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81.
Chaves-Olarte E Freer E Parra A Guzmán-Verri C Moreno E Thelestam M 《The Journal of biological chemistry》2003,278(10):7956-7963
Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease. Several toxin A-negative strains from C. difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect. Ultrastructural analysis indicated these toxins induce a rounding cytopathic effect and filopodia-like structures. Toxin B variants glucosylated R-Ras, and transfection with a constitutively active mutant of this GTPase protected cells against their cytopathic effect. Treatment of cells with toxin B variants induced detachment from the extracellular matrix and blockade of the epidermal growth factor-mediated phosphorylation of extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins. Treatment with toxin B variants also induced a transient activation of RhoA probably because of inactivation of Rac1. Altogether, these data indicate that the cytopathic effect induced by toxin B variants is because of cell rounding and detachment mediated by R-Ras glucosylation, and the induction of filopodia-like structures is mediated by RhoA activation. Implications for the pathophysiology of C. difficile-induced diarrhea are discussed. 相似文献
82.
Warm temperatures activate TRPV4 in mouse 308 keratinocytes 总被引:7,自引:0,他引:7
Mammalian survival requires constant monitoring of environmental and body temperature. Recently, several members of the transient receptor potential vanilloid (TRPV) subfamily of ion channels have been identified that can be gated by increases in temperature into the warm (TRPV3 and TRPV4) or painfully hot (TRPV1 and TRPV2) range. In rodents, TRPV3 and TRPV4 proteins have not been detected in sensory neurons but are highly expressed in skin epidermal keratinocytes. Here, we show that in response to warm temperatures (>32 degrees C), the mouse 308 keratinocyte cell line exhibits nonselective transmembrane cationic currents and Ca2+ influx. Both TRPV3 and TRPV4 are expressed in 308 cells. However, the warmth-evoked responses we observe most closely resemble those mediated by recombinant TRPV4 on the basis of their electrophysiological properties and sensitivity to osmolarity and the phorbol ester, 4alpha-phorbol-12,13-didecanoate. Together, these data support the notion that keratinocytes are capable of detecting modest temperature elevations, strongly suggest that TRPV4 participates in these responses, and define a system for the cell biological analysis of warmth transduction. 相似文献
83.
Molinari P Ambrosio C Riitano D Sbraccia M Grò MC Costa T 《The Journal of biological chemistry》2003,278(18):15778-15788
Fusion proteins between heptahelical receptors (GPCR) and G protein alpha-subunits show enhanced signaling efficiency in transfected cells. This is believed to be the result of molecular proximity, because the interaction between linked modules of one protein chain, if not constrained by structure, should be strongly favored compared with the same in which partners react as free species. To test this assumption we made a series of fusion proteins (type 1 and 4 opioid receptors with G(o) and beta(2) adrenergic and dopamine 1 receptors with G(sL)) and some mutated analogs carrying different tags and defective GPCR or Galpha subunits. Using cotransfection experiments with readout protocols able to distinguish activation at fused and non-fused alpha-subunits, we found that both the GPCR and the Galpha limb of one fusion protein can freely interact with non-fused proteins and the tethered partners of a neighboring fusion complex. Moreover, a bulky polyanionic inhibitor can suppress with identical potency receptor-Galpha interaction, either when occurring between latched domains of a fused system or separate elements of distinct molecules, indicating that the binding surfaces are equally accessible in both cases. These data demonstrate that there is no entropy drive from the linked condition of fusion proteins and suggest that their signaling may result from the GPCR of one complex interacting with the alpha-subunit of another. Moreover, the enhanced coupling efficiency commonly observed for fusion proteins is not due to the receptor tether, but to the transmembrane helix that anchors Galpha to the membrane. 相似文献
84.
Spisni E Bianco MC Griffoni C Toni M D'Angelo R Santi S Riccio M Tomasi V 《Journal of cellular physiology》2003,197(2):198-204
A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2. 相似文献
85.
Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages 总被引:7,自引:0,他引:7
Porporatto C Bianco ID Riera CM Correa SG 《Biochemical and biophysical research communications》2003,304(2):266-272
Chitosan is a linear polymer of N-acetyl-D-glucosamine and deacetylated glucosamine widely used as a wound-healing accelerator in clinical and veterinary medicine. Chitosan enhances the functions of inflammatory cells such as macrophages (Mphi), inducing the production of cytokines as well as the expression of activation markers, Fc receptors and mannose receptor. In this work we studied the effects of chitosan on the arginine metabolic pathways of both resident and inflammatory (proteose-peptone elicited) rat Mphi. Our results show that low molecular weight (LMW) chitosan activated moderately both the inducible nitric oxide synthase (iNOS) and arginase pathways in resident Mphi. In inflammatory Mphi treated with chitosan instead, the arginase activity was strongly enhanced. Supernatants of chitosan-stimulated Mphi enhanced the proliferation of the rat cell line C6. These findings suggest that the healing activity of chitosan could rely on the enhanced arginase activity observed in a wound-associated inflammatory milieu. 相似文献
86.
Zito F Costa C Sciarrino S Poma V Russo R Angerer LM Matranga V 《Developmental biology》2003,264(1):217-227
Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-beta superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis. 相似文献
87.
The directionality of matrix deposition in vivo is governed by the ability of a cell to direct vesicularflow to a specific target site. Osteoblastic cells direct newly synthesized bone matrix proteins toward the bone surface. In this study, we dissect the molecular mechanisms underlying the polarized trafficking of matrix protein in osteoblasts. We demonstrate using TEM, immunocytochemistry, and cDNA analysis, the ability of osteoblastic cells in culture to form tight junction-like structures and report the expression of the tight junction associated proteins occludin and claudins 1-3 in these cells. We identify intercellular contact sites and the leading edge of migratory osteoblasts as major target sites of vesicular trafficking in osteoblasts. Proteins required for this process, rsec6, NSF, VAMP1, and syntaxin 4, as well as the bone matrix protein, osteopontin, localize to these sites. We demonstrate that osteoblasts in vivo possess VAMP1 and, furthermore, report the expression of two VAMP1 splice variants in these cells. In addition, osteoblasts express the NSF attachment protein alpha-SNAP and the t-SNARE SNAP23. Thus, cell-to-cell contact sites and the leading edge of migratory osteoblasts contain a unique complement of proteins required for SNARE mediated membrane fusion. 相似文献
88.
89.
Callebaut I Curcio-Morelli C Mornon JP Gereben B Buettner C Huang S Castro B Fonseca TL Harney JW Larsen PR Bianco AC 《The Journal of biological chemistry》2003,278(38):36887-36896
The three iodothyronine selenodeiodinases catalyze the initiation and termination of thyroid hormone effects in vertebrates. Structural analyses of these proteins have been hindered by their integral membrane nature and the inefficient eukaryotic-specific pathway for selenoprotein synthesis. Hydrophobic cluster analysis used in combination with Position-specific Iterated BLAST reveals that their extramembrane portion belongs to the thioredoxin-fold superfamily for which experimental structure information exists. Moreover, a large deiodinase region imbedded in the thioredoxin fold shares strong similarities with the active site of iduronidase, a member of the clan GH-A-fold of glycoside hydrolases. This model can explain a number of results from previous mutagenesis analyses and permits new verifiable insights into the structural and functional properties of these enzymes. 相似文献
90.
Nisole S Said EA Mische C Prevost MC Krust B Bouvet P Bianco A Briand JP Hovanessian AG 《The Journal of biological chemistry》2002,277(23):20877-20886
The multivalent pseudopeptide HB-19 that binds the cell-surface-expressed nucleolin is a potent inhibitor of human immunodeficiency virus (HIV) infection by blocking virus particle attachment and thus anchorage in the plasma membrane. We show that cross-linking of surface-bound HB-19A (like HB-19 but with a modified template) results in aggregation of HB-19A with surface nucleolin. Consistent with its specific action, HB-19A binding to different types of cells reaches saturation at concentrations that have been reported to result in inhibition of HIV infection. By using Chinese hamster ovary mutant cell lines, we confirm that the binding of HB-19A to surface nucleolin is independent of heparan and chondroitin sulfate proteoglycans. In vitro generated full-length nucleolin was found to bind HB-19A, whereas the N-terminal part containing the acidic amino acid stretches of nucleolin did not. The use of various deletion constructs of the C-terminal part of nucleolin then permitted the identification of the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif, RGG, as the domain that binds HB-19A. Finally, a synthetic peptide corresponding to the last C-terminal 63 amino acids was able to inhibit HIV infection at the stage of HIV attachment to cells, thus suggesting that this domain could be functional in the HIV anchorage process. 相似文献