The kinetic mechanism of manganese-containing superoxide dismutase from Deinococcus radiodurans: a specialized enzyme for the elimination of high superoxide concentrations.

Deinococcus radiodurans (Drad), a bacterium with an extraordinary capacity to tolerate high levels of ionizing radiation, produces only a manganese-containing superoxide dismutase (MnSOD). As MnSOD has been shown to remove superoxide radical with varying efficiency depending upon its cellular origin, a comparison of the Drad MnSOD efficiency with that of both human and Escherichia coli MnSODs was undertaken. Pulse radiolysis studies demonstrate that, under identical ratios of enzyme to superoxide radical, the dismutation efficiencies scaled as Drad MnSOD > E. coli MnSOD > human MnSOD. Further, Drad MnSOD is most effective at high superoxide fluxes found under conditions of high radioactivity. A mechanism is postulated to account for the differences in the activities of the MnSODs that considers the release of peroxide as not always an optimal process.

The effect of superoxide anion on autoregulation of cerebral blood flow.

BACKGROUND AND PURPOSE: Recent studies have suggested that autoregulation of cerebral blood flow (CBF) is impaired after traumatic and ischemic brain injury. Given that the levels of superoxide anion (O2*-) are increased in these conditions, we postulate that O2*- contributes to the impairment of CBF autoregulation. METHODS: CBF was monitored with laser Doppler flowmetry during increases in blood pressure. RESULTS: During the control period, CBF was well autoregulated after the increase in mean arterial pressure (MAP) from 98+/-3 to 140+/-6 mm Hg. The autoregulation index (AI; DeltaCBF/DeltaMAP) averaged 0.25+/-0.02 (n=6). O2*- in the brain was then increased by subdural perfusion of xanthine/xanthine oxidase (different concentrations) and catalase. Low concentrations of O2*- decreased basal CBF by 10+/-1.6% but had no effect on autoregulation (AI, 0.19+/-0.02; n=6). Higher concentrations of O2*- (0.2 mmol/L xanthine and either 3 or 20 mU xanthine oxidase) increased basal CBF by 30+/-2% and 42+/-4%, respectively, and impaired autoregulation of CBF (AI, 0.55+/-0.03 and 0.76+/-0.02; n=6). Inclusion of superoxide dismutase in the O2*(-)-generating system restored autoregulation (AI, 0.28+/-0.05; n=6). Neither inhibition of NO synthase nor the addition of deferioxamine had any effect on the ability of higher concentrations of O2*- to impair autoregulation of CBF (AI, 0.65+/-0.07 and 0.72+/-0.05 respectively; n=6). O2*- also increased the activity of KCa channels in cerebral vascular smooth muscle cells (VSMCs; n=8). CONCLUSIONS: These results suggest that O2*- increases basal CBF and impairs autoregulation of CBF, likely through the activation of KCa channels in cerebral VSMCs.

Estallido respiratorio de los fagocitos / Phagocyte respiratory burst

El estallido respiratorio de los fagocitos, producido por la NADPH-oxidasa, complejo enzimático que cataliza la formación de radical superóxido, constituye una de las fuentes endógenas más importantes de especies reactivas del oxígeno en el organismo. Este sistema comprende un complejo flavocitocromo b558 unido a membrana, y factores citosolicos p47phox, p67phox, p40phox y la pequeña GTPasa Rac2, que se trastocan a la membrana plasmática donde experimentan un proceso de ensamblaje que conforma el sistema enzimático activo. El conocimiento de las interacciones proteina-proteína que permiten el ensamblaje y el mecanismo de acción enzimático, ha permitido detectar los cambios que transcurren en el estado activo. La importancia de la NADPH oxidasa se muestra en la enfermedad granulomatosa crónica, patología transmitida por herencia, en la cual un componente de la NADPH oxidasa está ausente o defectivo. Tales individuos padecen infecciones recurrentes crónicas y severas debido a la incapacidad de sus neutrófilos para destruir microbios

The phagocyte respiratory burst produced by the NADPH oxidase, enzyme complex which catalyzes the production of superoxide radical, is one of the main endogenous sources of reactive oxygen species in the body. NADPH oxidase consists of a membrane-bound flavocytochrome b558 complex, and cytosolic factors p47phox, p67phox, p40phox and the small GTPase Rac, which translocate to the membrane to assemble the active complex following cell activation. A great deal of current research involves understanding the protein protein interactions involved in the assembly and enzyme mechanism and how these change with the activation state. The importance of the NADPH oxidase is illustrated by the inherited condition Chronic Granulomatous Disease in which a component of the respiratory burst oxidase is absent or defective. Affected individuals suffer from recurrent, chronic and severe infections due to the inability of their neutrophils to kill microbes

Enhancement of amphotericin B activity against Candida albicans by superoxide radical.

This study aimed to examine the involvement of oxidative damage in amphotericin B (AmB) activity against Candida albicans using the superoxide (O2-) generator paraquat (PQ). The effects of PQ on AmB activities against growth, viability, membrane permeability and respiration were examined in a wild-type parent strain (K) and a respiration-deficient mutant (KRD-19) since PQ-induced superoxide generation depends on respiration. In the parent strain, the minimal inhibitory concentration (MIC) of AmB, 0.25 microg/ml, tested with a liquid culture was lowered to 0.025 microg/ml by 1 mM PQ. Such a PQ-induced decrease in the MIC value of AmB was minimal in the mutant. Similar PQ-induced enhancement of AmB activity toward the parent strain was also observed with growth on an agar medium. In viability tests, when candidal cells were exposed to AmB (0.1 microg/ml) for I h, the lethality of AmB was enhanced by 1 mM PQ only in the parent strain. Exogenous superoxide dismutase and catalase failed to diminish the enhancing effect of PQ on the growth inhibitory activity of AmB in the parent strain, suggesting an interaction between superoxide and AmB in candidal cells. The enhancement of AmB activity by PQ, observed preferentially in the wild-type strain, can be explained by extensive superoxide generation depending on respiration. These results suggest that oxidative damage induced by superoxide is involved in AmB activity against C. albicans.

Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria.

1 In this study, we have used isolated brain mitochondria to investigate the effects of superoxide anions (O(2)(-)) on mitochondrial parameters related to apoptosis, such as swelling, potential, enzymatic activity, NAD(P)H, cytochrome c release, and caspase activity. 2 Addition of the reactive oxygen species (ROS) generator KO(2) produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca(2+) independent. 3 Calcium induced mitochondrial swelling only at high concentrations and in the presence of succinate. This correlated with the increase in O(2)(-) production detected with hydroethidine in mitochondrial preparations exposed to Ca(2+) and the fact that ROS were required for Ca(2+)-induced mitochondrial swelling. 4 Superoxide anions, but not Ca(2+), decreased citrate synthase and dehydrogenase enzymatic activities and dropped total mitochondrial NAD(P)H levels. 5 Calcium, but not O(2)(-), triggered a rapid loss of mitochondrial potential. Calcium-induced Deltapsi(m) dissipation was inhibited by Ruthenium Red, but not by CSA. 6 Calcium- and superoxide-induced mitochondrial swelling released cytochrome c and increased caspase activity from isolated mitochondria in a CS A-sensitive manner. 7 In summary, superoxide potently triggers mitochondrial swelling and the release of proteins involved in activation of postmitochondrial apoptotic pathways in the absence of mitochondrial depolarization.

Photocatalysis-dependent inactivation of Lactobacillus phage PL-1 by a ceramics preparation.

A ceramics preparation (Cleansand-205), which was coated with a mixture of the oxides of Si, Al, Ti, and Ag, was found to inactivate Lactobacillus phage PL-1 suspended in a buffer solution. The inactivation of phage was dependent on the amounts of Cleansand-205 added, and the reaction obeyed almost first-order reaction kinetics. The phage inactivation was considerably accelerated by the presence of light.

Are aldehydes in heat-sterilized peritoneal dialysis fluids toxic in vitro?

OBJECTIVE: Chemical analysis of several brands of peritoneal dialysis fluids (PD fluids) has revealed the presence of 2-furaldehyde, 5-HMF (5-hydroxymethylfuraldehyde), acetaldehyde, formaldehyde, glyoxal, and methylglyoxal. The aim of this study was to investigate if the in vitro side effects caused by glucose degradation products, mainly formed during heat sterilization, are due to any of these recently identified aldehydes. DESIGN: Cell growth media or sterile filtered PD fluids were spiked with different concentrations of thealdehydes. MEASUREMENTS: In vitro side effects were determined as the inhibition of cell growth of cultured mouse fibroblasts or stimulated superoxide radical release from human peritoneal cells. RESULTS: Our results demonstrate that the occurrences of 2-furaldehyde, 5-HMF, acetaldehyde, formaldehyde, glyoxal, or methylglyoxal in heat-sterilized PD fluids are probably not the direct cause of in vitro side effects. In order to induce the same magnitude of cell growth inhibition as the heat-sterilized PD fluids, the concentrations of 2-furaldehyde, glyoxal, and 5-HMF had to be 50 to 350 times higher than those quantified in the PD fluids. The concentrations of acetaldehyde, formaldehyde, and methylglyoxal observed in the heat-sterilized PD fluids were closer to the cytotoxic concentrations although still 3 to 7 times lower. CONCLUSION: Since none of these aldehydes caused in vitro toxicity at the tested concentrations, the toxicity found in PD fluids is likely to be due to another glucose degradation product, not yet identified. However, it is possible that these aldehydes may still have adverse effects for patients on peritoneal dialysis.

Spinal anaesthesia but not general anaesthesia enhances neutrophil biocidal activity in hip arthroplasty patients.

The purpose of this study was to compare neutrophil cidal activity during general or spinal anaesthesia. Assays were performed on neutrophils extracted from the blood of patients after surgery had been under way for one hour. First, the ability of the neutrophils to kill a standard laboratory strain of S. aureus was examined. Neutrophils extracted from the blood during surgery in the spinal anaesthetic group and incubated with the staphylococci for one hour killed twice as many bacteria than those from two groups of patients that received halothane or isoflurane general anaesthesia (P < 0.05). This effect persisted, to a lesser extent, in the spinal group neutrophils after two hours of incubation with the bacteria. Second, neutrophils from patients under the same conditions of surgery and anaesthesia were tested to examine the effect of the different anaesthetic techniques on neutrophil biocidal mechanisms. Neutrophils extracted during surgery in the spinal group released more superoxide in response to phorbol-12-myristate-13-acetate (PMA) than those from both groups of patients that received general anaesthesia (P < 0.05). It is concluded that there is an increased state of reactivity of the neutrophil cell membrane NADPH oxidase system in patients receiving spinal anaesthesia than in patients receiving general anaesthesia.

[Indices of structure and function of erythrocyte membranes in rats with vitamin E deficiency and their correction with antioxidant drugs]. / Pokazateli struktury i funktsii membran érytrotsitov krys s E-vitaminnoi nedostatochnost'iu i ikh korrektsiia preparatami antioksidantov.

It is found that the amount of saturated fatty acids grows, while that of unsaturated--falls in the erythrocyte membranes of rats maintained on a diet without vitamin E. In this case transmembrane calcium transport catalyzed by Ca2(+)-ATPase (EC 3.6.1.3) is not broken and activity of antioxidant enzyme superoxide dismutase (EC 1.15.1.11) in lysate of erythrocytes falls. The found shifts are corrected with administration of antioxidant preparations.

Modulation of human peripheral blood monocyte superoxide release by interferon-gamma and lipopolysaccharide.

Reactive oxygen species (ROS) have generated increasing interest for their possible role in a wide variety of diseases. Interferon-gamma (IFN-gamma), a potent immunoregulatory lymphokine, is likely involved in control of ROS metabolism. In this study, the superoxide release of cultured human peripheral blood monocytes (PBM) after exposure to IFN-gamma and lipopolysaccharide (LPS) was examined. Compared with controls, adherent monocytes cultured with 80 units of IFN-gamma for 48 hours demonstrated fourfold increased spontaneous and twofold increased PMA stimulated release of superoxide anion. In addition, the enhanced superoxide release was both dose and time dependent. Further experiments showed that bacterial LPS in concentrations as low as 4 ng/mL markedly reduced monocyte superoxide release and abrogated the enhancing effects of IFN-gamma.

Periodontitis associated with Papillon-Lefèvre syndrome.

The predominant subgingival microflora, host immune response, and genetic history of a 14-year-old girl with Papillon-Lefèvre Syndrome (PLS) are reported. The patient had high counts of Actinobacillus actinomycetemcomitans and surface translocating bacteria. She had significantly raised levels of antibodies to five of the bacterial species studied with the levels to A. actinomycetemcomitans remaining high after antibiotic therapy. The polymorphonuclear leukocytes (PMN) also released significantly increased amounts of O2 compared to controls. The data presented support a role for A. actinomycetemcomitans and PMN dysfunction in the pathogenesis of PLS.

Sobre los compuestos de coordinaciòn como agentes antitumorales. IV: acciòn sobre el radical superóxido / On coordination copounds as antitumorigenic agents: IV: action on superoxide radical

Como modelaciòn de la actividad protectora de la superoxidodismutasa, se estudiò cinéticamente el sistema O2 Cu (II) mediante la determinaciòn de seudoconstantes de velocidad en la reacciòn de 17 compuestos de algunas tio y semicarbazonas de cobre (II) con el radical superóxido. Se correlacionaron estas seudoconstantes de velocidad con la citotoxidad in vitro de los 17 compuestos, así como de cada familia de compuestos por separado. La mejor correlaciòn se logró en el caso de las semicarbazonas. Las tiosemicarbazonas, por su parte, resultaron ser las más citotóxicas. Se interpretan estos resultados a partir de las características del enlace quìmico

Sobre los compuestos de coordinaciòn como agentes antitumorales: IV: acciòn sobre el radical superóxido / On coordination compounds as antitumorigenic agents: IV: action on superoxide radical

Como modelaciòn de la actividad protectora de la superoxidodismutasa, se estudiò cinéticamente el sistema O2 Cu (II) mediante la determinaciòn de seudoconstantes de velocidad en la reacciòn de 17 compuestos de algunas tio y semicarbazonas de cobre (II) con el radical superóxido. Se correlacionaron estas seudoconstantes de velocidad con la citotoxidad in vitro de los 17 compuestos, así como de cada familia de compuestos por separado. La mejor correlaciòn se logró en el caso de las semicarbazonas. Las tiosemicarbazonas, por su parte, resultaron ser las más citotóxicas. Se interpretan estos resultados a partir de las características del enlace quìmico

Studies on the mechanism of inhibition of chemotactic tripeptide stimulated human neutrophil polymorphonuclear leucocyte superoxide production by chloroquine and hydroxychloroquine.

The effect of chloroquine and hydroxychloroquine on neutrophil superoxide release stimulated by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was examined. Both drugs caused time and dose dependent inhibition of superoxide release but had no effect on equilibrium binding of [3H]FMLP to its receptor. Preliminary experiments suggest that these drugs may exert their inhibitory effect on superoxide release by inhibiting the FMLP stimulated hydrolysis of phosphoinositides.