Serum effects on DNA repair in human cells

Freshly isolated human lymphocytes were used to determine how serum supplements affect cellular capacity to repair UV damage. Repair capacity was always found to be greatest in medium supplemented with autologous plasma. Variability in repair capacity among individuals was greater in serum supplemented medium than in unsupplemented medium. Thus, in vitro cellular responses will most accurately represent in vivo responses if autologous serum factors are present in the culture medium. This is of particular importance in studies attempting to correlate DNA repair capacity with age or susceptibility to carcinogenesis.     

Fungal metabolism and detoxification of the nitropolycyclic aromatic hydrocarbon 1-nitropyrene.

Nitropolycyclic aromatic hydrocarbons are ubiquitous environmental pollutants, many of which are potent mutagens in bacterial and mammalian cells and carcinogenic to rodents. In this study, we investigated the fungal metabolism of 1-nitropyrene and determined the mutagenic activity of the metabolites toward Salmonella typhimurium TA98, TA98NR, and TA100. Cunninghamella elegans metabolized 1-nitropyrene to form glucoside conjugates of 6-hydroxy-1-nitropyrene and 8-hydroxy-1-nitropyrene. The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by application of UV absorption, 1H-nuclear magnetic resonance, and mass spectroscopy. Mutagenicity assays performed on samples extracted from incubations of C. elegans with 1-nitropyrene indicated that mutagenic activity decreased with time. Consistent with the loss in mutagenic activity, the glucoside conjugates of 6- and 8-hydroxy-1-nitropyrene were nonmutagenic in the Salmonella reversion assay. The results indicate that the fungus C. elegans metabolizes 1-nitropyrene to detoxified products.     

Systemic and renal hemodynamic responses to vascular blockade of vasopressin in conscious dogs with ascites

A role for arginine vasopressin has been implicated in the compensatory control of arterial blood pressure in several animal models with reported increases in plasma levels of arginine vasopressin. A threefold elevation in plasma vasopressin has been reported in conscious dogs following constriction of the inferior vena cava. In the present study, infusion of the arginine vasopressin antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine] Arg8-vasopressin into conscious dogs with chronic caval constriction did not decrease mean arterial blood pressure. However, the dose of infused antagonist completely blocked the pressor response to 2 micrograms of exogenous vasopressin. Also the antagonist produced no effect on heart rate, plasma renin activity, or urinary volume and electrolyte excretions. A slight, transient increase (P less than or equal to 0.05) was observed in creatinine clearance and in PAH clearance following antagonist infusion, suggesting a possible decrease in renal vascular resistance. These data suggest that the direct vasoconstrictor actions of vasopressin contribute minimally, if at all, to blood pressure maintenance following chronic caval constriction. Alternatively, blockade of endogenous vasopressin receptors at the level of peripheral arterioles may have resulted in no depressor response due to a masking of this response by other compensatory hormonal and neural pressor systems.     

Effect of a cold stimulus on the synthesis of uncoupling protein in brown adipose tissue of rats and newborn rabbits

Rats are known to respond to a cold stimulus by increasing the activity and amount of the uncoupling protein in brown adipose tissue. A 48 h cold stimulus was found to increase the synthesis of uncoupling protein 3.g-fold in 4–5 week old rats whereas no change was observed with newborn rabbits. The lack of response in the latter case may reflect a difference between rabbits and rats or that synthesis is already maximal in newborn rabbits.     

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies

Summary Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).     

Variation in the oxytocin content of caprine corpora lutea across the breeding season

Ovarian tissues were obtained from cyclic goats during the early, mid and late breeding season. Immunoreactive oxytocin was measured by RIA in tissue extracts after chromatography on octadecylsilica cartridges. Luteal oxytocin concentrations were significantly greater during the early breeding season than during the mid or late breeding season. Oxytocin is luteolytic in goats. High concentrations of luteal oxytocin may be related to the high incidence of short estrus at the onset of the breeding season.     

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions

Summary Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with⁵¹Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, managanese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H₂O₂ became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress. Research supported by American Lung Association Fellowship Training Grant and Research Training Grant, the R. J. Reynolds Corporation, and National Institutes of Health Grants HL29784 and 1 HL 23805.     

Liposome-entrapped antioxidant enzymes prevent lethal O2 toxicity in the newborn rat

Newborn rat pups delivered and maintained in greater than 95% O2 had a lethal time for 50% of population (LT50) of 13 days. Daily intraperitoneal injection per se did not alter the mortality due to inhalation of O2. Survival after 13.5 days of exposure increased from 40 +/- 7% of pups that received buffered saline only to 95 +/- 4% of pups that received liposomes containing superoxide dismutase and catalase. Daily injection of control liposomes containing no enzymes also caused a less dramatic, though significant, increase in survival from 40 +/- 7 to 71 +/- 7% of O2-exposed pups, indicating that the lipid vehicle, as well as the liposome-entrapped antioxidant enzymes, contributed to the protective effect. The component of the protective effect that could be attributed to the antioxidant enzymes was lost if inactive catalase was used in liposome preparation.     

Alterations in magnetic characteristics produced by intracellular particles

Comparisons were made of the magnetic susceptibility in tissue containing intracellular particles with respect to control tissue. Twenty animals, Sprague Dawley rats, were utilized of which ten were injected with FeTPPS₄-acetate particles under one micron in size. Magnetic susceptibility measurements were performed on tumor tissue from the injected and control animals. Studies showed an average susceptibility ratio of 0.79 in the tumors of the control group while in the injected group there was a susceptibility ratio of 1.25 in the tumors of the injected group as compared to the liver tissue in the injected group (p<0.001).     

Human liver sulphamate sulphohydrolase. Determinations of native protein and subunit Mr values and influence of substrate agylcone structure on catalytic properties.

Human sulphamate sulphohydrolase was purified at least 20,000-fold to homogeneity from liver with a three-step four-column procedure, which consisted of a concanavalin A-Sepharose/Blue A agarose coupled step, and Bio-Gel HT step and then a CM-Sepharose step. The procedure was also used to purify enzyme from kidney and placenta. The subunit Mr of liver, kidney and placenta sulphamate sulphohydrolase was assessed to be 56,000 by using SDS/polacrylamide-gel electrophoresis. The native protein Mr of enzyme from all three tissue sources was assessed by gel-permeation chromatography to be approx. 120,000 on Sephacryl S-300 and 100,000 on Fractogel TSK. It is probable that the native enzyme results from dimerization of subunits. Kinetic parameters (km and kcat.) of human liver sulphamate sulphohydrolase were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparin and heparan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, are turned over up to 372000 times faster than the monosaccharide substrate 2-sulphaminoglucosamine. Aglycone structures that influence substrate binding and/or enzyme activity were penultimate-residue C-6 carboxy and C-2 sulphate ester groups and a post-penultimate 2-sulphaminoglucosamine residue. The C-4 hydroxy group of the 2-sulphaminoglucosamine under enzymic attack is involved in binding of substrate to enzyme. The presence of C-6 sulphate ester on the non-reducing end 2-sulphaminoglucosamine stimulates sulphamate bond hydrolysis and substrate affinity if the adjacent monosaccharide residue is idose or 2-sulphoidose, but strongly inhibits hydrolysis if the adjacent monosaccharide residue is iduronic acid. Sulphamate sulphohydrolase is an exoenzyme, since activity toward internal sulphamate bonds was not detected. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure. The presence of aglycone C-2 sulphate ester and aglycone C-6 carboxy groups and C-6 sulphate ester groups on the 2-sulphaminoglucosamine residue under attack considerably affect the pH response. Structurally complex substrates had two pH optima. Incubation temperature and buffer ionic strength markedly influenced pH optima and enzyme activity. Cu2+ and SO4(2-)ions are potent inhibitors of enzyme activity.