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Biochemical Analysis of Interactions between Outer Membrane Proteins That Contribute to Starch Utilization by Bacteroides thetaiotaomicron 总被引:3,自引:0,他引:3
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An early step in the utilization of starch by Bacteroides thetaiotaomicron is the binding of starch to the bacterial surface. Four starch-associated outer membrane proteins of B. thetaiotaomicron that have no starch-degrading activity have been identified. Two of these, SusC and SusD, have been shown by genetic analysis to be required for starch binding. In this study, we provide the first biochemical evidence that these two proteins interact physically with each other. Both formaldehyde cross-linking and nondenaturing gel electrophoresis experiments showed that SusC and SusD interact to form a complex. Two other proteins encoded by genes in the same operon, SusE and SusF, proved not to be essential for starch utilization and actually decreased starch binding when they were present along with SusC and SusD. Consistent with this, nondenaturing gel analysis revealed that in a strain producing SusC, SusD, and SusE, the SusCD complex was partially destabilized. The strain producing SusC, SusD, and SusE also grew more slowly on starch than a strain producing SusC, SusD, SusE, and SusF (mu(max), 0.29 and 0.37/h, respectively). Thus, SusE appears to interact with the SusCD complex. SusE also interacts with SusF, because SusE was less susceptible to proteinase K digestion when SusF was present, and nondenaturing gel analysis detected a complex formed by these two proteins. Our results indicate that SusC, SusD, SusE, and SusF form a protein complex in the outer membrane but that SusE and SusF are dispensable members of this complex. 相似文献
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Lin Chen Gladys J. Keitany Xiaohong Peng Claire Gibson Isaac Mohar Marissa Vignali Ian N. Crispe Fusheng Huang Ruobing Wang 《PloS one》2014,9(7)
Pre-erythrocytic malaria vaccines, including those based on whole-parasite approaches, have shown protective efficacy in animal and human studies. However few pre-erythocytic antigens other than the immunodominant circumsporozoite protein (CSP) have been studied in depth with the goal of developing potent subunit malaria vaccines that are suited for use in endemic areas. Here we describe a novel technique to identify pre-erythrocytic malaria antigens that contribute to protection elicited by whole-parasite vaccination in the mouse model. Our approach combines immunization with genetically attenuated parasites and challenge with DNA plasmids encoding for potential protective pre-erythrocytic malaria antigens as luciferase fusions by hydrodynamic tail vein injection. After optimizing the technique, we first showed that immunization with Pyfabb/f−, a P. yoelii genetically attenuated parasite, induces killing of CSP-presenting hepatocytes. Depletion of CD8+ but not CD4+ T cells diminished the killing of CSP-expressing hepatocytes, indicating that killing is CD8+ T cell-dependent. Finally we showed that the use of heterologous prime/boost immunization strategies that use genetically attenuated parasites and DNA vaccines enabled the characterization of a novel pre-erythrocytic antigen, Tmp21, as a contributor to Pyfabb/f− induced protection. This technique will be valuable for identification of potentially protective liver stage antigens and has the potential to contribute to the understanding of immunity elicited by whole parasite vaccination, as well as the development of effective subunit malaria vaccines. 相似文献
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A Population Density Approach That Facilitates Large-Scale Modeling of Neural Networks: Analysis and an Application to Orientation Tuning 总被引:9,自引:0,他引:9
We explore a computationally efficient method of simulating realistic networks of neurons introduced by Knight, Manin, and Sirovich (1996) in which integrate-and-fire neurons are grouped into large populations of similar neurons. For each population, we form a probability density that represents the distribution of neurons over all possible states. The populations are coupled via stochastic synapses in which the conductance of a neuron is modulated according to the firing rates of its presynaptic populations. The evolution equation for each of these probability densities is a partial differential-integral equation, which we solve numerically. Results obtained for several example networks are tested against conventional computations for groups of individual neurons.We apply this approach to modeling orientation tuning in the visual cortex. Our population density model is based on the recurrent feedback model of a hypercolumn in cat visual cortex of Somers et al. (1995). We simulate the response to oriented flashed bars. As in the Somers model, a weak orientation bias provided by feed-forward lateral geniculate input is transformed by intracortical circuitry into sharper orientation tuning that is independent of stimulus contrast.The population density approach appears to be a viable method for simulating large neural networks. Its computational efficiency overcomes some of the restrictions imposed by computation time in individual neuron simulations, allowing one to build more complex networks and to explore parameter space more easily. The method produces smooth rate functions with one pass of the stimulus and does not require signal averaging. At the same time, this model captures the dynamics of single-neuron activity that are missed in simple firing-rate models. 相似文献
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Cristina Herrera Jéssica Kele A. Macêdo Andrés Feoli Teresa Escalante Alexandra Rucavado José María Gutiérrez Jay W. Fox 《PLoS neglected tropical diseases》2016,10(4)
The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms. 相似文献
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ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins. 相似文献
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gag, vif, and nef Genes Contribute to the Homologous Viral Interference Induced by a Nonproducer Human Immunodeficiency Virus Type 1 (HIV-1) Variant: Identification of Novel HIV-1-Inhibiting Viral Protein Mutants
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Paola DAloja Eleonora Olivetta Roberta Bona Filomena Nappi Daniela Pedacchia Katherina Pugliese Giuliana Ferrari Paola Verani Maurizio Federico 《Journal of virology》1998,72(5):4308-4319