Oxidation of dihydronicotinamide adenine dinucleotide by a flavin derivative of polyethylenimine

A modified Polyethylenimine has been prepared that has riboflavin attached to it, as well as hydrophobic groups. The catalytic efficiency toward oxidation of6 dihydronicotinamide adenine dinucleotide (NADH) by this flavopolymer is more than 100-fold greater than observed with small-molecular riboflavin. The products of the reaction in aerobic solution have been established to be the oxidized nicotinamide NAD⁺ and hydrogen peroxide. The kinetics fit a Michaelis-Menten mechanism. Thus, the versatility of modified polyethylenimines as catalysts has been extended from hydrolyses and decarboxylations to oxidation-reduction reactions also.     

Flash photolysis of flavins. V. Oxidation and Disproportionation of flavin radicals

Flash photolysis techniques have been utilized to investigate the reactivity patterns of flavin radical species. Rate constants for disproportionation were found to be la the following order: lumiflavin>FMN>FAD and neutral radicals>anionic radicals. Neutral flavin radicals react with oxygen at a rate which is at least 10⁴ times slower than the anionic species. No evidence for an intermediate complex or adduct is obtained in this reaction. The pK values for the ionization of the neutral flavin radicals are in the order FAD>FMN>riboflavin=limiflavin. The rates of reaction of ferricyanide with flavin radicals are essentially independent of pH, whereas benzoquinone reacts slightly more slowly (5 times) with the neutral flavin radical than with the anionic form. Cytochromec reacts at least ten times more slowly with flavin radicals than does ferricyanide.     

Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.     

Indices of oxidative stress. 2. Lipid peroxides

Two methods of the determination of lipid peroxidation products have been compared which are based on Fe(II) oxidation by them at acid pH values in the presence of xylenol orange which binds Fe(III) have been compared. The first method uses cumene hydropeoxide as an internal standard. In the second one, lipid peroxides are previously reduced by triphenylphosphine and these substances content is measured as a difference of the production of complexes with xylenol orange and iron ions in the control (with reduction) and experimental sample (without reduction). The optimization of measurement conditions is described. The levels of lipid peroxides in goldfish tissues assayed simultaneously by two methods were similar. The method with cumene hydroperoxide needs less amounts of biological material; moreover, there is no necessity in a calibration curve. Effects of hyperoxia on lipid peroxide levels in goldfish tissues were studied with the cumene method. Within the first hours of hyperoxia this index increased 13-times in the liver and 2-times in the brain and muscle. The further exposure rebounded this parameter to the initial level. Levels of lipid peroxides positively correlated with levels of end products of lipid peroxidation (thiobarbiturate acid reactive substances) in the goldfish tissues. The method of quantification of lipid peroxides with cumene is recommended for wide using in biological investigations.     

Oxidation of organoarsenicals and antimonite by a novel flavin monooxygenase widely present in soil bacteria

Arsenic can be biomethylated to form a variety of organic arsenicals differing in toxicity and environmental mobility. Trivalent methylarsenite (MAs(III)) produced in the methylation process is more toxic than inorganic arsenite (As(III)). MAs(III) also serves as a primitive antibiotic and, consequently, some environmental microorganisms have evolved mechanisms to detoxify MAs(III). However, the mechanisms of MAs(III) detoxification are not well understood. In this study, we identified an arsenic resistance (ars) operon consisting of three genes, arsRVK, that contribute to MAs(III) resistance in Ensifer adhaerens ST2. ArsV is annotated as an NADPH-dependent flavin monooxygenase with unknown function. Expression of arsV in the arsenic hypersensitive Escherichia coli strain AW3110Δars conferred resistance to MAs(III) and the ability to oxidize MAs(III) to MAs(V). In the presence of NADPH and either FAD or FMN, purified ArsV protein was able to oxidize both MAs(III) to MAs(V) and Sb(III) to Sb(V). Genes with arsV-like sequences are widely present in soils and environmental bacteria. Metagenomic analysis of five paddy soils showed the abundance of arsV-like sequences of 0.12–0.25 ppm. These results demonstrate that ArsV is a novel enzyme for the detoxification of MAs(III) and Sb(III) and the genes encoding ArsV are widely present in soil bacteria.     

The Formation of Phosphatidylcholine Oxidation Products by Stimulated Phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814?m/z) and bishydroperoxides (846?m/z) of PAPC was observed. However, the major oxidized product occurred at 828?m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E²)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.     

Prostaglandin biosynthesis can be triggered by lipid peroxides.

Studies of ferriheme cyclooxygenase, using two different assay systems, show that a variety of peroxides can trigger a rapid acceleration of cyclooxygenase activity to produce prostaglandins. Lipid hydroperoxides formed by lipoxygenase were the most potent activators tested, followed by prostaglandin G₂, which was slightly less potent. Peroxides nonspeciflcally generated during arachidonate autoxidation were as potent as the enzymatically formed lipid peroxides. These findings have important implications for cell function since any process which generates peroxides may activate the cyclooxygenase. Thus the balance between formation and removal of cellular lipid peroxides sets a peroxide tone that can regulate the rate of prostaglandin formation in cells.     

Cyanide Formation from Oxidation of Glycine by a Pseudomonas Species

With whole cells of a hydrogen cyanide-producing bacterium strain C, of the genus Pseudomonas, it was found that the oxygen necessary for the oxidation of glycine to cyanide could be replaced by various artificial electron acceptors. The order of reactivity was: oxygen > phenazine methosulphate > methylene blue > 2,6-dichlorophenolindophenol > ferricyanide. Cyanide production was inhibited by pyrrolnitrin, a well-known inhibitor of many flavine enzymes. The molar ratio of added glycine to cyanide produced was found to be 1.09. With whole bacteria the apparent K(m) (glycine) for the cyanide production was found to be 5.0 x 10(-4) M.     

Clustering of apoptotic cells via bystander killing by peroxides.

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell-to-cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo-, gamma-ray, and gene therapy of cancer.