The effect of antioxidants on electrocatalytic activity of cytochrome P450 3A4

The electrochemical analysis of cytochrome P450 3A4 catalytic activity has shown that vitamins C, A and E influence reduction of cytochrome P450 3A4. These data suggest a possibility of cross effects and interference of vitamins-antioxidants with drugs metabolised by cytochrome P450 3A4, during complex therapy of patients. These vitamins demonstrate antioxidant properties that lead to the increase of the cathodic current corresponding to heme reduction of this functionally significant hemoprotein. Ascorbic acid (0.028–0.56 mM) stimulated the cathodic peak (an electrochemical signal) of cytochrome P450 3A4. In the presence of diclofenac (Voltaren), a typical substrate of cytochrome P450 3A4, the increase in the catalytic current suggesting electrocatalysis and stimulating action of ascorbic acid was observed. In the presence of vitamins A and E the dose-dependent increase in the catalytic current of cytochrome P450 3A4 was observed in the range of vitamin concentrations from 10 to 100 μM. The maximal increase of 229 ± 20 and 162 ± 10% was observed at 100 μM vitamin A and vitamin E, respectively. In contrast to vitamin A, vitamin E in the presence of the cytochrome P450 inhibitor itraconazole did not increase the catalytic current. The latter implies existence of some substrate properties in vitamin E. The electrochemical approach for the analysis of catalytic activity of cytochrome P450 3A4 and studies of the effect of biologically active compounds on electrocatalysis is the sensitive and effective sensor approach, allowing to use low concentration of protein on an electrode (up to 10–15 mol/electrode), to carry out the analysis without involvement of protein redox partners, and to reveal drug-drug or drug-vitamins interaction in pre-clinical experiments.     

The study of markers of herpes virus infections in myocardial ischemia

A group of patients with ischemic heart disease (IHD), who underwent surgical aorta-coronary vascular shunting, was examined in this investigation. Low titers of HSV-1 specific IgG were detected in all patients, the obtained values being consistent with similar data obtained in healthy subjects of the same age. Negative PCR of HSV-I DNA in blood and biopsy results were obtained. None of the patients demonstrated typical clinical pattern of infectious disease caused by herpes simplex virus. These data are evidence of the absence of the HSV-1 correlation with coronary atherosclerosis in patients with the IHD diagnosis. The significance of HCMV specific IgG titers and HCMV DNA detected in blood plasma in 87.7% cases is probably attributed to existence of connection of HCMV infection markers revealing in patient' blood with IHD diagnosis and coronary atherosclerosis. Besides, the HCMV DNA presence in biopsy taken from myocardium or vascular wall with lesion is revealed in 100% cases. The cytomegalovirus markers in tissue lesions with the help of specific antiserums marked to HSMV recombinant proteins are also revealed in 100% cases. This fact indicates the connection between pathological atherosclerotical process in IHD and cytomegalovirus infection.     

Efficacy of therapeutic and prophylactic actions of ultralow doses of antibodies to gamma-interferon in experimental murine model of herpes virus]

The process of the disease due to herpes simplex virus types 1 and 2 (HSV-1 and 2) was studied on white uninbred mice weighing 10 to 12 g. The animals were infected intracerebrally or intraperitoneally. Intraperitoneal contamination of the animals with MS strain of HSV-2 was used for the experimental model of the herpes simplex infection. The prophylactic antiherpes action of ultralow doses of the human gamma-interferon antibodies (ULD of anti-IFN-gamma) at a course of its intragastral administration was evaluated. The preparation was shown to have a significant (p < 0.05) protective effect in a dose of 10 LD50, evident from a 10-fold decrease of the HSV-2 accumulation in the brain, a lower percentage of the animal deaths and an increase of the average lifespan of the animals by 3.3 days. The study of the therapeutic action of ULD of anti-IFN-gamma at a course of its intragastral administration showed that the preparation had no significant positive effect on the disease process in the animals infected with HSV-2 in a dose of 10 LD50. However, a positive effect associated with delayed virus replication in the brain was observed in the study on the therapeutic effect of ULD of anti-IFN-gamma after its intragastral administration to the mice infected with a sublethal dose of the virus.     

Oxidative and SOS-response in Escherichia coli and their interference]

Interference between the oxidative and SOS responses in Escherichia coli was studied. The oxidative response involves both reactive oxygen scavenging system and DNA repair systems which are distinct from either the SOS or adaptive response to alkylating agents. The oxyR gene is a positive regulatory gene for the oxidative response and controls at least 9 proteins which are induced by treatment with H2O2. This gene is not a portion of the SOS regulon that involves at least 17 different genes in E. coli and controls the SOS response--another inducible and nonspecific repair activity. The SOS response was measured in E. coli PQ37 by means of a sfiA: :lacZ operon fusion according to "SOS Chromotest" in a completely automated system "Bioscreen C" (Labsystems, Finland). Our data have shown that: 1) H2O2 was a potent inducer of sfiA gene--one of the SOS genes; 2) there was strong negative effect of the oxidative response on the subsequent induction of the SOS response. In common with our previous findings it should be concluded that there is an interference between the SOS response--on the one hand, and the adaptive and oxidative responses--on the other. The nonspecific heat shock response is proposed to be a main key in these interferences.     

Herpes simplex virus type 1 and 2 (HSV-1,2) DNA detection by PCR during genital herpes

The oligonucleotide primers for herpes simplex virus type 1 and 2 DNA detection are developed. In examined group of patients with genital herpes the virus of type 2 and type 1 was detected in 63% and 26% cases, respectively. The mixed infection of both types is revealed in 11% of the patients.     

Heat shock inhibits the induced expression of the SOS genes and SoxRS regulons in Escherichia coli

Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression of sfiA and soxS genes induced by oxidative agents H2O2, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined in E. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ. The expression of these genes induced by cell treatment with H2O2, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion O2-, it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H2O2 is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O2-.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Potentiation of YC-1 activation of soluble guanylate cyclase by NO donors and the increase of the synergistic effect of YC-1 on the NO-dependent activation of the enzyme by 1,2,3-triazolyl-1,2,5-oxadiazole derivatives

The influence of (1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole derivatives: 4-amino-3-(5-methyl-4-ethoxycarbonyl-(1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole (TF₄CH₃) and 4,4′-bis(5-methyl-4-ethoxycarbo-nyl-1H-1,2,3-triazol-1-yl)-3,3′-azo-1,2,5-oxadiazole (2TF₄CH₃) on stimulation of human platelet soluble guanylate cyclase by YC-1, NO donors (sodium nitroprusside, SNP, and spermine NONO) and on a synergistic increase of NO-dependent activation of the enzyme in the presence of YC-1 has been investigated. Both compounds increased guanylate cyclase activation by YC-1, potentiated guanylate cyclase stimulation by NO donors and increased the synergistic effect of YC-1 on the NO-dependent activation of soluble guanylate cyclase. The similarity in the properties of the examined 1,2,3-triazol-1-yl-1,2,5-oxadiazole derivatives with that of YC-1 and a possible mechanism underlying the recognized properties of compounds used are discussed.     

Connectivity between soxS gene expression and adaptive resistance to the SoxRS-regulon inducers in E. coli cells

Development of the adaptive response (AR) to the SoxRS-inducers-menadione (O2(-.)-donor), dinitrosyl-iron complex (NO donor) and their simultaneous action was studied in E. coli. Two AR parameters were used: an increasing in viability and decreasing in the soxS gene (SoxRS-regulon) expression in adapted cells. It was shown that namely peroxynitrite (ONOO-), being formed inside the cells from O2-. and NO, was the most cytotoxic agent among the drugs tested. On the one side, an increase in resistance to menadione treatment was selectively demonstrated in adapted E. coli delta oxyR mutant cells, defective in OxyR-regulon activity. On the other side, a decrease in soxS gene expression was marked in the experiments with menadione, as well So, an AR to O2-. superoxide anion was selectively regulated by the SoxRS DNA-repair pathway. OxyR-regulon that is selectively activated by the most redox-cycling agents and controls AR to these agents doesn't provide development of the AR to O2-..