, AND H. ORIN HALVORSON. Properties of a purified halophilic malic dehydrogenase. J. Bacteriol. 90:316-326. 1965.-The malic dehydrogenase (MDH) from Halobacterium salinarium required high concentrations of monovalent ions for stability and activity. Studies of inactivation rates at different salt concentrations suggested that approximately 25% NaCl (w/v) is required to stabilize MDH. From 50 to 100% reactivation, depending on the salt concentration present during inactivation, could occur in 2.5 to 5 M NaCl or KCI. The optimal salt concentration for activity of MDH was a function of the pH, and ranged from 1 to 3 M NaCl or KCl. The effect of salt concentration on the pH-activity curves occurred chiefly below pH 7.0. Inactivation of MDH with heat or thiol reagents showed that the enzyme was more labile in the state induced by absence of salt. The activation of MDH by salts was attributed to a decreased rate of dissociation of MDH and reduced nicotinamide adenine dinucleotide (NADH2). The inactivation of the enzyme in the absence of salt could be largely prevented by the presence of NADH2. The S20,. of MDH decreased threefold at low salt concentrations. The enzyme was assumed to be in its native compact configuration only in the presence of a high concentration of salt.
Purpose: The purpose of this study is to evaluate the role of the cell survival gene clusterin in radiation-induced cell death in human LNCaP and PC-3 prostate cancer models.Experimental Design: Radiation sensitivities were compared in parental and clusterin-overexpressing LNCaP cells and in PC-3 cells and tumors treated with antisense or mismatch clusterin oligonucleotides.Results: Clusterin-overexpressing LNCaP cells were less sensitive to irradiation with significantly lower cell death rates (23% after 8 Gy) compared with parental LNCaP cells (50% after 8 Gy) 3 days after irradiation. Clusterin expression in PC-3 cells after radiation was found to be up-regulated in a dose-dependent manner in vitro by 70% up to 12 Gy and in vivo by 84% up to 30 Gy. Inhibition of clusterin expression in PC-3 cells using antisense oligonucleotides (ASOs) occurred in a sequence-and dose-dependent manner and significantly enhanced radiation-induced apoptosis and decreased PC-3 cell growth rate and plating efficiency. Compared with mismatch control oligonucleotide treatment, clusterin ASO treatment enhanced radiation therapy and significantly reduced PC-3 tumor volume in vivo by 50% at 9 weeks. In addition, TUNEL staining revealed increased number of apoptotic cells in clusterin ASOtreated and irradiated PC-3 tumors, compared with treatment with mismatch control oligonucleotides plus radiation. Conclusions:These findings support the hypothesis that clusterin acts as a cell survival protein that mediates radioresistance through the inhibition of apoptosis. In vivo results further suggest that inactivation of clusterin using ASO technology might offer a novel strategy to improve results of radiation therapy for prostate cancer patients.
Spores of Bacillus megaterium QM B1551 germinated, elongated, and resporulated (microcycle sporogenesis) in simple chemically defined media which permitted no cell division. The second-stage spores thus produced were heat-stable and required heat activation for germination. The original amount of spore deoxyribonucleic acid tripled before completion of the cycle. Acetate and a small amount of a tricarboxylic acid cycle intermediate were the minimal organic metabolic requirements for microcycle sporogenesis. During this cycle, germinated cells oxidized acetate only after a delay, whether or not glucose was initially present. Spores that were germinated in the absence of a carbon source first oxidized an endogenous substrate, and then developed the ability to oxidize acetate.
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