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991.
Shiflett SL Ward DM Huynh D Vaughn MB Simmons JC Kaplan J 《The Journal of biological chemistry》2004,279(12):10982-10990
We identified VTA1 in a screen for mutations that result in altered vacuole morphology. Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA. These results suggest a VPS defect for Deltavta1 cells. Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting. Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane. Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells. We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white. Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity. A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment. This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport. Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment. 相似文献
992.
S100P stimulates cell proliferation and survival via receptor for activated glycation end products (RAGE) 总被引:14,自引:0,他引:14
Arumugam T Simeone DM Schmidt AM Logsdon CD 《The Journal of biological chemistry》2004,279(7):5059-5065
S100P is a member of the S100 protein family that is expressed in several malignant neoplasms. Currently the effects of this molecule on cell function are unknown. In the present study we investigated the biological effects and mechanisms of action of S100P using NIH3T3 cells. Expression of S100P in NIH3T3 cells led to the presence of S100P in the culture medium, increased cellular proliferation, and enhanced survival after detachment from the culture substrate or after exposure to the chemotherapeutic agent 5-flurouracil. The proliferation and survival effects of S100P expression were duplicated in a time- and concentration-dependent manner by the extracellular addition of purified S100P to wild-type NIH3T3 cells and correlated with the activation of extracellular-regulated kinases (Erks) and NF-kappaB. To determine the mechanisms involved in these effects, we tested the hypothesis that S100P activated RAGE (receptor for activated glycation end products). We found that S100P co-immunoprecipitated with RAGE. Furthermore, the effects of S100P on cell signaling, proliferation, and survival were blocked by agents that interfere with RAGE including administration of an amphoterin-derived peptide known to antagonize RAGE activation, anti-RAGE antibodies, and by expression of a dominant negative RAGE. These data suggest that S100P can act in an autocrine manner via RAGE to stimulate cell proliferation and survival. 相似文献
993.
Ehrenman K Yang G Hong WP Gao T Jang W Brock DA Hatton RD Shoemaker JD Gomer RH 《The Journal of biological chemistry》2004,279(2):837-847
Developing Dictyostelium cells form structures containing approximately 20,000 cells. The size regulation mechanism involves a secreted counting factor (CF) repressing cytosolic glucose levels. Glucose or a glucose metabolite affects cell-cell adhesion and motility; these in turn affect whether a group stays together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase reduces a wide variety of aldehydes to the corresponding alcohols, including converting glucose to sorbitol. The levels of this enzyme previously appeared to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in the ability of cell lysates to reduce both glyceraldehyde and glucose in an NADPH-coupled reaction. Counterintuitively, alrA- cells grow normally and have decreased glucose levels compared with parental cells. The alrA- cells form long unbroken streams and huge groups. Expression of AlrA in alrA- cells causes cells to form normal fruiting bodies, indicating that AlrA affects group size. alrA- cells have normal adhesion but a reduced motility, and computer simulations suggest that this could indeed result in the formation of large groups. alrA- cells secrete low levels of countin and CF50, two components of CF, and this could partially account for why alrA- cells form large groups. alrA- cells are responsive to CF and are partially responsive to recombinant countin and CF50, suggesting that disrupting alrA inhibits but does not completely block the CF signal transduction pathway. Gas chromatography/mass spectroscopy indicates that the concentrations of several metabolites are altered in alrA- cells, suggesting that the Dictyostelium aldehyde reductase affects several metabolic pathways in addition to converting glucose to sorbitol. Together, our data suggest that disrupting alrA affects CF secretion, causes many effects on cellular metabolism, and has a major effect on group size. 相似文献
994.
Sharma M Shubert DE Sharma M Lewis J McGarrigle BP Bofinger DP Olson JR 《Chemico-biological interactions》2003,146(3):237-249
Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium. 相似文献
995.
Abstract We examined the effect of queen size on the probability of new colony establishment in the ant Pogonomyrmex occidentalis. Large queens are significantly more likely to survive than small queens through the initial stages of colony founding. These differences in individual fitness correlates have corresponding effects on colony fitness. In species in which individual queens vary in fitness, sexual allocation ratios should incorporate the individual fitness functions. 相似文献
996.
997.
Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus
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Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the "holdfast," which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export. 相似文献
998.
Morris DH 《Molecular and cellular biochemistry》2003,246(1-2):83-90
Although epidemiologic studies suggest a role for alpha-linolenic acid (ALA) in the prevention of coronary heart disease and certain types of cancer, the findings of clinical studies suggest that ALA is inferior biologically to the n-3 long-chain fatty acids because its bioconversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is limited in humans and because the magnitude of its biologic effects is smaller than that of EPA and DHA. This paper reviews several methodologic issues that may confound the findings of clinical studies and complicate our interpretations of them: the ALA and EPA + DHA dietary enrichment levels; the choice of tissue; the choice of lipid species; and the method of reporting fatty acid composition. Although the ALA enrichment levels used in most clinical studies can be achieved by consuming ground flaxseed, flaxseed oil, canola oil and other ALA-rich plants as part of a typical dietary pattern, the EPA + DHA enrichment levels are not practical and can only be obtained from fish oil supplements. The lack of consistency in the choice of lipids species and the reporting of data makes it difficult to compare outcomes across studies. The choice of tissue (blood) for analysis is a limitation that probably cannot be overcome. The use of practical ALA and EPA+ DHA dietary enrichment levels and some standardization of clinical study design would allow for greater comparisons of outcomes across studies and ensure a more realistic analysis of how individual n-3 fatty acids differ in their biologic effects in humans. 相似文献
999.
Carton I Hermans D Eggermont J 《American journal of physiology. Cell physiology》2003,285(4):C935-C944
An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way. Rho; actin; swelling 相似文献
1000.
Cholesteryl ester transfer protein (CETP) activity is regulated, in part, by lipoprotein composition. We previously demonstrated that CETP activity follows saturation kinetics as cholesteryl ester (CE) levels in the phospholipid surface of donor particles are increased. We propose here that the plateau of CETP activity occurs because the surface concentration of CE in the acceptor becomes rate limiting. This hypothesis was tested in CETP assays between synthetic liposomes whose CE content was varied independently. As donor CE increased, CETP activity followed saturable kinetics, but the slope of the first-order portion of the curve and the maximum achievable CE transfer rate were linearly related to the acceptor's surface CE concentration. These findings, plus studies with free cholesterol-modified LDL, strongly suggest that CE-rich donor liposomes can measure the CETP-accessible CE in acceptor lipoproteins. CETP activity from CE-rich liposomes to multiple control LDLs ranged 1.8-fold despite equivalent CETP binding capacity, suggesting that LDLs vary widely in their capacity to present CE to CETP. Thus, CETP activity depends on the surface availability of substrate lipids in the donor and acceptor. Donor liposomes with high CE content can be used to assess how subtle changes in composition alter the substrate potential of plasma lipoproteins. 相似文献