Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Autolysis of psychrophilic bacteria from marine fish.

Two psychrophillic bacterial isolates of marine fish origin unable to grow at 20 degrees C or above were found to be distinguishable on the basis of autolysis at elevated temperature in various buffer systems. Isolate OP2 exhibited autolysis at 30 degrees C and above, while isolate OP7 underwent autolysis only at 35 degrees C and above. Tris buffer at pH 7.0 and 8.0 and at 35 degrees C significantly protected isolate OP2 from autolysis and failed to do so with isolate OP7. At pH 5.0, suspension phosphate buffer resulted in significantly greater autolysis of both isolates than did suspension in succinate buffer.     

Platelet immune complex interaction in pathogenesis of Kawasaki disease and childhood polyarteritis.

The role of platelets in the pathogenesis of vasculitis and the formation of coronary artery aneurysms was studied in 19 children with Kawasaki disease and five with polyarteritis. All patients with Kawasaki disease developed thrombocytosis in the third week of illness. The peak platelet count was significantly correlated (p less than 0.005) with the subsequent development of coronary artery aneurysms. The rise in platelet count was associated with the appearance in the circulation of a factor that induced aggregation and serotonin release in normal platelets. This factor was shown to be of high molecular weight, and its activity was lost at low pH--features suggestive of an immune complex. Immune complexes, detected by precipitation with polyethylene glycol, also appeared in the circulation as the platelet count increased. These complexes induced platelet aggregation, and there was a significant correlation (p less than 0.001) between the concentrations of IgG and IgA in the polyethylene glycol precipitated material and the platelet aggregating activity. Similar platelet aggregating activity was also detected in patients with polyarteritis but followed a different time course, persisting in the circulation for several months in association with continued disease activity. These findings imply that different mechanisms have a role in distinct phases of Kawasaki disease. The initial feverish phase (probably infective) is probably followed by an immune complex vasculitis that occurs when antibodies to the initiating agent appear in the circulation. The immune complexes aggregate platelets and induce release of serotonin. Platelet derived vasoactive mediators may increase vascular permeability and facilitate further deposition of complexes in the tissues.     

Antiepileptic effects of amphetamine may require GABA (benzodiazepine) activity

Secondary components of visual evoked potentials (slow negative wave-SNW, and photically-evoked sensory afterdischarge-SAD) are known to be precursors of experimentally activated wave-spike discharges, similar to wave-spikes of petit mal epilepsy. Both SNW and SAD may be potently suppressed wither by amphetamine or GABAergic compounds such as diazepam and sodium valproate. A hypothesis was tested in the present study, that amphetamine-induced suppression of wave-spike discharges may require GABA-benzodiazepine activity for its expression.Electrocortical activity was recorded and averaged in unrestrained albino rats with chronically implanted epicortical electrodes. SNW and SAD obtained in habituated rats in the predrug state were potently suppressed by amphetamine (1 mg/kg, i.p.). Fifteen minutes after amphetamine injection, a challenging drug (metrazol, picrotoxin, convulsant benzodiazepine, Ro 5-3663, or imidazodiazepine, Ro 15-1788) was administered intraperitoneally. Subconvulsive doses of metrazol (10 mg/kg) reversed amphetamine suppression; imidazodiazepine (20 mg/kg) and picrotoxin (1.5 mg/kg) reliably opposed the SNW suppression; convulsant benzodiazepine, Ro 5-3663 (2 mg/kg), showed modest and nonsignificant effect in the same direction. It is proposed that the antiepileptic potency of amphetamine may be associated with its ability, apparently via modulatory effect of norepinephrine, to facilitate the activation of benzodiazepine-GABA receptors.     

Changes in the concentration of metalloproteins in the blood plasma in hypoxia and hyperbaric oxygenation

Dynamics of hemoglobin, ceruloplasmin concentration, the changes of chemiluminescence in blood plasma and kinetics of rat erythrocyte heat denaturation during consequent exposition of high altitude hypoxia and hyperbaric oxygenation have been studied. Severe hypoxia causes the decrease of extraerythrocyte hemoglobin and oxidase activity of ceruloplasmin. Reoxygenation results in significant increase of blood plasma chemiluminescence with simultaneous increase of extraerythrocyte hemoglobin level and with modification of surface structure of the erythrocyte membranes. Possible pathways of activation of oxygen-dependent of free-radical processes during reoxygenation are discussed.     

Lack of endotoxin in Borrelia hispanica and Treponema pallidum

Borrelia hispanica from infected guinea pigs and Treponema pallidum from testicular syphilomas of rabbits were assayed for the presence of endotoxin with the Limulus lysate test. A suspension of Borrelia, containing 1.3 X 10(8) spirochetes/ml, was nonreactive both when it was tested as intact organisms, and when tested after disruption of the spirochetes by sonication. Eight different suspensions of treponemes, ranging from 0.6 X 10(9) to 3 X 10(9) treponemes/ml, were negative at a 1:10 dilution and were no more active than control suspensions of normal rabbit testes. Therefore, it was concluded that T. pallidum, as well as the Borrelia, possessed no endotoxin.     

Clinical trial of chromium and yeast supplements on carbohydrate and lipid metabolism in diabetic men

Chromium (Cr) deficiency in experimental animals and in humans sustained by prolonged total parenteral nutrition has been shown to cause diabetes mellitus. Prior trials in humans indicated that Cr supplements, in either inorganic or organic form, may improve carbohydrate utilization. We report here a clinical double-blind, random cross-over trial of inorganic chromium trichloride, a brewer’s yeast that contained Cr as glucose-tolerance-factor (GTF), a brewer’s yeast extract without GTF, and a placebo. Forty-three outpatient diabetic men received three of these supplements for 4 months each. Subgroups included 21 ketosis-prone, 7 ketosis-resistant non-obese, and 15 ketosis-resistant obese men. Cr levels were followed pre- and post-treatment in hair, red blood cells, plasma, and urine. Response of carbohydrate metabolism to treatment was assessed in terms of change in insulin requirements, fasting plasma glucose, plasma cholesterol, and triglycerides, as well as the change in plasma glucose, glucagon, and insulin or C-peptide levels in response to a standard meal. In some men, these parameters were also measured after iv tolbutamide. Both the inorganic and organic oral Cr supplements increased measurable body pools of Cr in hair and red blood cells by about 25%. However, fasting plasma glucose and lipids and the glucose response to either the standard meal or to tolbutamide were not significantly altered by any of the treatments.     

Maternal smoking and trisomy among spontaneously aborted conceptions

In a study of spontaneous abortions, we found an apparently robust association of trisomy with smoking that varies with maternal age. Among women under age 30, smoking either before or at the time of conception is less common in women aborting trisomic conceptions than in controls delivering at 28 weeks or later. Among older women, smoking is more common in women aborting trisomic conceptions than in controls. Our results point to an effect of smoking on the frequency of trisomic abortions that varies with age, and they suggest that the causes of recognized trisomic abortions differ in younger and older women.     

Lactic acid translocation: terminal step in glycolysis by Streptococcus faecalis

Streptococcus faecalis obtains metabolic energy chiefly from the conversion of glucose to lactic acid; the present experiments deal with the mechanism of lactic acid translocation across the cytoplasmic membrane. Efflux of [(14)C]lactate from preloaded cells was accelerated by raising the external pH, and also by the ionophores nigericin and valinomycin. These results suggest that lactate leaves the cell by an electroneutral process, presumably as lactic acid. Further evidence was obtained by studying the entry of [(14)C]lactate into nonmetabolizing cells. It appears that the membrane is essentially impermeable to the lactate anion, but allows passage of lactic acid. The most persuasive evidence is that, upon establishment of a pH gradient such that the cytoplasm was alkaline, l-[(14)C]lactate accumulated in the cells against the concentration gradient. Accumulation was transient, and dissipated in parallel with the collapse of the pH gradient. The concentration gradient attained at the peak was a function of the pH difference. Ionophores which are known to collapse a pH gradient, such as nigericin and valinomycin, abolished accumulation of l-lactate. We infer that lactic acid translocation, whether into the cells or outward, is an electroneutral process and for that reason the distribution of lactic acid across the membrane is a function of the pH of cytoplasm and medium. The specificity of translocation and its kinetic parameters suggest that it is mediated by a carrier of low specificity.