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V Khramtsov

Publications and source records attributed to V Khramtsov.

8 recordsLinked to original sources
PubMed2001

Unique in vivo applications of spin traps.

The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to instrument sensitivity and particularly to the relative stability of these radicals and their nitrone adducts, as well as toxicity limitations with dosing. Our aim here is to review the strengths and weaknesses of both traditional and in vivo ESR spin trapping and to describe new approaches that couple the strengths of spin trapping with methodologies that promise to overcome some of the problems, in particular that of radical adduct decomposition. The new, complementary techniques include: (i) NMR spin trapping, which monitors new NMR lines resulting from diamagnetic products of radical spin adduct degradation and reduction, (ii) detection of *NO by ESR with dithiocarbamate: Fe(II) "spin trap-like" complexes, (iii) MRI spin trapping, which images the dithiocarbamate: Fe(II)-NO complexes by proton relaxation contrast enhancement, and (iv) the use of ESR to follow the reactions of sulfhydryl groups with dithiol biradical spin labels to form "thiol spin label adducts," for monitoring intracellular redox states of glutathione and other thiols. Although some of these approaches are in their infancy, they show promise of adding to the arsenal of techniques to measure and possibly "image" oxidative stress in living organisms in real time.

Animals↗
PubMed2000

Plant rac proteins induce superoxide production in mammalian cells.

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution.

3T3 Cells↗
PubMed1999

NMR spin trapping: detection of free radical reactions using a phosphorus-containing nitrone spin trap.

This study employs (31)P-nuclear magnetic resonance (NMR) to probe for changes in molecular structure arising from reactions between free radicals and a phosphorus-containing nitrone spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). A number of biologically relevant free radical reactions were detected: a) reactions of DEPMPO with ( small middle dot)OH resulted in a new (31)P-NMR resonance at 27.05 ppm (shifted from the parent compound at 23.67 ppm); evidence suggests that this species is a diamagnetic hydroxy-pyrrolidone reduction product; b) (31)P-NMR spectra of DEPMPO/( small middle dot)CH(3) reactions resulted in peaks at 24.54, 30.83, and 32.31 ppm, while DEPMPO/( small middle dot)CH(2)OH produced peaks at 24.05, 30.80 and 32.52 ppm; in the presence of excess ascorbate, only resonances between 30 and 32 ppm were evident, which we have tentatively assigned to the hydroxylamine isomers of their respective adducts; and c) reaction of DEPMPO with O(2)( small middle dot-), produced by xanthine/xanthine oxidase or stimulated neutrophils, resulted in a single line, indistinguishable from DEPMPO/( small middle dot)OH reaction products. We conclude that NMR spin trapping is a useful approach for detecting free radical reaction pathways. It may have future applications for human free radical biology and imaging. Magn Reson Med 42:228-234, 1999.

Cyclic N-Oxides↗
PubMed1998

Oxygen radical generation and enzymatic properties of mitochondria in hypoxia/reoxygenation.

The time-dependence of oxygen radical formation and development of enzymatic dysfunction after hypoxia/reoxygenation was investigated in isolated rat liver mitochondria. Generation of oxygen radicals was studied by electron paramagnetic resonance (EPR) spectroscopy using the spin trap DMPO (5,5-dimethyl-l-pyrroline-N-oxide). The spin adduct DMPO-OH was found to be formed from the primarily generated adduct of DMPO with the superoxide anion radical (DMPO-OOH). Hypoxic storage followed by reoxygenation at room temperature resulted in an increased decay rate of the DMPO-OH spin adduct while its steady state concentration remained unchanged. This finding strongly suggests an increased rate of DMPO-OH formation which originally derived from enhanced superoxide anion radical production due to hypoxia/reoxygenation. The enhanced superoxide radical formation seems to be due to dysfunction of respiratory chain enzymes, resulting in increased levels of reductive components. In agreement with that, we found the decrease of respiration control and ATP synthesis activity at a similar time scale as that for DMPO-OH adduct formation. The increase of superoxide radical formation and of the reductive capacity of mitochondria was accompanied by a decrease in membrane order at the polar interface. Oxidative phosphorylation was completely abolished after 30 min of hypoxic storage, whereas ATP synthesis decreased significantly after 15 min of hypoxia.

Adenosine Triphosphate↗
PubMed1996

Studies of human and rat blood under oxidative stress: changes in plasma thiol level, antioxidant enzyme activity, protein carbonyl content, and fluidity of erythrocyte membrane.

Previously by selection and inbreeding of Wistar rats susceptible or resistant to the cataractogenic effect of galactose the S and R rat strains were developed as a model of oxidative stress. It was earlier found S rats have elevated level of OH-radical generation and enhanced lipid peroxidation compared with that for R rats. Our data show that superoxide dismutase and catalase activities in the blood of S rats are twice lower than corresponding values of R rats. Increased level of disulfides and protein carbonyl groups was obtained in the blood plasma of S-rats. It was found that fluidity of erythrocyte membrane measured by photobleaching method is definitely higher for S rats compared with that for S rats. Similar effect of decreased catalase activity and increased disulfide content has been observed for kyphoscoliosis patients.

Animals↗
PubMed1996

Determination of rate constants of the reactions of thiols with superoxide radical by electron paramagnetic resonance: critical remarks on spectrophotometric approaches.

Two new EPR approaches were developed for determination of rate constants of reaction glutathione (GSH), N-(2-mercaptopropionyl) glycine (MPG), dihydrolipoic acid (BNL), and tetranor-dihydrolipoic acid (TNL) with superoxide radical. In both cases the competition between thiols and spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for superoxide radical was used. In the first method the dependence of amplitude of EPR spectrum of DMPO-OOH spin adduct on concentration of thiols in a superoxide-generating system was studied. In the second approach the changes in reduced thiol concentration due to reaction with superoxide radical were measured by nitroxide biradical containing disulfide bond. Observed rate constants were the following: GSH, 1.8 x 10(5) M-1s-1; MPG, 2.2 x 10(5) M-1s-1; TNL, 1.2 x 10(5) M-1s-1; BNL, 2.5 x 10(5) M-1s-1; DHL, 4.8 x 10(5) M-1s-1. The determination of the rate constants of reaction of superoxide radical with thiols by spectrophotometrical cytochrome C assay could result in an underestimation of the values due to the reduction of cytochrome C by thiols. Use of epinephrine for this purpose could lead to an overestimation of experimental rate constants because the adrenochrome formed in the reaction of epinephrine with superoxide radical reacts with thiols.

Adrenochrome↗