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31.
BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells 总被引:5,自引:0,他引:5
Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H2 O2 treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H2 O2 -induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H2 O2 but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H2 O2 exposure and NGF rescue could reflect activation in part of a common antioxidant pathway. 相似文献
32.
M. Teresa García-López Ibon Alkorta M. José Domínguez Rosario González-Mu?iz Rosario Herranz Nils L. Johansen Kjeld Madsen Henning Th?gersen Peter Suzdak 《Letters in Peptide Science》1995,1(6):269-276
Summary In order to enforce different spatial orientations in the C-terminal hexapeptide of neurotensin (NT8–13) and to gain information about the importance of the 10–11 peptide bond for binding to NT receptors, the Pro10-Tyr11 fragment has been replaced with (2R,8S,8aR)-, (2S,8S,8aR)-, (2S,8S,8aS)-, (2S,8R,8aS)- and (2R,8R,8aS)-8-amino-2-benzyl-3-oxoindolizidine-2-carboxylic acid. Molecular dynamics calculations and energy minimization studies have shown that, contrarily to the Pro-Tyr moiety, none of these indolizidines display a tendency to adopt type I and III -turns, but those having (8S,8aR) or (8R,8aS) stereochemistry essentially adopt extended conformations and the (8S,8aS) stereoisomer prefers a nonstandard folding. The four diastereomeric NT8–13 analogues incorporating (8S,8aR) or (8R,8aS) indolizidines displayed binding affinities for the brain NT receptor similar to that of [Ala11]-NT8–13 and only five- to ninefold lower than that of the corresponding analogue, [Phe11]NT8–13. Although this slight decrease could be attributed to differences in conformational behavior between these constrained NT8–13 analogues and [Phe11]NT8–13 or NT8–13, it is not clear whether the -turn around Pro10-AA11 (AA=Phe, Tyr) is conserved upon receptor binding. An excessive restriction in the motions of the aromatic side chain, imposed by the highly steric constraint of the indolizidine moiety, emerges as an alternative explanation. The findings reported here demonstrate the possibility of replacing the Pro10-Tyr11 dipeptide in NT8–13 with a non-peptide residue without affecting considerably the affinity for brain NT receptors. 相似文献
33.
Rosario DAgata Fernando Cassorla Abraham K. Munabi Veronica Mericq Marie C. Gelato Barry D. Albertson D.Lynn Loriaux. 《Steroids》1984,43(2):201-207
The luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH (LHRHa) inhibits rat testicular testosterone secretion. To determine whether LHRHa decreases serum testosterone concentrations solely by inhibiting gonadotropin secretion or, in addition, by influencing directly testicular testosterone biosynthesis, we examined the effects of LHRHa on the activities of 5 key testicular steroidogenic enzymes. Thirty hypophysectomized, hCG treated rats were given either LHRHa (1 μg sc/day) or saline during 7 days. The LHRHa treated animals exhibited a significant decrease of serum testosterone when compared to the control group (498 ± 37 ng/dl vs 2044 ± 105 ng/dl, mean ± SEM, P 〈0.001). 17-Hydroxyprogesterone serum levels were also decreased in the LHRHa treated rats (61 ± 6 ng/dl vs 93 ± 7 ng/dl, P 〈0.005), while serum progesterone levels were similar in both groups of animals. These changes in steroid concentrations were associated with decreases in the musomal enzyme activities of 17-hydroxylase (37 ± 9 vs 654 ± 41 pmol/mg protein/min, P 〈0.001), 17, 20-desmolase (103 ± 9 vs 522 ± 47 pmol/mg protein/min, P 〈0.001), 3β-hydroxysteroid dehydrogenase (1.7 ± 0.02 vs 4.1 ± 0.1 nmol/mg protein/min, P 〈0.001), aromatase (95 ± 7 vs 228 ± 6 pmol/mg protein/ min, P 〈0.001) and 17-ketosteroid reductase (167 ± 9 vs 290 ± 18 pmol/mg protein/min, P 〈0.01) in the LHRHa treated animals. These findings indicate that LHRHa can inhibit directly rat testicular testosterone biosynthesis. 相似文献
34.
Guinea pig antithrombin III has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography. The final product was homogeneous as judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of antithrombin III was determined by progressive inactivation of thrombin coagulant and amidolytic activity. Heparin cofactor activity was demonstrated by immediate inactivation of thrombin by antithrombin III in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin III occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities of heparin. 相似文献
35.
A method has been developed for the measurement of DNA synthesis in vivo using the incorporation of multilabeled, non-radioactive thymidine. Simultaneous intraperitoneal injection of hexalabeled thymidine and tritiated thymidine into a normal adult rat resulted in the incorporation of both labeled nucleosides into the DNA of cells undergoing replication. The DNA of several tissues and organs was analysed, including liver, thymus, spleen, bone marrow, and small intestine. Following extraction with hot trichloroacetic acid, acid hydrolysis, and thin-layer chromatography of the hydrolysates, the isotopic compositions of the thymine products were determined by field ionization mass spectrometry and by scintillation counting. The relative incorporation of radioactive and stable isotope-labeled thymidine was similar in all tissues, and corresponded to the ratio of the two labeled nucleosides in the injected material. These results indicate the feasibility of utilizing thymidine multilabeled with stable isotopes for measurement of cellular proliferation rates in conjunction with cancer therapy. 相似文献
36.
Rosario Donato 《Journal of neurochemistry》1982,39(1):125-131
Abstract: The nonlinearity of single components of the Scatchard plot of S-100 binding to synaptosomal particulate fractions (SYN) and the observation that dilution of the 125 I-labeled S-100 site complex results in a greater extent of dissociation of the tracer in the presence than in the absence of an excess of unlabeled S-100 suggest that sites change their binding behavior depending on fractional occupancy. To study this aspect of the interaction in more detail, 125 I-labeled S-100 binding experiments were conducted in the presence of, or after preincubation of SYN with various concentrations of, unlabeled S-100. The results indicate that: (a) S-100 synaptosomal sites do change their binding behavior depending on fractional occupancy; and (b) the nonrapid equilibrium between bound S-100 and the medium, which has been referred to as the formation of a tight complex between S-100 and its binding sites, is related to the activation of high-affinity sites. However, no univocal interpretation of these data in terms of binding model can be offered at present, as the binding models currently employed in the analysis of ligand-site interactions can each account for only part of the results described in this report. In any case, data obtained by studying 125 I-labeled S-100 binding to untreated SYN at 2°C and to prefixed SYN at 37°C indicate that the physical state of membranes influences both the extent of the interaction and the binding behavior of the sites. 相似文献
37.
Maria P. Abbracchio R. Mark Evans J. Daniel Heck Orazio Cantoni Max Costa 《Biological trace element research》1982,4(4):289-301
The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl2 were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated
10-fold more63Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of63Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance
medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni2+-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased63Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni2+. Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both
the63Ni2+ uptake and cell detachment caused by Ni2+, while dialyzed (amino acid-free) serum was 3–5-fold less effective than undialyzed serum at reducing63Ni2+ uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed
serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel
uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific
amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni2+. Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl2 or 1 mM NiCl2 masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular
weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake. 相似文献
38.
Summary The effect of sampling conditions on the levels of adenine nucleotides, pyridine nucleotides, glycolytic intermediates and related metabolites in yeast has been studied. A systematic examination of the conditions for harvesting has shown that it can be best accomplished by rapid filtration. Delays in the handling for removal of the medium, as is usual in the process of obtaining a number of data reported in the literature, lead to important changes in some of the metabolites examined. It is also shown that when a washing is imperative it can be carried out with a methanol-water mixture (50/50, v/v) cooled at –40° without loss of intracellular concentrations of non-readily diffusible metabolites.On the basis of this experience the outline of a generally applicable procedure is presented. 相似文献
39.
40.
Hao Wu Lei Sun Fabian Blombach Stan J.J. Brouns Ambrosius P. L. Snijders Kristina Lorenzen Robert H. H. van den Heuvel Albert J. R. Heck Sheng Fu Xuemei Li Xuejun C. Zhang Zihe Rao John van der Oost 《Proteins》2010,78(3):705-713
The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc. 相似文献