On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium

In aerobic reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium, O2- production is mediated by the tetrazolium, not the phenazine. Thus, superoxide dismutase inhibited reduction of the tetrazolium, but when ferricytochrome c was substituted for the tetrazolium its reduction was not affected by this enzyme. Furthermore, NADH plus the phenazine did not accelerate the oxidation of epinephrine to adrenochrome unless the tetrazolium was present, and under those circumstances superoxide dismutase did inhibit adrenochrome formation. When the tetrazolium and ferricytochrome c were present simultaneously, addition of superoxide dismutase was seen to accelerate the reduction of the cytochrome. This is explainable by the reduction of O2- by the reduced phenazine, which thus competes with cytochrome c for the available O2-. When the O2- was eliminated by superoxide dismutase, more of the reduced phenazine was available for the direct reduction of cytochrome c.     

Vanadate and molybdate stimulate the oxidation of NADH by superoxide radical

Vanadate or molybdate strongly accelerate the cooxidation of NADH, or of reduced nicotinamide mononucleotide, by the xanthine oxidase plus xanthine reaction. Superoxide dismutase eliminated the effect of vanadate or molybdate, while catalase was without effect. It follows that vanadate or molybdate accelerate the oxidation of dihydropyridines by O-2. A stoichiometry of 4 NADH oxidized per O-2 introduced suggests a chain reaction for which a mechanism is proposed. These results provide an explanation for the reported stimulation, by vanadate, of NADH oxidation by biological membranes.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Direct scanning active enzyme gel chromatography with halted flow for detecting molecular weight heterogeneity of active enzymes

As an extension of the method of scanning active enzyme chromatography we have introduced the option of halting flow while the enzyme is still present on the column. Once the flow has been halted, continuous monitoring of the absorbancy profile provides information on the relative distribution of the enzyme activity. When the baseline is relatively stable, one can detect very low levels of enzyme activity and thus monitor for the presence of small amounts of active enzyme of molecular weight different from that of the predominant form. This gives a significant improvement over regular active enzyme chromatography in the qualitative detection of molecular weight heterogeneity, by in effect reducing the noise level of the experiment. The method has been applied to several systems including aldolase from rabbit muscle, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase of bovine heart and hexokinase and glucose-6-phosphate dehydrogenase from yeast.     

The scavenging of superoxide radical by manganous complexes: in vitro

Dialyzable manganese has been shown to be present in millimolar concentrations within cells of Lactobacillus plantarum and related lactic acid bacteria. This unusual accumulation of Mn appears to serve the same function as Superoxide dismutase (SOD), conferring hyperbaric oxygen and Superoxide tolerance on these SOD-free organisms. The form of the Mn in the lactic acid bacteria and the mechanisms whereby it protects the cell from oxygen damage are unknown. This report examines the mechanisms by which Mn catalytically scavenges O₂^?, both in the xanthine oxidase/cytochrome c SOD assay and in a number of in vitro systems relevant to the in vivo situation. In all the reaction mixtures examined, Mn(II) is first oxidized by O₂^? to Mn(III), and H₂O₂ is formed. In pyrophosphate buffer the Mn(III) thus formed is re-reduced to Mn(II) by a second O₂^?, making the reaction a true metal-catalyzed dismutation like that catalyzed by SOD. Alternatively, if the reaction takes place in orthophosphate or a number of other buffers, the Mn(III) is preferentially reduced largely by reductants other than O₂^?, such as thiols, urate, hydroquinone, or H₂O₂. H₂O₂, a common product of the lactic acid bacteria, reacted rapidly with Mn(III) to form O₂, apparently without intermediate O₂ release. Free hexaquo Mn(II) ions were shown by electron spin resonance spectroscopy and activity assays in noncomplexing buffers to be poorly reactive with O₂^?. In contrast, Mn(II) formed complexes having a high catalytic activity in scavenging O₂^? with a number of organic acids, including malate, pyruvate, propionate, succinate, and lactate, with the Mn-lactate complex showing the greatest activity.     

Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase high-pressure liquid chromatography with electrochemical detection

We describe an analytical procedure for the simultaneous determination of 5-hydroxyindole derivatives using high-pressure liquid chromatography with electrochemical detection. The procedure clearly resolves 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol, and 5-hydroxyindole-3-acetic acid. The C-18 extraction column methodology and high-pressure liquid chromatography-electrochemical detection parameters have been developed to provide a rapid, sensitive, and reproducible quantitative determination of these 5-hydroxyindoles with picogram sensitivity. Chromatograms obtained from the analysis of whole normal mouse brain by the present technique clearly resolve the 5-hydroxyindoles and appear to be uncomplicated by interfering substances.     

Ultrasensitive silver-stain method for the detection of proteins in polyacrylamide gels and immunoprecipitates on agarose gels

The highly sensitive silver-stain procedure for the detection of proteins in polyacrylamide gels has been revised and simplified using a single-step silver ion reduction after suitable treatment of proteins with bifunctional aldehyde. Washing steps were eliminated and excellent reproducibility of results was achieved. Sensitivity obtained using this procedure was at least equal to that obtained with the original one. Use of the present silver-staining methods has been extended to the quantitative analysis of immunoprecipitates on agarose gels, with a good increase of sensitivity and excellent increase of resolution when compared to the Coomassie blue stain.     

Influence of dietary selenium on the mutagenic activity of perfusate and bile from rat liver, perfused with 1,1-dimethylhydrazine

The mutagenic effect of 1,1-dimethylhydrazine (UDMH) was studied in the liver perfusion/cell culture system. Male Wistar rats, fed a selenium-deficient diet with or without selenium supplementation in the drinking water, were used as liver donors. UDMH caused an increased mutation frequency in Chinese hamster V79 cells exposed in the perfusate. The effect was statistically significant with both selenium-deficient and selenium-supplemented livers. With selenium-deficient livers, a significant mutagenic effect was also obtained when V79 cells were treated with bile collected after the administration of UDMH. Bile flow and bile acid excretion were not affected by UDMH treatment of selenium-deficient or selenium-supplemented livers. There was a tendency towards reduced C-oxygenation of N,N-dimethylaniline in microsomes from selenium-deficient livers perfused with UDMH. The lactate/pyruvate ratio in the perfusate was increased by UDMH, the effect being more pronounced with selenium-deficient than selenium-supplemented livers.     

Sedimentation characteristics of subcellular vesicles associated with internalized insulin and those bound with intracellular glucose transport activity

Comparative studies were made on the sedimentation characteristics of microsomal vesicles associated with internalized [¹²⁵I]iodoinsulin and those bound with intracellular glucose transport activity. Upon linear sucrose density gradient centrifugation, the internalized hormone formed a peak slightly, but significantly, on the higher density side of the peak of intracellular glucose transport activity. After a long centrifugation, the peak of ¹²⁵I activity became lower and broader than that of glucose transport activity. Internalized ¹²⁵I activity was also found in the medium-density microsomal fraction, which had little glucose transport activity. Accumulation of ¹²⁵I activity in the medium-density fraction and that in the low-density fraction were both completed in approximately 10 min. Under basal conditions, little, if any, insulin binding activity was detectable in either the medium- or low-density microsomal fractions; in contrast, some glucose transport activity was always present in the low-density fraction. These results indicate that the subcellular distribution of internalized insulin and of intracellular glucose transport activity are different, suggesting that the pathways of intracellular processing of the insulin receptor and the glucose transport mechanism are different.     

Characterization of canine T-lymphocyte subpopulations: the detection of T mu and T gamma lymphocytes with homologous and heterologous immunoglobulins and the requirements for Fc receptor expression

Erythrocyte antibody (EA) rosette techniques employing sheep red blood cells sensitized with canine (homologous) and rabbit (heterologous) IgM and IgG antibodies were used to determine the number of cells with Fc receptors for IgM (Tμ) and IgG (Tγ) among T lymphocytes isolated from peripheral blood and lymph nodes of dogs. The percentages of Tμ and Tγ lymphocytes detected were found to be independent of the species origin of sensitizing antibody. Among peripheral blood T lymphocytes there were 53.0 ± 2.7% Tμ cells and 18.4 ± 3.6% Tγ cells. T lymphocytes obtained from lymph nodes were 62.1 ± 5.4% Tγ and 15.7 ± 2.6% Tγ. The number of Tμ cells detected increased from 20.0% when freshly isolated to 49.1 ± 4.1% after in vitro culture for 2–16 hr. The expression of the Fc-μ receptor in culture was inhibited by cycloheximide, demonstrating a requirement for active protein synthesis. In contrast, the number of Tγ lymphocytes detected did not vary between freshly isolated cells and those which had been cultured for 16 hr. Expression of the Fc-γ receptor during this time period was not inhibited by cycloheximide.     

Copper increases superoxide dismutase activity in rat liver

Superoxide dismutase (SOD) activity in rat liver cytosol and submitochondrial fractions was characterized as enzymatic and nonenzymatic (due to the SOD-like activity of copper) by four approaches: (i) aerobic NBT²⁺ (nitroblue tetrazolium) photoreduction in the absence of EDTA; (ii) aerobic NBT²⁺ photoreduction in the presence of 10^?4m EDTA; (iii) anaerobic NBT²⁺ photoreduction; and (iv) o-dianisidine photooxidation. Under normal conditions nonenzymatic SOD activity has been observed only in the intermembrane space. The single subcutaneous injection of rats with CuSO₄ solution (5 mg Cu/kg body wt) led to (i) an elevation of the copper level in all submitochondrial fractions; (ii) an increase in enzymatic SOD activity in only cytosol and intermembrane spaces; (iii) the appearance of a new electrophoretic SOD activity band in the intermembrane space preparations; and (iv) the appearance of nonenzymatic SOD-like activity in the outer and inner mitochondrial membranes, and a twofold increase in lipid hydroperoxides. This suggests that the increased nonenzymatic copper in vivo has a prooxidant effect, and does not catalyze the dismutation of O₂^? as it has been shown in in vitro experiments [E. M. Russanov S. G. Ljutakova, and S. I. Leutcher (1982) Arch. Biochem. Biophys.215, 220–229]. The peculiarities of the SOD activity in the intermembrane space are explained by the lysosomal localization of the granular CuZnSOD.     

A fluorometric procedure for measuring the neuraminidase activity: its application to the determination of this activity in influenza and parainfluenza viruses

A fluorometric procedure for quantitating the amount of N-acetylneuraminic acid enzymatically released by the neuraminidase activity from N-acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with beta-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD+; finally, the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.     

Multiple molecular forms of phosphorylase phosphatase associated with particulate glycogen and extracted from the cytosol of dog liver.

The glycogen pellet of dog liver extracts contains a phosphorylase phosphatase which has characteristics different from those of the phosphatases extracted from the cytosol. The phosphatase associated with glycogen is characterized by a M, of 51,000, a half maximal inhibition at 0.3 mM ATP (Hill coefficient : 2) and a Ki for Mg2+ of 1 mM. Treatment with urea or mercaptoethanol of the phosphatase associated with glycogen does not influence the activity, the Mr or the half maximal inhibition by ATP, but a decrease of the Hill coefficient for ATP is observed. A similar treatment of the phosphatases extracted from the high speed supernatant results in a decrease of the Mr of the spontaneously active form from 215,000 to 43,000, without an effect on the Ki for ATP (7 micronM), but accompanied by an increase in activity. The ATP-Mg dependent form of the phosphatase from the high speed supernatant (Mr : 138,000 ; Ka for ATP in the presence of 0.1 mM Mg2+ : 0.3 micronM), is denatured by urea or mercaptoethanol. The phosphatase associated with particulate glycogen cannot be found in the supernatant, nor the phosphorylase phosphatases present in the supernatant in the glycogen pellet. When all the glycogen is mobilized (starvation, glucagon) the phosphatase specifically associated with glycogen cannot be found as such in the cytosol. No activation of synthase beta can be detected neither with the phosphatases extracted from the cytosol nor with the enzyme released from the glycogen pellet.     

Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O₂^? at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of O₂^?, which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O₂^? and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to O₂^?, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂O₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O₂^? and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated O₂^? with k₃ values of 0.1 to 1.4 m^? · s^?1 and H₂O₂ with k₄ values of 0.009 to 4.3 m^?1 · s^?1.     

An extracellular 5'-nucleotidase with both monoesterase and diesterase activity from Micrococcus sodonensis.

Bovine γ-globulin was separated into four fractions by chromatography on cellulose phosphate. The chromatographic distribution was similar to that reported for human and dog γ-globulin. More than 80% of a nonspecific phagocytosis stimulating factor (leucokinin) present in the serum was isolated in γ-globulin fraction IV. Bocine red blood cells and polymorphonuclear leukocytes bind γ-globulin without appreciable selectivity for any of the four chromatographic fractions, but they do selectively bind the phagocytosis stimulating factor. Splenectomy caused no observable change in either the chromatographic distribution or phagocytosis stimulating activity of bovine serum γ-globulin. The tetrapeptide tuftsin stimulates phagocytosis by bovine neutrophiles, but on a molar basis the activity of tuftsin was only 10% that of the phagocytosis stimulating factor. If the factor exerts its effect, as has been proposed, by having a phagocytosis stimulating peptide cleaved from it by an enzyme on the leukocyte membrane, that peptide must differ in structure from tuftsin. This conclusion is supported by the inability of trypsin to liberate an active peptide from bovine serum.     

Modulation of natural killer activity in mice following infection with Listeria monocytogenes

Mice that received a sublethal, intraperitoneal dose of viable Listeria monocytogenes, virulent strain 10403, exhibited a systemic increase in natural killer (NK) activity. The kinetics of the response differed with respect to the various effector cell populations analyzed. Resident peritoneal cells and peripheral blood leukocytes demonstrated high NK activity on Days 3, 7, and 10. Peak spleen and bone marrow NK activity was observed on Day 3, returning to normal levels by Day 7. In contrast, peritoneal exudate cells, elicited with proteose peptone, expressed enhanced NK activity for 60 days following infection with viable Listeria. Augmented NK activity was detected with all cell types as early as 12 hr after infection. The intraperitoneal injection of nonviable antigenic preparations derived from L. monocytogenes, strain 10403, resulted in the enhancement of peritoneal and splenic NK activity. In contrast, mice that received an intraperitoneal injection of avirulent Listeria, strain 19113, failed to express enhanced levels of NK activity. The genetic trait of anti-listerial resistance which is associated with non-H-2 linked genes was of no importance with respect to enhanced NK activity. Listeria-resistant C57BL/6J and Listeria-susceptible DBA/2J mice both produced systemic augmentation of NK activity following infection. NK activity was not abrogated by macrophage depletion or by treatment with anti-Thy 1.2 serum plus complement. These results confirm the potent immunostimulatory capacity of virulent Listeria for NK activity and provide further insight into the kinetics of this response in various lymphoid compartments. The protracted augmentation of NK activity of elicited peritoneal exudate cells as compared to nonelicited peritoneal cells in Listeria-primed mice suggests that the influx of inflammatory cells may provide NK-enriched and/or accessory populations for immunopotentiation of NK activity in inflammatory sites.     

Putative superoxide dismutase activity of iron-EDTA: a reexamination

It has been reported that iron-EDTA complexes mimic the action of superoxide dismutase, displaying 0.01% of the activity of the enzyme (Halliwell, B., 1975, FEBS Lett., 56, 34–38). This was purportedly directly confirmed by J. G. McClune, J. A. Fee, G. A. McCluskey, and J. T. Groves, 1977, J. Amer. Chem. Soc., 99, 5220–5222. A reexamination of the behavior of this compound has demonstrated that it does not catalyze the dismutation of O₂^?, but rather inferferes with assays for superoxide dismutation activity, which are based on the reductions of nitroblue tetrazolium or of cytochrome c. The sources of this interference have been examined. Investigators engaged in searching for mimics of superoxide dismutase are urged to be wary of similar artifacts.     

Threshold of plasma testosterone required for normal mating activity in male sheep

The finer control of mating activity by testosterone in male sheep was investigated using the castrated ram (wether) as an experimental model. Adult wethers that had been castrated before puberty were injected with graded doses of testosterone propionate (TP) and mating behavior was assessed in standardized libido trials at various times during treatment. Doses of 1 to 2 mg TP/day elicited mounting behavior in wethers but did not result in intromission or ejaculation. On the other hand, TP doses of 4 mg/day or greater stimulated the complete mating response which included intromission and the ejaculatory reflex. The threshold dose of TP required for complete mating activity (4 mg/day) produced plasma testosterone levels which were lower than those normally observed in intact rams. The results of this study indicate that the behavioral aspects (arousal mechanisms) of mating in rams have a lower testosterone threshold than intromission and ejaculation (consummatory mechanisms). Also, since complete mating activity was stimulated in wethers having relatively low plasma testosterone levels, this may explain why there is no apparent relationship between plasma testosterone and mating drive in intact rams.