Formation of reduced nicotinamide adenine dinucleotide peroxide

Incubation of NADH at neutral and slightly alkaline pH leads to the gradual absorption of 1 mol of H+. This uptake of acid requires oxygen and mainly yields anomerized NAD+ (NAD+), with only minimal formation od acid-modified NADH. The overall stoichiometry of the reaction is: NADH + H+ + 1/2O2 leads to H2O + NAD+, with NADH peroxide (HO2-NADH+) serving as the intermediate that anomerizes and breaks down to give NAD+ and H2O2. The final reaction reaction mixture contains less than 0.1% of the generated H2O2, which is nonenzymically reduced by NADH. The latter reaction is inhibited by catalase, leading to a decrease in the overall rate of acid absorption, and stimulated by peroxidase, leading to an increase in the overall rate of acid absorption. Although oxygen can attack NADH at either N-1 or C-5 of the dihydropyridine ring, the attack appears to occur primarily at N-1. This assignment is based on the inability of the C-5 peroxide to anomerize, whereas the N-1 peroxide, being a quaternary pyridinium compound, can anomerize via reversible dissociation of H2O2. The peroxidase-catalyzed oxidation of NADH by H2O2 does not lead to anomerization, indicating that anomerization occurs prior to the release of H2O2. Chromatography of reaction mixtures on Dowex 1 formate shows the presence of two major and several minor neutral and cationic degradation products. One of the major products is nicotinamide, which possibly arises from breakdown of nicotinamide-1-peroxide. The other products have not been identified, but may be derived from other isomeric nicotinamide peroxides.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Radioreceptor assay for epidermal growth factor.

An established cell line of human lung fibroblasts with a high number of surface receptorsfor mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. ¹²⁵I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.     

Purification of riboflavin-binding proteins from bovine plasma and discovery of a pregnancy-specific riboflavin-binding protein.

Riboflavin-binding proteins have been purified from bovine plasma using flavinyl agarose beads. At least three major protein bands, migrating in regions assigned to the beta- and gamma-globulins of plasma, are observed by cellulose acetate electrophoresis. These proteins coelute from a calibrated Sephadex G-100 column in the volume corresponding to a molecular weight of approximately 150,000; a small amount of another riboflavin-binding protein (molecular weight approximately 37,000) is also present. Polyacrylamide gel electrophoresis of the proteins, with detection by autoradiography of those having tightly bound [2-14C]riboflavin, reveals one protein band which is present only in preparations from pregnant cows. This protein has been purified to apparent homogeneity by storing the mixture of riboflavin-binding proteins at 8 degrees C for 3 weeks, which precipitates the other, less stable proteins. Hence, bovine plasma, like that of the laying hen, contains a number of riboflavin-binding proteins, one of which correlates with pregnancy.     

Identifying human cells capable of metabolizing various classes of carcinogens

Human cells that appear capable of metabolizing various classes of carcinogens have been identified using one of two methods: metabolism of tritiated benzo(a)pyrene to aqueous-acetone soluble forms or inhibition of cellular DNA synthesis. Each of the assay systems was optimized and the results on 15 human epithelial cell lines were compared. One or more cell lines were found to activate each of four classes of carcinogens examined: polycyclic hydrocarbons, aromatic amines, heterocyclic hydrocarbons, and nitrosamines. Cells that appeared capable of metabolizing polycyclic hydrocarbons or aromatic amines by these methods were also found to produce metabolites which were cytotoxic to cocultivated human xeroderma pigmentosum fibroblasts after a 48-hr exposure to the carcinogen.     

Renal function in patients receiving long-term lithium therapy.

Renal function was assessed in 42 stable outpatients who had been taking lithium for an average of 4 1/2 years. Impaired ability to concentrate the urine was found in 61% of the 41 patients who provided a urine sample for an osmolality measurement, and a moderate reduction in creatinine clearance was present in 12% of the entire group; 1 patient showed both defects. Urine microscopy revealed an excess of cells in 40%. It is suggested that lithium therapy produces a self-limiting lesion of the distal nephron that does not usually progress to chronic renal failure. The lesion is not dangerous, except that it may predispose to acute neurotoxic effects in the event of intercurrent illness or dehydration.     

Ammonium-limited growth and uptake by Oscillatoria agardhii in chemostat cultures

Growth of Oscillatoria agardhil was studied in ammonium-limited chemostat cultures, at various dilution rates (=growth rates, μ). The uptake kinetics for ammonium of nitrogen (ammonium or nitrate)-limited chemostat cultures also was investigated. The kinetics of ammonium-limited growth could be adequately described by both the Monod and Droop equations, and were closely similar to the nitrate-limited growth kinetics of this species. The uptake kinetics for ammonium showed similarities as well as differences with the uptake kinetics for nitrate. The similarities were apparent in the uptake capacity values for ammonium and nitrate , which were identical, high and independent of μ. The differences were to be found in the half-saturation constants for ammonium uptake and nitrate uptake , the former being hardly influenced by μ. A consitutive, high affinity, system is likely to operate in the uptake and assimilation of ammonium by nitrogen-limited O. agardhii. The use of ammonium uptake parameters in studies of growth-limiting factors in nature can provide information as to whether a nitrogen-limitation prevails in natural habitats of this species.     

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.