Showing papers in "Ukraïns'kyĭ biokhimichnyĭ zhurnal in 2009"

The role of alpha7 nicotinic acetylcholine receptors in B lymphocyte activation

Abstract: The involvement of nicotinic acetylcholine receptor (nAChR) a7 subtype in B lymphocyte activation has been investigated. B lymphocytes were magnetically separated from the spleens of C57Bl/6J mice. The purified lymphocytes were treated with fluorescently labeled IgM-, CD40-, CD16/32 or CD23-specific antibodies and unlabeled alpha7-specific antibody and examined by flow cytometry. The alpha7-specific antibody binding interfered with that of anti-CD40 but not of anti-IgM, anti-CD16/32 or anti-CD23 suggesting that alpha7 nAChRs are located close to CD40. B lymphocyte activation either in vitro with anti-CD40 or in vivo by immunization with cytochrome c resulted in increased alpha7 nAChR expression. Anti-CD40-induced B lymphocyte proliferation studied by [3H]thymidine incorporation was increased upon alpha7 nAChR inhibition with methyllicaconitine, choline or antibiotic gentamicin, as well as in the presence of the inhibitor of acetylcholine synthesis hemicholine-3. Mice injected with both cytochrome c and methyllicaconitine responded with IgM anti-cytochrome c antibodies faster than those injected with cytochrome c alone, while the secondary IgG responses were similar. It is concluded that alpha7 nAChRs negatively control CD40-mediated B lymphocyte proliferation but did not affect the IgM-IgG class switch or memory B cell activation. Endogenous acetylcholine may be regarded as an auto/paracrine regulator of B lymphocyte activation.

Screening of epigenetic and genetic disturbances of human chromosome 3 genes in colorectal cancer.

Abstract: DNA microarray technology comprising NotI-linking clones was used in a large-scale study of genetic and epigenetic changes in colorectal cancer. Analysis of samples from 24 patients revealed methylation, deletions, and amplifications in 137 of 181 NotI clones. For 27 genes/loci, these changes occurred in more than 30% of the tumor samples, suggesting that these genes are involved in the development of colorectal cancer. An analysis of the methylation status of CpG island of the ITGA9 gene/loci by bisulfite sequencing confirmed the NotI microarray data on the gene/loci methylation in colorectal cancer. Aberrations in 19 genes/loci were unknown previously. Their characterization may help ascertain the mechanisms responsible for colorectal cancer development and identify novel diagnostic and prognostic markers.

Fluorescent derivatives of diphtheria toxin subunit B and their interaction with Vero cells

Abstract: Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell.

Nonalcoholic fatty liver disease: biochemical and therapeutic considerations.

Abstract: Nonalcoholic fatty liver disease (NAFLD) is a rapidly emerging chronic liver disease and is reported to affect up to 70-80% of overweight and obese individuals. NAFLD represents a spectrum of liver diseases that range from simple hepatic steatosis, to a more severe and treatment resistant stage that features steatosis plus inflammation, termed nonalcoholic steatohepatitis (NASH), which may in turn progress to hepatic fibrosis, cirrhosis, and sub-acute liver failure. Thus, NAFLD and its subsequent complications create a significant health burden, and currently there is no effective treatment strategy. The biochemical mechanisms that underlie NAFLD are unclear at this time, but there is evidence that insulin resistance is a major contributing factor. In addition, circulating concentrations of inflammatory cytokines (e.g., TNF-alpha, IL-6) as well as decreased antiinflammatory factors (e.g., adiponectin, IL-10) are not only implicated in the development of insulin resistance and type 2 diabetes, but are also related to NAFLD. Such inflammatory mechanisms are fundamental in the progression of NAFLD toward higher risk cirrhotic states. This review outlines the leading theories of pathogenesis of NAFLD and highlights the potential role of exercise in treating and preventing NAFLD. Regular exercise can reverse insulin resistance, suppress low-grade systemic inflammation, and attenuate inflammatory markers associated with NAFLD. Thus, exercise has the potential to become an effective treatment and prevention modality for NAFLD and NASH.

[Lactate dehydrogenase, adenosine deaminase and thymidine phosphorylase activity of blood and tissues in breast cancer].

Abstract: Lactate dehydrogenase (LDH), adenosine deaminase and thymidine phosphorylase activity was analyzed in the blood serum, primary tumor and adjacent uninvolved breast tissues from 49 women with adenocarcinoma and from 10 ones with benign adenofibroma. The LDH activity was increased in both cancerous and adjacent tissues. Serum LDH level reflects cell membrane alterations not only in the tumor node cells but also to a greater extent--in the surrounding unmalignant tissues. The discovered changes in nucleosides catabolic enzyme's activity in patients with breast cancer are correlated with LDH activity and its level in the blood serum.

[Construction of immune library of murine immunoglobulin genes and screening of single-chain Fv-antibodies specific to diphtheria toxin B subunit].

Abstract: The aim of this work was to obtain the panel of recombinant single-chain Fv-antibodies against diphtheria toxin B subunit, the main diagnostic and pathogenic antigen of Corynebacterium diphtheriae. For this purpose we have constructed the immune library of murine immunoglobulin genes. A number of scFv specific to diphtheria toxin B subunit were acquired from the obtained library after one round of selection by phage-display. ScFv encoding DNA-fragments of eight clones were subcloned into plasmid pET-22b(+). It was shown that selected scFv were highly specific to diphtheria toxin B subunit, with affinity constant for different clones ranged from 10(7) to 10(9) M(-1).

[Toxin-neutralizing properties of antibodies to diphtheria toxin recombinant subunits A and B and a new method of their estimation].

Abstract: Development of complications during diphtheria depends to a large extent on toxin-neutralizing antibodies level in the patient's blood. Active immunization of people with diphtheria anatoxin is widely used for diphtheria prevention and passive immunization with hyperimmune antitoxic horse serum is used for diphtheria treatment. A traditional component of anti-diphtheria vaccines--diphtheria anatoxin has a number of serious disadvantages, which are mainly associated with complicated procedure of its production. Thus, the search for new antigen substances, which can effectively stimulate protective humoral response to diphtheria toxin, is an urgent task in anti-diphtheria vaccine development. Furthermore, one of the most important objects is the development of new in vitro methods for estimation of diphtheria toxin-neutralizing polyclonal and monoclonal antibodies, which allow to avoid using active diphtheria toxin and toxin-sensitive laboratory animals. Comparative studies of toxin-neutralizing antibodies induction after immunization of laboratory animals with recombinant subunits A and B of diphtheria toxin were carried out. The new method for detection of protective antibodies in serum was proposed. This method is based on the ToBI test (Toxin Binding Inhibition test); namely on the property of anti-diphtheria antibodies to inhibit the biding of toxin subunit B fused with enhanced green fluorescent protein (EGFP) to the sensitive to diphtheria toxin Vero cells. The ability of subunit B to induce toxin-neutralizing antibodies in laboratory animals (rabbits and guinea pigs) was confirmed by the intradermal test, which is traditionally used to detect protective antitoxic antibodies in the serum, and by flow cytometry method, developed for this purpose. The results suggest that diphtheria toxin recombinant subunit B may be used for the induction of the protective immune response. The new developed approach for estimation diphtheria toxin-neutralizing antibodies is more ethical and safe and can substitute successfully the traditional methods.

[Structure and functions of glutathione S-transferase P1-1].

Abstract: Glutathione S-transferase P1-1 (GSTP1-1) is a multifunctional enzyme which protects the cell from the influence of genotoxic factors and apoptosis. Contemporary knowledge about the GSTP1 molecule structure and the enzyme functions are summarized in this review. Also P1 isoform is compared to other glutathione S-transferases and structure-function relationships are emphasized. Novel functions of GSTP1 in cell signaling modulation, NO storage and metabolism are highlighted in this paper.

[Spin-catalysis in the processes of photo- and bioactivation of molecular oxygen].

Abstract: Rational explanation of the mechanisms of bioactivation of molecular oxygen by enzymes is impossible without understanding more simple mechanisms of the O2 photoactivation in collision complexes of gases and solvents. Production of peroxides in oxidases and more complicated oxidation processes by molecular oxygen are spin forbidden reactions and bioactivation of dioxygen is connected with enzymatic spin-catalysis by acceleration of the triplet-singlet (T-S) quantum transitions. Internal magnetic perturbations in the free oxygen molecule and in O2 complexes with solvents or with coenzyme in biopolymers bear characteristic entirely similar features and removal of spin prohibition on T-S transitions is quantified by some common physical mechanisms. An account of specific spin-orbit coupling (SOC) in the open pi(g)-shell of dioxygen permits to explain the T-S transitions intensity of the red atmospheric band in the O2, molecule and the selective enhancement of the radiative alpha1delta(g)-->X3Sigma(g)- transition intensity in various solvents (P). Charge transfer contribution P+ O2- leads to increase of SOC between the T-S oxygen states that enhances the O2 (alpha1delta(g)) quenching. Similar T-S transitions mechanisms and SOC enhancement is realized upon dioxygen activation by enzymes of the glucoseoxidase type. Three electronic mechanisms of reductive bioactivation of O2 by oxidases coenzymes are considered on the basis of physical mechanisms of the O2 photoactivation. They include intermediate stages of the superoxide and peroxide ion formation. Study of the O2 activation mechanisms by T-S transitions in enzymatic complexes of dioxygen binding open new potentialities in biotechnology and medicine.

Characteristics of thiamine triphosphatase from neural cells plasma membranes

Abstract: The kinetic parameters of the ThTP hydrolysis by synaptic plasma membranes isolated from rat brain were investigated. It was shown that the ThTPase reaction pH optimum was 7.4, the apparent K(m) was 52 microM and the apparent affinity constant for Mg2+ was 1.9 mM. The comparative analysis of the indicated parameters was done for the ThTPase activity of membrane bound (the data of present work and literature data) and cytosolic (literature data) proteins. The analysis allows us to suppose that thiamine-binding protein described earlier is the single ThTPase activity carrier in neural cells plasma membranes. It was shown that the active site of the enzyme that catalyzes the ThTP hydrolysis in neural cells plasma membranes is associated with the inside membrane surface.

[Anti-inflammatory effect of N-stearoylethanolamine in experimental burn injury in rats].

Abstract: The biochemical mechanisms of anti-inflammatory effect of endocannabinoid congener N-stearoylethanolamine (NSE) was studied on the model of experimental burn in rats. The animals after the thermal burn of the skin received per os during 7 days the water suspension of NSE in a doze 10 mg/kg of body weight. In the other groups of rats the suspension was applied to the wound (the concentration of NSE was 10 mg/ml). It was shown for the first time that NSE accelerated the process of burn wound healing by the inhibition of proinflammatory cytokines (TNFalpha, IL-6) production. NSE caused the normalization of the iNOS and cNOS activity and of nitrite content in plasma, erythrocytes, liver and spleen of rats. NSE also modified the antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) activity and diminished the level of lipid peroxidation. The discovered anti-inflammatory NSE properties suggest the possibility of its usage for burn treatment.

Effect of homocysteine on the structure and functions of human placenta trophoblasts

Abstract: Elevated level of homocysteine in blood serum of pregnant women is the risk factor for placental malfunction and fetal abnormalities. Our study has shown the activation of apoptosis, inhibition of proliferation, destruction of placental trophoblast and activation of the transsulfuration pathway under elevated homocysteine level in the incubation medium in the range of 20-80 microM. The activation of the transsulfuration pathway indicates that placenta may to some extent withstand elevated homocysteine level.

[Influence of hydrogen sulfide, dithionite, sulfite, thiosulfate and sulfate anions on human platelet aggregation].

Abstract: The influence of hydrogen sulfide, dithionite, sulfite, thiosulfate, and sulfate anions on human platelet aggregation was investigated in vitro. It was established that sulfite, thiosulfate, and sulfate did not influence the platelet aggregation induced by ADP, collagen, or epinephrine in the concentrations range 10-1000 microM. Hydrogen sulfide and dithionite inhibited platelet aggregation induced by ADF or collagen in a dose-dependent manner. The action of hydrogen sulfide began in concentration of 100 microM and the action of dithionite began in concentration of 1000 microM. They did not influence epinephrine-induced platelet aggregation.

[Analysis of signaling pathways activated by nicotinic acetylcholine receptors in B-lymphocyte-derived cells].

Abstract: The expression of nicotinic acetylcholine receptors (nAChRs) in the chicken pre-B-lymphoma DT40 cell line was investigated DT40 cells were shown to express at least alpha7-containing nAChRs; their amount increased upon incubation with 10 microM nicotine Addition of 10 microM choline favoured the inclusion of 3-[45dimethylthiazol-2-yl]-25-diphenyltetrazolium bromide (MTT); the effect of choline was inhibited by 25-25 nM methyllicaconitine (MLA) or 10-100 nM alpha-cobra-toxin indicating the alpha7 nAChR role in maintaining the proliferative potential of DT40 cells Nicotine and choline potentiated the effect of 05 microM ionomycin, which suppresses cell viability via Ca2+ ions influx Contrariwise, the suppressive effect of 1 microM hydrogen peroxide, mainly affecting cell mitochondria, was weakened by choline, but was increased by 25 nM MLA MEK1/2 and PKC kinases activity was necessary for maintaining the proliferative potential of DT40 cells MLA increased the effect of the MEK1/2 kinase inhibitor (U0126), while suppressive effect of MLA itself was decreased The presence of CaMKII kinase inhibitor (KN-62) also decreased MLA effect MLA favoured cell survival in the presence of PKC inhibitor (chelerythrine) These data indicate that MEK1/2 and CaMKII kinases are involved in alpha7-containing nAChR signaling in DT40 cells and that PKC plays a key role in this process

Activity of hydrogen sulfide production enzymes in kidneys of rats

Abstract: An experimental research of activity and kinetic descriptions of enzymes participating in formation of hydrogen sulfide in the kidney of rats has been carried out. It was established that cystein, homocystein and thiosulphate are the basic substrates for hydrogen sulfide synthesis. The higest activity for hydrogen sulfide production belongs to thiosulfate-dithiolsulfurtransferase and cysteine aminotransferase, less activity is characteristic of cystathionine beta-synthase and cystathio-nine gamma-lyase. The highest affinity to substrate is registered for thiosulfate-dithiolsulfurtransferase and cystathionine gamma-lyase. It is discovered that the substrate inhibition is typical of all hydrogen sulfide formation enzymes, although this characteristic is the most expressed thiosulfat-dithiolsulfurtransferase.

Evaluation of hepatotoxicity of novel maleimide derivative with cytostatic activity and its effect on peroxidation process and antioxidant system in the liver

Abstract: Novel maleimide derivative 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2.5-dione (MI-1) with cytostatic activity does not damage rat liver cells after intragastric administration. It is confirmed by alanine- and aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase activities in blood serum. Preliminary treatment with MI-1 partially prevents from liver cell damage caused by CoCl2. the content of thiobarbituric-active products, protein carbonyl groups, reduced glutathione and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in the liver cell after one- and ten-days treatment with novel maleimide derivative have been studied. It has been determined that one-day administration of MI-1 has not caused significant changes of peroxidation process and antioxidant system in the liver cells. After ten days treatment the activity of glutathione-S-transferase has been increased, superoxide dismutase--two times decreased, but other parameters have not been significantly changed. Ten days injection of CoCl2 provokes some manifestations of oxidative stress in the liver cells that has been partially leveled by preliminary treatment with maleimide derivative.

Possible pathways involved in activation of catalase and superoxide dismutase with sodium nitroprusside in yeast Saccharomyces cerevisiae.

Abstract: The effect of nitric oxide (*NO) on biological systems depends very much on many circumstances. Nitric oxide can activate redox sensitive pathways that in many cases results in an increase of antioxidant potential of the cell. However, the direct effects of nitric oxide on the activity of principal antioxidant enzymes such as catalase and superoxide dismutase (SOD) have not been studied. In the present work we exploited the yeast model to elucidate a possibility of regulation of the mentioned activity by NO-donor sodium nitroprusside (SNP). We demonstrated that nitric oxide spontaneously generated at SNP decomposition increased the activity of catalase and SOD 1.3 times. Using inhibitors of mRNA (actinomycin D) and protein (cycloheximide) synthesis, the strain deficient in Yap1p, a master regulator coordinating yeast adaptive response to oxidative stress, we have found that these enzymes are up-regulated via synthesis of new molecules at transcription and translation levels. This response is mediated by Yap1p. Despite the increase of SOD activity in yeast cells possibly includes the activation of the present apoprotein by Ccs1p, the ways of nitric oxide regulation of Ccs1p activity are still unclear.

Hydrogen peroxide inhibits acetylcholinesterase of myometrium sarcolemma

Abstract: The action of hydrogen peroxide on acetylcholinesterase enzymatic activity in myometrium sarcolemma fraction is investigated. Hydrogen peroxide (0.1-26 microM), depending on the concentration, suppressed the activity. Acetylcholinesterase proved to be highly sensitive to the action of H2O2, making Ki = 2.4 +/- 0.4 microM, nH = 0.65 +/- 0.08 (n = 4-5). It is established, that hydrogen peroxide in the range of 1.6 - 6.4 microM essentially reduce V(0,max) and K(M). In the presence of dithiothreitole (a reducer of SH-groups of the membrane surface) the investigated substance effect considerably decreased.

Expression of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-3 and VEGF mRNA in rat liver, lung and heart: effect of methyl tertbutyl ether.

Abstract: The main goal of this work was investigation of the effect of methyl tertbutyl ether, ecologically dangerous chemical compound, on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) and vascular endothelial growth factor (VEGF) mRNA in different rat organs. Expression of PFKFB-3 and VEGF is a hypoxia inducible factor (HIF)-dependent process which significantly increases under hypoxia, in malignant tumors and other pathology. In this study we have shown that PFKFB-3 and VEGF mRNA expression in the liver, lung, and heart changes in rats, treated with methyl tertbutyl ether for two months, in organ-specific manner. Expression of alternative splice variants of PFKFB-3 mRNA as well as VEGF mRNA also changes in organ-specific manner in rats, treated with methyl tertbutyl ether. The effect of methyl tertbutyl ether on the expression of PFKFB-3 and VEGF mRNA and its alternative splice variants is dose-dependent. Results of this investigation clearly demonstrated that methyl tertbutyl ether affects the expression of PFKFB-3, a key regulatory enzyme of glycolysis, as well as VEGF, very important factor of angiogenesis, in an organ-specific and dose-dependent manner.

[Tyrosine nitration as regulatory post-translational modification of proteins].

Abstract: Review highlights new pathways of signal transduction from nitric oxide (NO) that is a recognized secondary messenger in regulatory cascades of phylogenetically distant species. Indirect (via Ca2+ and cGMP) and direct (protein S-nitrosylation and tyrosine nitration) pathways of NO signal transduction are presented. S-nitrosyation as new paradigm in signal transduction and protein functions regulations is discussed. The importance of animal and plant protein tyrosine nitration in vivo and the potential regulatory significance of this posttranslational modification are emphasized. Also review describes tree mechanisms of protein tyrosine nitration: via peroxynitrite, peroxydase and haem-peroxidase. Particular attention is paid to cytoskeleton proteins tyrosine nitration, particularly tubulines, because microtubules are involved into modulation of signal transduction from various messengers.

Influence of gold and silver nanoparticles on ATPase activity of native and rehydrated cells of Escherichia coli

Abstract: The expressed changes in the bacteria cells' plasma membrane ATPase activity values cells of Escherichia coli, which are used in the manufacturing of vaccines against cattle colienterotoxemia, have been revealed under the influence of lyophilization/rehydration process. Influence of gold (average size 20, 30, 45 nm) and silver (30 nm) nanoparticles in different concentrations on ATPase activity of native and rehydrated (delyophilized) bacteria cells has been studied. Gold and silver nanoparticles stimulated ATPase activity of native and rehydrated (delyophilizated) cells of the investigated strains. Obtained data show, that application of examined metal nanoparticles of certain sizes and concentrations as protectors and stimulators of main physiological-biochemical properties of production strains' cells under the conditions of lyophilization and long storage stress in technologies of different immunobiological preparations manufacturing is very promising.

Detection of antiadenoviral antibodies by surface plasmon resonance

Abstract: A possibility to detect antiadenoviral antibodies by surface plasmon resonance (SPR) was demonstrated. Immobilization on the surface of a sensor of the hexone or degraded purified adenovirus of one of the types (Ad 2) allows finding antibodies to the hexone antigenic determinants of wide specificity common for human adenoviruses of different types. Optimum conditions for immobilization of the antigen and formation of the complex antigen-antibody are determined. The comparative assays of the levels of antibodies in rabbit antisera obtained to the hexone and adenoviruses of different types (1, 2 and 6) by SPR and ELISA was analyzed. The biosensor was used to detect antiadenoviral antibodies in the blood sera of children with aggravation of chronic nonspecific broncho-pulmonary diseases. The sensitivity of SPR in comparison with ELISA was 86.9%, in comparison with the method of fluorescing antibodies (MFA)--89.5%.

Effect of stress on the content of free radical oxidation products in subcellular brain fractions in rats of pubertal age

Abstract: The purpose of the present work is to study the influence of immobilization stress of different intensity on the content of protein and lipid oxidation products in subcellular brain fractions in rats of different age. It has been shown that 30-minute immobilization of 1.5-month, 2-month- and 12-month-old rats was not accompanied by accumulation of protein and lipid oxidation products in subcellular brain fractions. A prolonged immobilization of 1.5-month-old rats was accompanied by manifestations of oxidative stress in subcellular brain fractions; such manifestations were not characteristic of the rats of older age groups.

Production of biologically active substances of indol nature by bacteria of Azotobacter genus

Abstract: A stimulating effect of Azotobacter vinelandii IMB B-7076 bacteria on germination of seeds and formation of germs of different plants has been shown. One of the factors, which determines such phenomenon is the accumulation of biologically active substances in culture medium. The researched bacteria of Azotobacter genus accumulate indoleacetic acid (IAA) in the culture medium. Other substances of indol nature have been identified in the culture medium of these bacteria. It has been shown, using the computer's program PASS that these substances cannot take part in the regulation of plants growth. However 1H-indole-3carboxaldehyde can be characterized by antagonistic activity.

Endoglucanase-producing micromycetes capable of plant wastes degrading.

Abstract: The screening study of endoglucanase producers among 58 strains 28 species 12 genera of mesophilous and thermotolerant micromycetes has been performed. Approximately 20% of investigated strains of micromycetes showed the highest endoglucanase activities (hydrolysis rates reached 0.40-0.57) during their submerged cultivation with different cellulose-containing subsrates. Such plant wastes as rye straw, wheat bran, husk of sunflower seeds, leaves and stems of Zostera marina were optimal for fungal growth and high yields of endoglucanase. As a result, 15 strains of mesophilous and thermotolerant micromycetes with high endoglucanase activity were selected among fungi of Aspergillus, Fusarium, Penicillium and Corynascus genera.

[Effect of salicylic acid on water potential, ethylene secretion and activity of antioxidative processes in the winter wheat leaves under drought conditions].

Abstract: Effect of plants treatment by salicylic acid on the water potential, ethylene emission, intensity of lipid peroxidation oxidation and enzymatic antioxidative activity in the leaves with contrasting drought-resistance of winter wheat cultivars was investigated. It is ascertain, that the treatment of plants by salicylic acid contributes to a decrease of water loss and intensity of lipid peroxidation, to an increase of ethylene synthesis and peroxidase, catalase, superoxide dismutase activity in the winter wheat leaves under drought conditions.

Role of vitamin E in regulation of cholecalciferol hydroxylation in hypovitaminosis D and hypervitaminosis D

Abstract: It is established, that dose-dependent influence of vitamin E on vitamin D3 metabolism, is conditioned by degree of cholecalciferol sufficiency. Under a condition of D-hypovitaminosis, contents of 25OHD3 in blood serum is 2-fold reduced and vitamin D3 25-hydroxylase enzymes activity increased in rat hepatocytes. Vitamin E (0.726-7.26 IU) significantly stimulated the effect of vitamin D3 (40 IU) in animals with D-hypovitaminosis and led to further increase of 25OHD3 content in the blood serum and activity of vitamin D3 25-hydroxylase enzymes in hepatocytes. In D-hypervitaminosis the contents of 25OHD3 in blood serum was more than 3-fold increased and vitamin D3 25-hydroxylase enzymes activity was inhibited. Vitamin E (0,726-7,26 IU) lowered the vitamin D toxicity, decreased contents of 25OHD3 in blood serum and activity of vitamin D3 25-hydroxylase enzymes in hepatocytes. High doses of vitamin E (36.3 IU) under these conditions demonstrated negative effect on vitamin D3 metabolism. The mechanism of vitaminE participation in the vitamin D3 metabolism under D-hypovitaminosis and D-hypervitaminosis may be its influence on the activity of different vitamin D3 25-hydroxylase systems of hepatocytes.

[Comparative effects of flavonoids on cell cycle passage and apoptosis induction in human acute lymphoblastic leukemia MT-4 cells].

Abstract: In the present study, 8 flavonoids of three major flavonoid subgroups, namely flavones, flavonols and flavanones were tested for their cytotoxic and apoptogenic effects in human acute lymphoblastic leukemia MT-4 cells in vitro. Apoptotic cells were identified by DNA flow cytometric analysis. The effects of the flavonoids on the cell cycle patterns and activation of caspase-3 were also examined. Among the flavonoids tested, 7,8-benzoflavone, flavone, quercetin, chrysin, and galangine were shown to be effective apoptosis inducers. At concentrations corresponding to ED50, the flavonoids mentioned above exerted varying degrees of caspase-3 activation in MT-4 cells. The flavonoid-treated cells demonstrated different cell cycle profiles with accumulation in either G0/G1 (flavone, morin) or G2/M (7,8-benzoflavone, naringenin, quercetin, apigenin) phase. The induction of apoptosis did not correlate with phase-specific effects of flavonoid assayed. The relationship between chemical structure and apoptogenic activity of flavonoids tested is discussed.

Cytotoxicity of the B subunit of diphtheria toxin to human histocytic lymphoma U937

Abstract: The B subunit of diphtheria toxin (DT) is responsible for interaction with receptor on the cell surface and translocation of the catalytically active A subunit across endosomal membrane into the cell cytosole. Receptor for DT and its B subunit is membrane-anchored precursor of heparin-binding epidermal growth factor-like growth factor (pro-HB-EGF), which under the action of metalloproteases turns into soluble form (sHB-EGF), which acts as a potent mitogen for different cell types. Since free B subunit of DT has no catalytic activity it is considered to be nontoxic. However its influence on the cells in vitro remains to be investigated. The aim of this study was to examine the influence of diphtheria toxin B subunit on viability of diphtheria-sensitive cells using B subunit recombinant analogues. It was shown that diphtheria toxin B subunit recombinant analogue at a concentration of 12.8 x 10(-7) M had a cytotoxic effect on the human histocytic lymphoma cell line U937, which expresses a large amount of sHB-EGF. Besides, the similar cytotoxic effect had a fusion protein which consisted of a B subunit and an enhanced green fluorescent protein (EGFP). However recombinant EGFP alone didnot influence the cell viability. Annexin-V-FITC/PI staining demonstrated that maximal cytotoxic effect had been elicited after 48 hours of cultivation. Cytotoxic test with trypan blue and propidium iodide staining excluded the direct influence of investigated proteins on the integrity of plasma membrane because of the ability of B subunit to pore formation. So, we offer a hypothesis that realization of cytotoxic effect of diphtheria toxin B subunit and its derivative on the U937 cell culture occurs via inhibition of mitogenic activity of sHB-EGF resulted in induction of apoptosis.

[Changes of mitochondria membrane potential of the uterine smooth muscle under Mg2+ and Ca2+ influence].

Abstract: Using the method of flow cytometry and potential-sensitive fluorescent dye TMRM the effect of divalent cations on the membrane potential of isolated myometrium mitochondria was studied. It was shown, that Mg2+ (7 mM) addition to the incubation medium induced mitochondrial membrane hyperpolarization. In the case of protonophore CCCP (1 microM) preliminary presence in the incubation medium, Mg2+ addition did not lead to membrane potential hyperpolarization. Addition of Ca24+ leads to membrane potential dissipation. Ca2+ -induced mitochondrial depolarization is the same as NaN3 (10 mM)-induced depolarization. It also was shown that Mg2+ (7 mM) preliminary presence in the incubation medium did not protect mitochondria from Ca2+ -induced depolarization.