters that Respond to Membrane Polarization.
Journal of the American Chemical Society, 2009Co-Authors: Andreas Hennig, Lucile Fischer, Gilles Guichard, Stefan MatileAbstract:Macrocyclic urea/amide hybrids are introduced as functional, Anion-Selective Membrane transporters in lipid bilayer Membranes. Six derivatives with varying side chains (aliphatic and aromatic) and conformations (parallel and antiparallel carbonyl dipoles) are investigated by fluorescence methods, among which the more active aromatic derivatives were selected for an in-depth study. Strong response of transport activity toward anion exchange and weak response toward cation exchange establish anion selectivity for all macrocycles. "Antiparallel" macrocycles that self-assemble into "antiparallel" nanotubes without macrodipole exhibit Hofmeister selectivity. Parallel macrocycles that self-assemble into parallel nanotubes with strong macrodipole are capable of overcoming the dehydration penalty of the Hofmeister bias. Both systems show additional chloride selectivity. The activity of antiparallel and parallel nanotubes in binary mixtures of bromide/perchlorate and chloride/thiocyanate is over- and underadditive, respectively (positive and negative AMFE). The activity of antiparallel nanotubes decreases rapidly with increasing Membrane polarization, whereas parallel nanotubes are inactivated at high and activated by Membrane potentials at low concentration. Hill coefficients of parallel nanotubes decrease significantly with Membrane polarization, whereas those of antiparallel nanotubes increase slightly. The overall unusual characteristics of parallel nanotubes call for a new transport mechanism, where macrodipole-potential interactions account for voltage sensitivity and anion-macrodipole interactions account for anion selectivity.
Luc Pinoy - One of the best experts on this subject based on the ideXlab platform.
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Influence of the type of anion Membrane on solvent flux and back diffusion in electrodialysis of concentrated NaCl solutions
Chemical Engineering Science, 2014Co-Authors: Thomas Rottiers, K. Ghyselbrecht, Boudewijn Meesschaert, B. Van Der Bruggen, Luc PinoyAbstract:Abstract The potential of using electrodialysis as a technology for separations in highly concentrated solutions is limited by back diffusion of ions and solvent flux from the diluate to the concentrate. Solvent flux dilutes the concentrate stream while simultaneously back diffusion forms a limitation to reach high ion concentrations. In this study the diffusion flux of sodium and chloride in two different combinations of ion-exchange Membranes, namely PC-SK/PC-SA with standard anion exchange Membranes and PC-SK/PC-MVA with monovalent selective anion Membranes, are studied for feed solutions with very high concentrations. It was found that diffusion fluxes of both ions are hardly influenced by the type of Anion-Selective Membrane and are solely concentration dependant. To assure an efficient separation concentration gradients must be small to minimize back diffusion. Furthermore, the quantitative influence of operating parameters on the solvent flux caused by osmosis and electro-osmosis was investigated. Electro-osmosis was found to be independent from the concentration gradient and proportional with the current density. Osmosis only depends on the concentration gradient. When the concentration gradient is below 1 mol L −1 the flux by osmosis was at least eight times smaller than by electro-osmosis. Membranes with high steric hindrance are therefore necessary to minimize the electro-osmotic flux.
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Influence of the type of anion Membrane on solvent flux and back diffusion in electrodialysis of concentrated NaCl solutions
Chemical Engineering Science, 2014Co-Authors: Thomas Rottiers, K. Ghyselbrecht, Boudewijn Meesschaert, Bart Van Der Bruggen, Luc PinoyAbstract:The potential of using electrodialysis as a technology for separations in highly concentrated solutions is limited by back diffusion of ions and solvent flux from the diluate to the concentrate. Solvent flux dilutes the concentrate stream while simultaneously back diffusion forms a limitation to reach high ion concentrations. In this study the diffusion flux of sodium and chloride in two different combinations of ion-exchange Membranes, namely PC-SK/PC-SA with standard anion exchange Membranes and PC-SK/PC-MVA with monovalent selective anion Membranes, are studied for feed solutions with very high concentrations. It was found that diffusion fluxes of both ions are hardly influenced by the type of Anion-Selective Membrane and are solely concentration dependant. To assure an efficient separation concentration gradients must be small to minimize back diffusion. Furthermore, the quantitative influence of operating parameters on the solvent flux caused by osmosis and electro-osmosis was investigated. Electro-osmosis was found to be independent from the concentration gradient and proportional with the current density. Osmosis only depends on the concentration gradient. When the concentration gradient is below 1molL-1 the flux by osmosis was at least eight times smaller than by electro-osmosis. Membranes with high steric hindrance are therefore necessary to minimize the electro-osmotic flux. ?? 2014 Elsevier Ltd.
Lucile Fischer - One of the best experts on this subject based on the ideXlab platform.
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Anion-macrodipole interactions: self-assembling oligourea/amide macrocycles as anion transporters that respond to Membrane polarization.
Journal of the American Chemical Society, 2009Co-Authors: Andreas Hennig, Lucile Fischer, Gilles Guichard, Stefan MatileAbstract:Macrocyclic urea/amide hybrids are introduced as functional, Anion-Selective Membrane transporters in lipid bilayer Membranes. Six derivatives with varying side chains (aliphatic and aromatic) and conformations (parallel and antiparallel carbonyl dipoles) are investigated by fluorescence methods, among which the more active aromatic derivatives were selected for an in-depth study. Strong response of transport activity toward anion exchange and weak response toward cation exchange establish anion selectivity for all macrocycles. “Antiparallel” macrocycles that self-assemble into “antiparallel” nanotubes without macrodipole exhibit Hofmeister selectivity. Parallel macrocycles that self-assemble into parallel nanotubes with strong macrodipole are capable of overcoming the dehydration penalty of the Hofmeister bias. Both systems show additional chloride selectivity. The activity of antiparallel and parallel nanotubes in binary mixtures of bromide/perchlorate and chloride/thiocyanate is over- and underadditive...
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Anion-Macrodipole Interactions: Self-Assembling Oligourea/Amide Macrocycles as Anion Transporters that Respond to Membrane Polarization.
Journal of the American Chemical Society, 2009Co-Authors: Andreas Hennig, Lucile Fischer, Gilles Guichard, Stefan MatileAbstract:Macrocyclic urea/amide hybrids are introduced as functional, Anion-Selective Membrane transporters in lipid bilayer Membranes. Six derivatives with varying side chains (aliphatic and aromatic) and conformations (parallel and antiparallel carbonyl dipoles) are investigated by fluorescence methods, among which the more active aromatic derivatives were selected for an in-depth study. Strong response of transport activity toward anion exchange and weak response toward cation exchange establish anion selectivity for all macrocycles. "Antiparallel" macrocycles that self-assemble into "antiparallel" nanotubes without macrodipole exhibit Hofmeister selectivity. Parallel macrocycles that self-assemble into parallel nanotubes with strong macrodipole are capable of overcoming the dehydration penalty of the Hofmeister bias. Both systems show additional chloride selectivity. The activity of antiparallel and parallel nanotubes in binary mixtures of bromide/perchlorate and chloride/thiocyanate is over- and underadditive, respectively (positive and negative AMFE). The activity of antiparallel nanotubes decreases rapidly with increasing Membrane polarization, whereas parallel nanotubes are inactivated at high and activated by Membrane potentials at low concentration. Hill coefficients of parallel nanotubes decrease significantly with Membrane polarization, whereas those of antiparallel nanotubes increase slightly. The overall unusual characteristics of parallel nanotubes call for a new transport mechanism, where macrodipole-potential interactions account for voltage sensitivity and anion-macrodipole interactions account for anion selectivity.
