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961.
962.
In the hydrogen peroxide (H2O2) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H2O2 in the absence and presence of 0.5 mM -cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H2O2 decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1–20 mM) enhanced cell viability in a dose-dependent manner, with 85% viable cells at 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. -Cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85 ± 3 to 71 ± 4% (p = 0.023) in presence of 3 mM pyruvate plus H2O2; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since -cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H2O2 apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm. 相似文献
963.
The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl2 caused the loss of cell viability in a dose-dependent manner. HgCl2-induced loss of cell viability was not affected by H2O2 scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide NG-nitro-arginine Methyl ester. HgCl2 did not cause changes in DCF fluorescence, an H2O2-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl2-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl2 resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl2-induced cell death is not associated with generation of H2O2 and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl2-induced cytotoxicity in human glioma cells. 相似文献
964.
A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined. The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa). Sequence analysis
of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous
to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated
cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain. A deletion mutant
of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability.
In terms of half-life at 70 °C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme,
XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain. The optimum temperature
of XynX-C was about 5–10 °C lower than that of XynX-TC.
Received: 12 January 2000 / Received revision: 24 April 2000 / Accepted: 1 May 2000 相似文献
965.
Degradation of focal adhesion proteins during nocodazole-induced apoptosis in rat-1 cells 总被引:4,自引:0,他引:4
Kook S Shim SR Kim JI Ahnn JH Jung YK Paik SG Song WK 《Cell biochemistry and function》2000,18(1):1-7
Nocodazole, a microtubule-disrupting agent, induced apoptosis in Rat-1 cells, as indicated by changes in cell morphology, DNA fragmentation, and eventual cell death. During nocodazole-induced apoptosis, normally flat cells became rounded in shape and detached from the extracellular matrix. These morphological changes appeared to be closely associated with degradation of focal adhesion proteins, including p130cas, p125(FAK) and paxillin. p130cas was also degraded in cells treated with staurosporine or etoposide, suggesting that degradation of focal adhesion proteins is a characteristic feature of apoptosis. Nocodazole-induced apoptosis was antagonized by Bcl-2: degradation of focal adhesion proteins was suppressed and cell viability was enhanced in bcl-2 over-expressing cells, even in the presence of nocodazole. Further study of the molecular mechanism of Bcl-2 activation should provide an understanding of the apoptosis induced by disruption of the microtubule network. 相似文献
966.
967.
968.
Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides. 相似文献
969.
Park HS Huh SH Kim Y Shim J Lee SH Park IS Jung YK Kim IY Choi EJ 《The Journal of biological chemistry》2000,275(12):8487-8491
Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death. 相似文献
970.
Ectopic expression of active processed form of atrial natriuretic peptide in skeletal myoblasts 总被引:1,自引:0,他引:1
Byun J Kim SH Kim SZ Heard JM Huh JE Choe YH Park SJ Jung EA Kim DK 《Biochemical and biophysical research communications》2000,270(2):637-642
Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells. The expression cassette, which was flanked by ITRs from AAV-2, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy. 相似文献