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41.
Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae-induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora. 相似文献
42.
A variety of biophysical methods used to study proteins requires protein modification using conjugated molecular probes. Cysteine is the main residue that can be modified without the risk of altering other residues in the protein chain. It is possible to label several cysteines in a protein using highly selective labeling reactions, if the cysteines react at very different rates. The reactivity of a cysteine residue introduced into an exposed surface site depends on the fraction of cysteine in the deprotonated state. Here, it is shown that cysteine reactivity differences can be effectively predicted by an electrostatic model that yields site-specifically the fractions of cysteinate. The model accounts for electrostatic interactions between the cysteinyl anion and side chains, the local protein backbone, and water. The energies of interaction with side chains and the main chain are calculated by using the two different dielectric constants, 40 and 22, respectively. Twenty-six mutants of Escherichia coli adenylate kinase were produced, each containing a single cysteine at the protein surface, and the rates of the reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) were measured. Cysteine residues were chosen on the basis of locations that were expected to allow modification of the protein with minimal risk of perturbing its structure. The reaction rates spanned a range of 6 orders of magnitude. The correlation between predicted fractions of cysteinate and measured reaction rates was strong (R = 92%) and especially high (R = 97%) for cysteines at the helix termini. The approach developed here allows reasonably fast, automated screening of protein surfaces to identify sites that permit efficient preparations of double- or triple-labeled protein. 相似文献
43.
Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion
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The membrane fusion events which initiate human immunodeficiency virus type 1 (HIV-1) infection and promote cytopathic syncytium formation in infected cells commence with the binding of the HIV envelope glycoprotein (Env) to CD4 and an appropriate coreceptor. Here, we show that HIV Env-coreceptor interactions activate Rac-1 GTPase and stimulate the actin filament network reorganizations that are requisite components of the cell fusion process. Disrupting actin filament dynamics with jasplakinolide or latrunculin A arrested fusion at a late step in the formation of Env-CD4-coreceptor complexes. Time-lapse confocal microscopy of living cells revealed vigorous activity of actin-based, target cell membrane extensions at the target cell-Env-expressing cell interface. The expression of dominant-negative forms of actin-regulating Rho-family GTPases established that HIV Env-mediated syncytium formation relies on Rac-1 but not on Cdc42 or Rho activation in target cells. Similar dependencies were found when cell fusion was induced by Env expressed on viral or cellular membranes. Additionally, Rac activity was specifically upregulated in a coreceptor-dependent manner in fusion reaction cell lysates. These results define a role for HIV Env-coreceptor interactions in activating the cellular factors essential for virus-cell and cell-cell fusion and provide evidence for the participation of pertussis toxin-insensitive signaling pathways in HIV-induced membrane fusion. 相似文献
44.
Gussakovsky EE Ionov MV Giller YE Ratner K Aripov TF Shahak Y 《Photosynthesis research》2006,87(3):253-265
Circularly polarized chlorophyll luminescence (CPL) may serve as a measure of chiral macroaggregates of the light-harvesting chlorophyll-protein complexes (LHC II) in both isolated chloroplasts and intact leaves (Gussakovsky et al (2000) Photosynth Res 65: 83–92). In the present work, we applied the CPL approach to study the effect of fast (1–2 min) thermal impacts on LHC II macroaggregates. The results revealed unexpected temperature-response kinetics, composed of initial bell-shaped changes in the CPL signal, followed by degradation down to a steady state (equilibrium). The bell-shape effect was dependent upon illumination, and vanished in the dark. A mathematical analysis of the temperature-response kinetics uniquely indicated that LHC II chiral macroaggregates may persist in both left- and right-handed forms. These forms differ in their response to high temperatures. Both forms are more thermostable in leaves than in isolated chloroplasts. The cooperative degradation of LHC II macroaggregates, which is induced by the thermal impact, is irreversible. It is therefore suggested that the native LHC II macroaggregates are stable, stationary, non-equilibrium, spatially heterogeneous (dissipative) structures. The dissipative properties probably allow the interconversion between left- and right-handed forms under perturbation by certain factors. Illumination probably serves as one such perturbation factor, initiating the interconversion of dark-adapted, left-handed to light-dependent, right-handed LHC II macroaggregates. The chiral heterogeneity of the LHC II macroaggregates is a newly revealed aspect which needs to be taken into consideration in future circular dichroism or CPL studies. 相似文献
45.
Engineering biomaterials to integrate and heal: the biocompatibility paradigm shifts 总被引:1,自引:0,他引:1
This article focuses on one of the major failure routes of implanted medical devices, the foreign body reaction (FBR)--that is, the phagocytic attack and encapsulation by the body of the so-called "biocompatible" biomaterials comprising the devices. We then review strategies currently under development that might lead to biomaterial constructs that will harmoniously heal and integrate into the body. We discuss in detail emerging strategies to inhibit the FBR by engineering biomaterials that elicit more biologically pertinent responses. 相似文献
46.
47.
Binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) with the primary receptor CD4 and one of two coreceptors, CXCR4 or CCR5, activates a signaling cascade resulting in Rac-1 GTPase activation and stimulation of actin cytoskeletal reorganizations critical for HIV-1-mediated membrane fusion. The mechanism by which HIV-1 Env induces Rac-1 activation and subsequent actin cytoskeleton rearrangement is unknown. In this study, we show that Env-mediated Rac-1 activation is dependent on the activation of Galpha(q) and its downstream targets. Fusion and Rac-1 activation are mediated by Galpha(q) and phospholipase C (PLC), as shown by attenuation of fusion and Rac-1 activation in cells either expressing small interfering RNA (siRNA) targeting Galpha(q) or treated with the PLC inhibitor U73122. Rac-1 activation and fusion were also blocked by multiple protein kinase C inhibitors, by inhibitors of intracellular Ca2+ release, by Pyk2-targeted siRNA, and by the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS). Fusion was blocked without altering cell viability or cell surface localization of CD4 and CCR5. Similar results were obtained when cell fusion was induced by Env expressed on viral and cellular membranes and when cell lines or primary cells were the target. Treatment with inhibitors and siRNA specific for Galpha(i) or Galpha(s) signaling mediators had no effect on Env-mediated Rac-1 activation or cell fusion, indicating that the Galpha(q) pathway alone is responsible. These results could provide a new focus for therapeutic intervention with drugs targeting host signaling mediators rather than viral molecules, a strategy which is less likely to result in resistance. 相似文献
48.
M. G. Laivenieks A. B. Tsiomenko E. N. Ratner B. A. Fikhte I. S. Kulaev 《Applied microbiology and biotechnology》1980,10(4):349-356
Summary During the dehydration of exponentially growing yeast cells for 24 h at 37° C, a 2–3 fold increase in the activity of acid phosphatase was observed. This increase is inhibited by cycloheximide and 2-deoxy-D-glucose and therefore is indicative of de novo synthesis. The presence of exogenous orthophosphate during drying does not affect the specific activity of this enzyme, thus indicating the constitutive character of the newly formed acid phosphatase.Freeze-etching showed some rearrangement of the plasmalemma structure of yeast cells during dehydration. 相似文献
49.
50.
Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum. 总被引:2,自引:0,他引:2
We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startlingly frequent level of homologous recombination, the use of cotransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation. 相似文献