Glucotoxicity is mediated by cytoplasmic distribution of RAP1 in pancreatic ß-cells.

Diabetes mellitus (DM) is a group of chronic metabolic disorders characterized by persistent hyperglycemia. In our study, we analyzed the level and location of RAP1 changes in the development of ß-cell dysfunction induced by glucotoxicity. We employed three pancreatic ß-cell lines, namely INS-1, 1.2B4, and NIT-1, as well as a streptozotocin-induced diabetes rat model. We demonstrate that after high glucose treatment, RAP1 is increased, probably through induction by AKT, allowing RAP1 to shuttle from the nucleus to the cytoplasm and activate NF-κB signaling. Furthermore, non-enzymatic post-translational modifications of RAP1, such as advanced glycation end products and carbonylation may affect the function of RAP1, such as activation of the NF-κB signaling. Taken together, we showed that RAP1 is a new player in the mechanism of glucotoxicity in pancreatic ß-cells.

[EFFECT OF L-ARGININE - NO ON PROOXIDANT-ANTIOXIDANT BALANCE IN ERYTHROCYTES OF RATS UNDER ALCOHOL INTOXICATION.]

It was shown changes in the activity of antioxidant enzymes (superoxide dismutase, catalase) and NO-synthase (NOS), in the content. of stable metabolic products of nitric oxide and levels of lipid peroxidation products in erythrocytes of rats under alcoholic intoxication. It was shown that animals with alcohol intoxication under of the admission of the main substrate NOS - L-arginine activity of antioxidant protection enzymes was increased in twice on the fond of TBA-positive products decrease contents. Established in hemolisate red blood cells in rats with alcohol inrotoxication value of total NOS activity decreases by 65% compared to control. Not selective inhibitor Nto-nitro-L-arginine methyl ester (L-NAME), which is a structural analog of L-arginine, reduced output level of total NOS activity by 23.4% in the control and 25% under conditions of pathology. The consumption of rats L-arginine NOS total activity increased in the two study groups. The results testify that L-arginine has antioxidant properties, whereas L-NAME exerts a slightly stabilizing influence.

Effects of the combined arginase and canavanine treatment on leukemic cells in vitro and in vivo.

It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. Administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. Thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities.

Reduction of radiation­induced nitrative stress in leucocytes and kidney cells of rats upon administration of polyphenolic complex concentrates from red wine.

The research has shown that exposure to ionizing radiation at the dose of 30 cGy leads to the activation of NO-synthase way of nitrogen oxide synthesis, as well as to the accumulation of its stable metabolites and 3'-nitrotyrosine modified proteins in rat peripheral blood leucocytes and the renal cortical layer. NO-synthase activity was preserved at the control value through the consumption of red wine natural polyphenolic complex concentrates by the irradiated animals. The content of proteins modified by tyrosine nitration decreased in the early period of post-radiation exposure due to the influence of the investigated concentrate. Thus the ability of red wine natural polyphenolic complex concentrates to prevent adverse changes in L-arginine/NO system and, therefore, inhibit the development of nitrative stress induced by low doses of ionizing radiation has been proved experimentally.

Inhibiting effect of agmatine on genetically programmed death of leukocytes in experimental diabetes mellitus.

This study is an attempt to elucidate of agmatine effects upon leukocyte apoptosis in experimental diabetes mellitus (EDM). We demonstrated the increase in numbers of the leukocytes with both early and late signs of apoptosis at diabetes. Further changes in the morphofunctional state of the leukocytes include the increased amount of fragmented DNA, elevated apoptotic index and violated ratio of p53 to Bcl-2 proteins. Agmatine has been shown to exert direct corrective effects on leukocyte apoptosis: the content of р53 and Bcl-2 proteins was normalized, apoptotic index was decreased, the process of nuclear DNA degradation was ceased, while the amount of cells with early and late signs of apoptosis was diminished.

[THE ALKALOID-FREE FRACTION FROM Galega officinalis EXTRACT PREVENTS OXIDATIVE STRESS UNDER EXPERIMENTAL DIABETES MELLITUS].

The effect of alkaloid-free fraction from Galega officinalis extract on the process of formation of reactive oxygen species and indicators of prooxidant-antioxidant balance was investigated in rat peripheral blood under conditions of experimental diabetes mellitus. It was shown that alkaloid-free fraction from Galega officinalis extract prevents oxidative stress development in rats with streptozotocin-induced diabetes, providing antioxidant and antiradical mobilization mechanisms to protect the blood system. In the case of extract application to animals with studied pathology, one can observe a reducing effect of reactive oxygen species generation in leukocytes, inhibition of proteins and lipids oxidative modification processes and increased activity of key enzymes of rat peripheral blood antioxidant system (superoxide dismutase, catalase and glutathione peroxidase). The revealed biological effect could be explained by the presence of biologically active substances with antioxidant properties in the extract composition (phytol and flavonoids).

[Effects of decarboxylated L-arginine on morphofunctional characteristics of the erythron in experimental diabetes mellitus in rats].

This paper reports the results of a study of the impact of the agmatine treatment on erythrocyte resistance to acid hemolytic, reticulocyte count and reticulocyte production index, erythrocyte surface architectonics in streptozotocin-induced diabetic rats. Our results indicate that treatment of diabetic rats with agmatine causes a suppression of erythropoiesis and increases the resistance of erythrocytes against HCl-induced hemolysis. It was shown a 10% increase in the number of young red blood cells and 35% reduction in the number of structurally transformed erythrocytes that are capable of restoring normal biconcave disc shape under physiological condition. Our data demonstrate an improvement of morphofunctional state of red blood cells in diabetes and reflect the positive effect of agmatine treatment on erythrone due to the glucose-lowering action.

[The effect of natural polyphenol complex of red grape wine on L-arginine/NO system in peripheral blood of rats under low doses of ionizing radiation].

The total activity of NO-synthase and content of stable metabolitic products of nitric oxide in the peripheral blood of rats under low doses of ionizing radiation and administration of natural polyphenol complex of grape was investigated. It was found that natural polyphenol compounds of grapes have the ability to correct radioinduced changes in L-arginine/NO system. It was noted that the action of X-radiation increased activity of NO-synthase in the peripheral blood of rats. However, the consumption of natural polyphenol complex of grape led to a decrease of this index to control values. An increase in NOS activity under irradiation leads to the increase of NO stable metabolites content, which is reflected in the accumulation of nitrite- and nitrate-anions in the peripheral blood of rats. The consumption of preparation of polyphenol complex this index decreased in the early period of the experiment, and on the third day after exposure, the total content of NO stable metabolites is slightly higher compared to the indices of control animals. Thus, the ability of natural polyphenol complex to cause attenuation of oxidative-nitrative stress caused by ionizing radiation was investigated experimentally.

[Quantitative changes of main components of erythrocyte membranes which define architectonics of cells under pttg gene knockout].

A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein--beta-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of erythrocyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed underpttg gene knockout.

[The effect of agmatine on L-arginine metabolism in erythrocytes under streptozotocin-induced diabetes in rats].

The effects of agmatine on oxidative and nonoxidative metabolic pathways of L-arginine were investigated both in plasma and erythrocytes under experimental diabetes mellitus. It was indicated, that agmatine prevents the development of oxidative-nitrosative stress in diabetic rats. After treatment of animals by agmatine NO-synthase methabolic pathway of L-arginine is depressed whereas arginase one increases in erythrocytes of rats with experimental diabetes mellitus.

[Participation of phosphatidylinositol-3'-kinase in signal transduction through galactosyl-containing glycoprotein receptors of segmentonuclear leukocytes under type 1 diabetes mellitus].

It is shown that reduction of beta,D-galactosyl-containing carbohydrate determinants ofglycoconjugates on theplasmatic membrane of segmentonuclear neutrophills of peripheral blood under type 1 diabetes mellitus (DM) is correlated with changes in aggregation of these cells and may cause their functional disorder. Changes in the parameters of ricin-induced neutrophil activation after inhibition of the phosphatidylinositol-3'-kinase (PI-3'-kinase) enzyme with wortmannin indicated that the functional state ofpolymorphonuclear leukocytes is mediated by signaling pathways in which PI-3'-kinase is involved. Thus, PI-3'-kinase-dependent signal networks are involved in the processes of signal transduction through galactosyl-containing glycoprotein receptors into neutrophilic leukocytes. Inertness of the intensity formation in time ofneutrophil granulocyte cell response on RCA-induced translocation of the p85alpha regulatory subunit of PI-3'-kinase from the cytosolic to the membrane fraction under type 1 DM is a consequence of changes in the number or structure of plasmatic galactosyl-containing glycoprotein receptors. The revealed changes may be etiologic premise of diabetic complications and chronic diseases that impair the functional condition of patients with type 1 DM.

[Role of PARP and protein poly-ADP-ribosylation process in regulation of cell functions].

This review focuses on the biological role of enzymes involved in posttranslational modification of proteins by their poly-ADP-ribosylation, a NAD-consuming process with an emerging key role in providing fundamental cell functions. To this end, detailed analysis of structural organization in relation to basic functions of the poly(ADP-ribose) polymerase-1 (PARP-1), the founding member of the PARP family, and other poly(ADP-ribose) polymerase isoforms is presented here. These include the current views on the role of PARP family enzymes and processes of poly-ADP-ribosylation of proteins in chromatin structure remodeling, DNA damage repair, regulation of gene expression, and integration of cellular signaling pathways. Considerable attention is paid to the involvement of PARP in cellular functions, particularly in cell division, intracellular transport of macromolcules, proteasomal protein degradation, immune response and caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). In the light of the remarkable successes that have been reported for treating inflammatory disorders and cancer with different classes of PARPs inhibitors, we discuss the prospects of targeting PARPs with therapeutic purposes.

[Activation of the phosphatidylinositol-3'-kinase pathway with lectin-induced signal through sialo-containing glycoproteins of leukocyte membranes under type 1 diabetes mellitus].

The influence of wortmannin and sialospecific lectins on the translocation of p85alpha regulatory subunit of phosphatidylinositol-3'-kinase (PI-3'-kinase) between membrane and cytosolic fractions of the mononuclear and polymorphonuclear leukocytes in healthy donors and patients with type 1 diabetes mellitus (DM) was investigated. It was found out that under type 1 DM PI-3'-kinase takes active part in the transduction of lectin-induced signal through membrane glycoprotein receptors that contain terminal sialic acids linked to subterminal carbohydrate residues with (alpha2-->6) glycosidic bond.

[Protective effect of grape wines in nitrosative stress caused by experimental diabetes mellitus].

It was found that under the diabetes-induced oxidative-nitrosative stress, red and white wines, which polyphenols are considered to be the main active components, decrease the level of nitrotyrosine-modified proteins toward the control level in sciatic nerve, dorsal root ganglia and spinal cord of animals with diabetes mellitus. A decrease of the activity of poly(ADP-ribose)polymerase-1 to the control level in the sciatic nerve of diabetic rats with red wine consumption was also shown. During the experiment the body weight of the control group and diabetic groups of rats with consumption of red wine was significantly increased by 52% and 19% accordingly. The present results allow us to assume an important role of red wine and possibility of production of its preparations for prevention and treatment of diabetic neuropathy.

[Effect of L-arginine and aminoguanidine on morphological characteristics of the lymphocyte plasma membranes during experimental diabetes mellitus].

Diameter, the surface area of lymphocytes and their nuclea as well as the reserve potential and regulatory properties of lymphocytes membranes in control rats, in rats with experimental diabetes mellitus before and after administration of L-arginine and aminoguanidine were investigated. Significant difference in size and the membrane fund of lymphocytes in animals with experimental diabetes mellitus was detected. In control lymphocytes, the membrane fund was 70% while during diabetes mellitus it was 40%. Such a difference might be one of the reasons of changes in properties and functional state of lymphocytes in diabetes. Administration of L-arginine and aminoguanidine to diabetic animals caused positive effect: the volume of lymphocytes diminished and the regulatory membrane properties of these cells became better.

[Particularities of arginase and NO-synthase pathways of L-arginine metabolism in leucocytes of peripheral blood of rats in conditions of chronic x-ray irradiation].

Arginine takes part in many metabolic cell processes. It is not only involved in the dynamic cycle of interconversion with prolin and glutamine but also serves as a precursor for protein, nitric oxide, creatine, polyamines, agmatine and urea synthesis. Particularities of arginase and NO-synthase pathways of L-arginine conversion under the X-ray radiation in the leucocytes-radiosensitive cells of peripheral blood, under the per os administration of L-arginine and L-NAME (NG-nitro-L-arginine methyl ester) to rats were detected. It was shown that both L-arginine (arginase and NO-synthase main substrate) and L-NAME (nonspecific inhibitor of NO-synthase) had a positive correct influence on the organism in case of decreasing of NO overproduction under the X-ray radiation.

[Disorder of membrane protein spectrum of erythrocytes in type 1 diabetes mellitus].

The research has shown substantial changes in the percentage correlation between plasmatic membrane proteins and erythrocyte cytoskeleton (alpha-spectrin, beta-spectrin, ankyrin, band 4.1) under type 1 diabetes mellitus. It has also established the difference in the content of erythrocyte membrane glycoproteins, i.e. band 3 proteins and glycophorine A, in healthy donors and patients with diabetes. Thus the content of glycophorine A decreased by 27%, while the content of band 3 protein increased by 23%. Under the pathology, changes in the structure of carbohydrate determinants of erythrocytes membrane glycoproteins were revealed by means of the lectin blot analysis. Type 1 diabetes mellitus is accompanied by an increase in the number of glycoproteins with mannose-containing carbohydrate determinants complementary to Con A and LCA, on the one hand, and a decrease in the number of glycoproteins with sialo- and galactose-containing oligosaccharides complementary to WGA and RCA lectins, on the other hand, on the erythrocyte membrane surface.

[Peculiarities of L-arginine metabolism in the blood leukocytes in experimental diabetes mellitus].

In experimental streptozotocin-induced diabetes, the activity of enzymes which have common substrate (L-arginine) changes in leucocytes of peripheral blood. It leads to misbalance between different pathways of arginine metabolism: NO-synthase (oxidative) pathway is activated, whereas arginase (nonoxidative) one is depressed. The injection of L-arginine under diabetes activated nonoxidative pathway, which can be seen in possible decrease in NO-synthase activity with unchanged arginase activity. After injection ofaminoguanidine in animals under diabetes the efficiency of both pathways in leucocytes decreases, which may be the basis of reconstitution of physiological pool to the relatively essential amino acid L-arginine.

[Effect of aminoguanidine on oxidative modification of proteins in experimental diabetes mellitus in rats].

It was shown that administration of aminoguanidine is accompanied by a decrease of the content of nitric oxide stable metabolites, as well as protein carbonyl groups in leukocytes and blood plasma in diabetic and control animals. Aminoguanidine is proposed to be used for pharmacological correction of NO biosynthesis. Aminoguanidine, being the selective iNOS inhibitor, antioxidant and the factor eliminating post-translational protein nitrozylation and oxidative modification, weaken the toxic effects of NO and positively modulates the pathological state caused by NO hyperproduction.

[Effect of nitric oxide upon apoptosis activation in immunocompetent blood cells in diabetes mellitus, type 1].

NO-synthase activity, the content of stable products of NO metabolism (nitrites and nitrates) as well as fragmented DNA as biochemical marker of apoptosis have been determined in segmentonuclear neutrophilic granulocytes and mononuclear leukocytes in blood of healthy donors and patients with type 1 diabetes mellitus. It was found that activation of apoptosis in polymorphonuclear and mononuclear blood leukocytes under diabetes mellitus was accompanied by an elevation of NO-synthase activity and the content of stable NO metabolites. This suggests an increase of proapoptotic action of nitric oxide in immunocompetent blood cells under the pathology studied.