Biomedical subjects

V V Khramtsov

Publications and source records attributed to V V Khramtsov.

At least 19 recordsLinked to original sources

PubMed2006

Fluorescence recovery under decaying photobleaching irradiation: concept and experiment.

A novel modification of photobleaching method for measurement of lateral diffusion is developed. In this approach fluorescence recovery kinetics is measured under decaying photobleaching irradiation, termed as fluorescence recovery under decaying photobleaching (FRDP). The time evolution of fluorescence intensity normalized to input irradiation starts from the photobleaching kinetics and transforms into the kinetics of fluorescence recovery at a later stage resulting in appearance of minimum. The analytical solution for the kinetics of fluorescence for Gaussian lineshape of laser beam and hyperbolic decay of irradiation in the first order approximation on bleaching rate was obtained. The accuracy of the analytical function was evaluated with exact numerical solution computed with finite differentiates method. The FRDP method was successfully applied to fluorescein solution in the glycerol/water mixture (80%) under various experimental settings using home-made experimental set-up. The FRDP approach demonstrated 25-30 fold enhancement in signal intensity over classical fluorescence recovery after photobleaching (FRAP) method at 3-5 fold increase in total irradiation. Among other advantages of the FRDP is the opportunity to perform measurements on varying time scales under constant size of the bleaching spot, including "safe" long time measurements. The potential extra advantage of FRDP method for analysis of complex diffusion in the biological system is discussed.

Fluorescence Recovery After Photobleaching↗

PubMed2001

NMR spin trapping: detection of free radical reactions with a new fluorinated DMPO analog.

Electron spin resonance (ESR) and nuclear magnetic resonance (NMR) spin trapping were used for detection of free radical reactions utilizing a new fluorinated analog of DMPO, 4-hydroxy-5,5-dimethyl-2-trifluoromethylpyrroline-1-oxide (FDMPO). The parent FDMPO spin trap exhibits a single 19F-NMR resonance at -66.0 ppm. The signal to noise ratio improved 10.4-fold compared to 31P-NMR sensitivity of the phosphorus-containing spin trap, DEPMPO. The spin adducts of FDMPO with .OH, .CH3, and .CH2OH were characterized. Competitive spin trapping of FDMPO with DMPO showed that both have similar rates of addition of .OH and C-centered radicals. The corresponding paramagnetic spin adducts of FDMPO were extremely stable to degradation. In the presence of ascorbate, reaction products from C-centered radicals resulted in the appearance of two additional 19F-NMR signals at -78.6 and -80 ppm for FDMPO/ .CH(3) and at -74.6 and -76.75 ppm for FDMPO/ .CH(2)OH. In each case, these peaks were assigned to the two stereoisomers of their respective, reduced hydroxylamines. The identification of the hydroxylamines for FDMPO/ .CH3 was confirmed by EPR and 19F-NMR spectra of independently synthesized samples. In summary, spin adducts of FDMPO were highly stable for ESR. For NMR spin trapping, FDMPO showed improved signal to noise and similar spin trapping efficiency compared to DEPMPO.

Cyclic N-Oxides↗

PubMed2001

Effect of selenolipoic acid on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase.

Seleno-organic compounds are known as efficient "scavengers" of peroxynitrite (PN). Here we studied the protective effect of selenolipoic acid (SeLA), the seleno-containing analogue of lipoic acid, on peroxynitrite-dependent inactivation of NADPH-cytochrome P450 reductase. 3-Morpholinosydnonimine hydrochloride (SIN-1) was used as a source of peroxynitrite. The reductase was irreversibly inactivated by PN generated from SIN-1. The inactivation occurred with the rate constant of about 3 x 10(4) M-1 s-1. The presence of SeLA at low concentration (0.5 microM) led to synergistic increase of the reductase inactivation by PN. Our results suggest the formation of a reactive derivative of SeLA in the reaction of SeLA with PN, probably selenolseleninate, that mediates the aggravation of reductase inactivation. In the presence of SeLA, the inactivation was reversible under the action of thiols, allowing us to conclude that the observed action of SeLA may be considered as protective.

Animals↗

PubMed2001

[Oxidative stress in pathogenesis of arterial hypertension in ISIAH rats].

The NO-synthase activity and the rate of NO production in the rat aortic wall and cerebellum were 2-1.5-fold higher in the ISIAH rats than in normotensive WAG rat strain. In contrast, the superoxide dismutase (SOD) activity was significantly reduced in the ISIAH rats. The blood level of reduced thiols was also much lower in the ISIAH rats. The findings suggest that oxidative stress may play a significant role in pathogenesis of stress-sensitive hypertension in the ISIAH rat strain.

Animals↗

PubMed2000

Biological applications of spin pH probes.

The determination of pH is one of the most important problems in the biochemistry of living organisms, since many of the vital processes of cells and cellular organelles depend on the local pH value. Amongst currently used experimental approaches for the measurement of pH, the application of spin pH probes in combination with EPR spectroscopy is a comparatively new and rapidly developing field. In this article we describe the background, advantages and limitations of the method of spin pH probes, and discuss its recent applications. Availability of a wide variety of pH-sensitive nitroxides with different ranges of pH-sensitivity, labeling group and lipophilicity facilitates their application to a variety of biological systems from subcellular organelles to complex organisms. The recent progress in low-field EPR-based imaging and spectroscopy-based techniques allows spin pH probes to be used for non-invasive in vivo pH measurement and pH-sensitive imaging.

Animals↗

PubMed1999

EPR detection of reactive oxygen species in hemolymph of Galleria mellonella and Dendrolimus superans sibiricus (Lepidoptera) larvae.

The formation of reactive oxygen species (ROS) in hemolymph and hemocytes of Galleria mellonella and Dendrolimus superans sibiricus larvae was studied by ESR spectroscopy using spin-trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H). The background level of ROS formation was detected in the intact hemolymph. The addition of dihydroxyphenylalanine (DOPA) into free cells of the hemolymph increased CP-H oxidation about two times for G. mellonella and about four times for D. superans sibiricus. This increase was completely inhibited by a specific inhibitor of phenoloxidase, phenylthiourea. The presence of exogenous superoxide dismutase (SOD) did not change CP-H oxidation in the hemolymph. The data obtained in hemocytes showed only a DOPA-induced increase in CP-H oxidation. Phagocytosis activators did not affect ROS formation in hemocytes of both insect species. SOD decreased DOPA-induced CP-H oxidation 20-30% in suspension of hemocytes of D. superans sibiricus only. Our results are in agreement with the contribution of superoxide radical and DOPA-derived quinones/semiquinones in the immune response of insects.

Animals↗

PubMed1999

Manifestation of oxidative stress in the pathogenesis of arterial hypertension in ISIAH rats.

The ISIAH rat strain with stress-sensitive arterial hypertension was intentionally selected to study the role of stress as a factor in the development of arterial hypertension. This study aimed to determine the role of reactive oxygen and nitrogen species (ROS and RNS) in the pathogenesis of hypertension in ISIAH rats. The nitric oxide concentrations measured by EPR were found to be significantly higher for hypertensive ISIAH rats compared with that for normotensive Wistar rats in both the aortic wall (2 times) and cerebellum (1.5 times). The activity of superoxide dismutase measured in the blood of ISIAH rats was found to be about 1.5 times lower compared with that of Wistar rats. These data support the suggestion that ROS and RNS, including superoxide radicals and nitric oxide, may play an important role in development of stress-induced hypertension in ISIAH rats. The tissue content of reduced thiols has been considered as a marker of oxidative damage. To study the tissue oxidative status we used an EPR method for quantitative determination of SH groups. The concentration of reduced thiols in the blood of ISIAH rats was much lower than that in Wistar rats (0.6 +/- 0.05 and 1.57 +/- 0.1 mM, respectively).

Animals↗

PubMed1998

Thiol-induced nitric oxide release from 3-halogeno-3,4-dihydrodiazete 1,2-dioxides.

In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.

Animals↗

PubMed1997

Quantitative determination and reversible modification of thiols using imidazolidine biradical disulfide label.

Earlier we reported an ESR method of quantitative determination of sulfhydryl groups. The method is based on the application of the imidazoline biradical disulfide label, R1S-SR1, which participates in the reaction of thiol-disulfide exchange followed by dramatic changes in ESR spectra. One of the disadvantages of the application of R1S-SR1 at physiological conditions is the requirement of excess of the biradical compared with thiol content which results in the consumption of the thiols and irreversible damage of the system under study. In the present paper we propose imidazolidine biradical disulfide reagent, R2S-SR2, for ESR determination of thiols and provide an experimental basis for its application. This label has the advantages of the previously used biradical disulfide, R1S-SR1, such as high sensitivity down to 1 microM of thiols even in opaque samples and could possibly be used for reversible modification of proteins and enzymes. The particular properties of the R2S-SR2 are pH-sensitivity of its ESR spectrum, higher stability of the imidazolidine radical fragment towards biological reductants and low concentration of the label sufficient for thiol determination at physiological conditions. The latter makes it possible to use ESR spectroscopy for non-invasive thiol measurements in biological systems, in vivo applications included.

Animals↗

PubMed1997

Modulation of the mitochondrial permeability transition by nitric oxide.

The influence of nitric oxide on mitochondrial permeability transition (MPT) phenomenon was studied. NO was generated by photolysis of S-nitroso-N-acetylcysteine, AcCys(NO), with green light (lambda = 550 nm). Two distinct effects of nitric oxide on rat liver mitochondria were identified. First, NO accelerated an onset of swelling in Ca2(+)-loaded mitochondria in a cyclosporin-A-sensitive manner acting as an inducer of permeability transition. This was, apparently, a result of irreversible alteration of mitochondrial function accompanying the inhibition of respiratory chain in the presence of calcium. Formation of ESR-visible iron-sulfur dinitrosyl complexes (g = 2.041) could also contribute to the irreversible changes resulting in MPT induction. Second, NO changed significantly the response of mitochondria to Ca2+/phosphate-induced MPT, acting as a regulator of permeability transition. In this case the action of nitric oxide led to division of the mitochondria into two subpopulations: one which underwent the rapid permeability transition and another in which the MPT was inhibited. The effect of NO on Ca2+/Pi-induced MPT was transient and resulted from reversible inhibition of cytochrome oxidase followed by the changes in transmembrane potential and Ca2+ distribution. The characteristic time of duration of these NO modulated effects depended on nitric oxide as well as on oxygen concentrations. With increasing NO at fixed oxygen concentrations, this time levelled off to reach a maximum value which was inversely related to the oxygen concentration. It is concluded that under physiological condition the duration of reversible NO effects on mitochondrial function could be determined by oxygen concentration.

Acetylcysteine↗

PubMed1997

Modelling of the prebiotic synthesis of oligopeptides: silicate catalysts help to overcome the critical stage.

On the basis of experimental studies of the initial stages of glycine oligomerization in aqueous suspension of zeolite and kaolinite catalysts, a model is suggested for the prebiotic synthesis of oligopeptides from alpha-amino acids. The formation of linear dipeptides by hydrolysis of one amide bond in the cyclic piperazinedione intermediate (formed from glycine spontaneously) is found to be the critical stage of the reaction. This stage is base catalyzed and its rate increases when pH of the medium goes up. The linear glycyl-glycine yield rises under effect of hydroxyl anions generated from different sources including insoluble silicates and soluble sodium bicarbonate. During prebiotic evolution silicates capable of cation-exchange can serve as local sources of the hydroxyl anions which dramatically accelerate formation of linear dipeptides from cyclic ones. Oligopeptides of higher molecular weight are then easily formed from the linear dipeptides at neutral pH, even in the absence of catalysts or sources of energy (e.g. such as light). The described catalytic synthesis could occur in the proximity of submarine hydrothermal vents.

Aluminum Silicates↗

PubMed1997

Synthesis and spin trapping applications of 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N.

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N (lTMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with alpha-protons of alkyl radicals trapped by lTMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (delta Bpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 +/- 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

Electron Spin Resonance Spectroscopy↗

PubMed1996

EPR and laser flash photolysis studies of the reaction of nitric oxide with water soluble NO trap Fe(II)-proline-dithiocarbamate complex.

Diethyl-dithiocarbamate (DETC) chelating ferrous iron is used during EPR detection of nitric oxide (.NO) as a precursor of spin traps for the quantification of released .NO both in vitro and in vivo. This method is based on the high affinity of water insoluble iron-dithiocarbamate complexes towards .NO. The nitrosyl-iron-dithiocarbamate complex NOFe(DETC)2 is formed with characteristic triplet EPR spectrum. In the present paper we have studied the reaction of water soluble Fe(II)-proline-dithiocarbamate complex, Fe(PDTC)2, with nitric oxide using both EPR spectroscopy and laser flash photolysis. It has been found that the treatment of the Fe(PDTC)2 complex either by authentic .NO dissolved in aqua buffer solutions or by different .NO donors results in the quantitative appearance of an EPR signal of a stable mononitrosyl complex NOFe(PDTC)2 with a g-factor giso = 2.04 and a hyperfine splitting constant aN = 12.5 G. The laser flash photolysis method has been used to study the kinetics of .NO trapping by Fe(PDTC)2. Nitric oxide release from a light sensitive .NO donor, piridazinofuroxan, was induced by a short flash of UV light generated by a XeCl eximer laser. The rate constant of the reaction of .NO with Fe(PDTC)2 was found to be (1.1 +/- 0.3).10(8) M-1s-1, manifesting a high efficiency of .NO trapping by Fe(PDTC)2.

Electron Spin Resonance Spectroscopy↗

PubMed1996

[Studies of in vitro and in vivo derived 1,2-diazetine and nitronylnitroxide as donors and acceptors of nitric oxide].

The series of nitronylnitroxyl radicals (NNR) were studied as paramagnetic scavengers of nitric oxide. These radicals react with NO with rate constants of (0.6-1.1).10(4) M-1.sec-1 forming stable iminonitroxyl radicals. They can be used to assay nitric oxide in solutions by EPR spectroscopy; the sensitivity of the method is 1 microM for the detection of NO concentration and 0.3 nM/sec for the measurements of the rates of NO generation for 1 h in 0.2 ml sample. To overcome fast reduction of the radicals in biological samples, charged NNR was incorporated into the inner volume of large unilamellar phosphatidylcholine liposomes thus decreasing the rates of NNR reduction about 1000-fold. The method was used for the NO synthase activity assay in rat cerebellum cytosol. NNR was used to study the kinetics of the decomposition of 3,4-dihydro-1,2-diazete 1,2-dioxides (DD). Several DD derivatives at 5-80 microM concentrations are very effective vasodilators in perfused rat tail artery. Intraperitoneal injection of several DD (40-200 micrograms/kg weight). in hereditary hypertensive rats (ISIAH-strain) significantly (by 30%) decreased systolic arterial blood pressure whereas similar effect of trinitroglycerin was detected at significantly higher dose (900 micrograms/kg weight).

Animals↗

PubMed1995

Kinetics of nitric oxide liberation by 3,4-dihydro-1,2-diazete 1,2-dioxides and their vasodilatory properties in vitro and in vivo.

Derivatives of 3,4-dihydro-1,2-diazete 1,2-dioxides (DD) have been investigated as NO donors in vitro and in vivo. Using nitronylnitroxides as spin traps for NO, these compounds were shown to decompose in water solutions at physiological pH and temperature, producing two molecules of NO per one DD molecule. Rate constants of DD decomposition were found to be in the range from 10(-8) to 6.5 x 10(-7) c-1 in water and between 3 x 10(-7) and 1.6 x 10(-5) c-1 in dimethylsulfoxide. In vitro experiments performed with perfused rat tail artery showed that some of DD derivatives are highly effective vasodilators in concentrations from 5 to 80 microM while standard NO donor 3-(4-morpholino)-sydnonimine, SIN-1, does not lead to arterial vasodilation in these concentrations. Significant (up to 30%) decrease of systolic arterial blood pressure was observed in hereditary hypertensive rats (ISIAH-strain) when some of DD were injected intraperitoneally in doses 40-200 micrograms/kg b.w., while the same effect of trinitroglycerin, TNG, was found at much higher dose equal to 900 micrograms/kg b.w.

Animals↗

PubMed1995

Reversible inhibition of NADPH-cytochrome P450 reductase by alpha-lipoic acid.

NADPH-cytochrome-P450 reductase both purified from rat hepatic microsomes and involved in microsomal fraction was inactivated by treatment with alpha-lipoic acid. Since alpha-lipoic acid contains disulfide bond, it reacts with SH-groups of the reductase via the reaction of thiol-disulfide exchange resulting in the loss of the enzyme reducing activity. NADP+ completely protected reductase from the inactivation. The modification of reductase was reversible: the modified enzyme was partially reactivated with dithiothreitol and dihydrolipoic acid in the case when cytochrome c was used as a substrate of reductase. In the case when inorganic substrate, K3Fe(CN)6, was used for assay the activity of modified reductase no reactivation was observed. It was found that the order of the reaction of inactivation of membrane-bound microsomal reductase is equal to 1.2 +/- 0.2, which is in an agreement with pseudo-first order kinetics, and the second-order-rate constant of 26 M-1min-1. The results have shown that well known therapeutic agent alpha-lipoic acid is an efficient inhibitor of both purified and microsomal reductase.

Animals↗

PubMed1994

Spin trapping of nitric oxide by nitronylnitroxides: measurement of the activity of no synthase from rat cerebellum.

Nitric oxide (NO) has been shown to be an important mediator in vasodilation, neurotransmission and cellular cytotoxicity. We investigated a new series of nitronylnitroxyl radicals (NNR) as spin traps for NO. It was found these radicals react with NO with rate constants of about 10(4) M-1c-1 forming stable iminonitroxides with dramatic changes in EPR spectra. To overcome fast reduction of the radicals (a few seconds in rat cerebella cytosol), NNR with charged trimethylammoniophenyl group (Ib) was incorporated into the inner volume of large unilamellar phosphatidylcholine liposomes. In this case the reduction of the radical Ib in rat cerebella cytosol is slow (ca. 1% per min). The rate of NO production by NO synthase from rat cerebellum measured by NNR, Ib, is in a reasonable agreement with that obtained by spectrophotometric method.

Amino Acid Oxidoreductases↗

PubMed1993

[Heterogeneous selection in a subdivided population with unstable deme sizes].

The influence of small deme sizes oscillations in a one-locus two-allele model is considered. The dependence of frequency deviations from the fluctuation numbers is obtained. The new method of selection coefficients and migration matrix estimation from the deme numbers and the allele frequencies observations is found.

Alleles↗