Thiazolium salt mimics the non-coenzyme effects of vitamin B1 in rat synaptosomes.

Long-term studies have confirmed a causal relationship between the development of neurodegenerative processes and vitamin B1 (thiamine) deficiency. However, the biochemical mechanisms underlying the high neurotropic activity of thiamine are not fully understood. At the same time, there is increasing evidence that vitamin B1, in addition to its coenzyme functions, may have non-coenzyme activities that are particularly important for neurons. To elucidate which effects of vitamin B1 in neurons are due to its coenzyme function and which are due to its non-coenzyme activity, we conducted a comparative study of the effects of thiamine and its derivative, 3-decyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium chloride (DMHT), on selected processes in synaptosomes. The ability of DMHT to effectively compete with thiamine for binding to thiamine-binding sites on the plasma membrane of synaptosomes and to participate as a substrate in the thiamine pyrophosphokinase reaction was demonstrated. In experiments with rat brain synaptosomes, unidirectional effects of DMHT and thiamine on the activity of the pyruvate dehydrogenase complex (PDC) and on the incorporation of radiolabeled [2-14C]pyruvate into acetylcholine were demonstrated. The observed effects of thiamine and DMHT on the modulation of acetylcholine synthesis can be explained by suggesting that both compounds, which interact in cells with enzymes of thiamine metabolism, are phosphorylated and exert an inhibitory/activating effect (concentration-dependent) on PDC activity by affecting the regulatory enzymes of the complex. Such effects were not observed in the presence of structural analogs of thiamine and DMHT without a 2-hydroxyethyl substituent at position 5 of the thiazolium cycle. The effect of DMHT on the plasma membrane Ca-ATPase was similar to that of thiamine. At the same time, DMHT showed high cytostatic activity against neuroblastoma cells.

[Nicotinamide influence on pancreatic cells viability].

The study was undertaken to investigate the modulating effect of nicotinamide (NAm) in different concentrations and under different glucose concentrations on the viability and oxidative stress induced by streptozotocin (STZ, 5 mmol/l) and hydrogen peroxide (H2O2, 100 micromol/l) on isolated rat pancreatic cells of the Langerhans islets in vitro. Cell viability did not depend on the concentration of glucose in the range of 5-20 mmol/l, and in subsequent studies we used glucose in concentration of 10 mmol/l to protect cells against its hypo- and hyperglycemic action. Cytoprotective effect of NAm in concentrations from 5 to 20 mmol/l on cells survival was the same. It was found that the destructive action of STZ and H2O2 during 24 hours on isolated cells of the pancreas resulted in the significant cell death. It was revealed that NAm in concentration of 5 mmol/l not only had cytoprotective effects against STZ and H2O2 but also partially reduced the level of oxidative stress in the investigated cells induced by these compounds. High concentration of NAm, 35 mmol/l, causes cytotoxic effect on the viability of pancreatic islet cells and increase of oxidative stress induced by STZ and H2O2. Most likely these effects could be associated with direct modulatory action of NAm on important effector mechanisms involved in cell death, including PARP-dependent processes, or/and indirectly, through metabolic and antioxidant effects of the compound.

[Existence of two different active sites on thiamine binding protein in plasma membranes of synaptosomes].

The current work is aimed at understanding the structure and functionality of thiamine binding protein (TBP) in neural cells plasma membranes. The influence of thiamine triphosphate on thiamine binding by TBP in synaptic plasma membranes (SPM) isolated from the rat brain was investigated. It was shown that thiamine triphosphate inhibits thiamine binding activity of SPM in concurrent manner (K(i) = 1.0 +/- 0.3 microM). At the same time thiamine had no effect on thiamine triphosphatase (ThTPase) activity at the concentration range 0.5-20 microM. Otherwise, ThTPase activation with the maximum at the concentration about 2.5 microM was observed. Further, the influence of classic thiamine antagonists (amprolium, oxythiamine and pyrithiamine) on both biological activities of TBP in SPM was studied. The IC50 value for inhibition of thiamine binding on SPM by amprolium comprised 50 +/- 4.0 microM. Still, this antagonist had no effect on ThTPase activity. For the oxythiamine inhibition of both TBP activities was detected. The values of IC50 were 125 +/- 28 and 1000 +/- 95 microM for thiamine binding and ThTPase activity, respectively. The values of IC50 for thiamine binding and ThTPase activity inhibition differed by more than one order of magnitude and comprised 2.2 +/- 0.2 and 43 +/- 9 microM, respectively. The obtained data indicate that the active sites on SPM responsible for thiamine binding and ThTPase activity have different sensitivity to thiamine antagonists. Our results allow us to suppose that different active protein sites are responsible for the specific binding and for thiamine phosphates hydrolysis by TBP of synaptic membranes.

[Characteristics of thiamine triphosphatase from neural cells plasma membranes].

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.

[Thiamine metabolism disorders in the rat brain in experimental alcoholism and a possibility of their correction by vitamin E].

The influence of the chronic consumption of alcohol on biochemical reactions of thiamine metabolism in the rat brain is investigated. It is shown that the content of thiamine diphosphate (ThDP) in the brain tissue does not change at these conditions, though there is an essential decrease in the thiamine-kinase activity. The ability of the isolated nerve terminals (synaptosomes) to absorb labelled thiamine also decreases under this condition. The specified disturbances are probably the reason for deceleration of exchange of free (uncombined with proteins) thiamine and its phosphates in nervous cells, that results in the observed reduction in activity of pyruvate dehydrogenase complex (PDC) due to inactivation by phosphorylation. Thiamine-binding and thiaminetriphosphatase activities of thiamine-binding protein (ThBP) in the structure of synaptic plasma membranes (SPM), isolated from the rat brain in various experimental groups, have been investigated. The increase, with respect to control, in the both enzymes activity in SPM, isolated from the brain of rats with chronic alcoholism has been shown. Kinetic researches testify to an increase of affinity of SPM (ThBP) for thiamine and thiaminetriphosphate in these conditions. When vitamin E was given to animals with a model of chronic alcoholism the normalization of PDC activity in nervous cells was observed, that can testify to the transient character of these changes. Inability of vitamin E to normalize biological activities of ThBP in PMS, that has been analyzed, can testify to more deep disturbances in the structure of SPM or thiamine binding protein in their structure.

[Characteristic metabolic disturbances in the rat tissues caused by long-term use of alcohol].

In the researches carried out on rats with models of chronic alcoholism and alcohol abstinence the most vulnerable to chronic action of alcohol biochemical parameters are revealed: a level of reduced glutathione (it was estimated by the content of free SH-groups in tissues), the content of thiamine diphosphate and thiaminekinase activity in a brain, the content of folic acid in the blood, the content of ubiquinone in the cardiac muscle, RNA/DNA relation in the liver. The data obtained demonstrate first of all the negative influence of alcohol on metabolism of sulfur-containing substances and processes of transmethylation. The results of our investigation have also shown that the part of metabolic changes caused by long-term usage of alcohol, can be caused by direct influence of ethanol or its metabolites on those or other enzymatic proteins or receptors, and their functions can quickly be normalized after the abolition of alcohol (NAD+ contents, alpha-ketoglutarate dehydrogenase activity and some others). More stable changes in other parts of metabolism, that were specified earlier, may be also a result of long-term alcohol consumption.

[Effect of iodine and cobalt salts on content of biologically active substances in spirulina biomass]. / Vliianie solei ioda i kobal'ta na soderzhanie biologicheski aktivnykh veshchestv v biomasse spiruliny.

Production technology of iodinized spirulina biomass at vertical panel airlifted photobioreactor has been developed. The influence of different concentrations of iodine ions at certain combinations with cobalt ions on iodine-accumulative properties of spirulina has been studied. It has been shown that it is possible to obtain spirulina biomass containing certain amount of organically bound iodine by varying cobalt and iodine ions combinations and by staged addition of ions to incubation medium. This technique of staged addition of iodine and cobalt salt allowed to adopt spirulina to their high amount in the incubation medium, to increase spirulina productivity during growth, and to produce biomass with large iodine content.

[State of GABA-benzodiazepine receptor complex in diabetic neuropathy: effect of nicotinamide and nicotinoyl-GABA]. / Stan HAMK-benzodiazepinovoho retseptornoho kompleksu za diabetychnoï neiropatiï: efekt nikotynamidu ta nikotynoïl-HAMK.

An increase in GABA uptake by isolated rat brain synaptic endings as well as a decrease of pharmacologically active GABA analogue muscimol specific binding have indicated a physiologically drastic failure in realization of GABA-mediated inhibitory effects in CNS induced by diabetic encephalopathy. In spite of the impairment of inhibitory function of GABAergic transmission in diabetes a crucial activation of benzodiazepine receptors was determined, as it is tested by the increase in specific binding of flunitrazepam by synaptic membranes. This increase may play an important role in endogenous control of neural activity associated with the factors undefined so far. Using the approach that GABA, and several synthetic GABA agonists, appear to increase the affinity of the benzodiazepine recognition sites for such ligands, presumably by some allosteric mechanism, the findings concerning the in vitro binding assay technique confirm at least some of the functional characteristics observed between GABA and benzodiazepine receptors in vivo under pathological conditions. Indeed, the absence of activating effect on the affinity of flunitrazepam specific binding in the presence of micromolar concentrations of exogenous GABA implicate diabetes-induced alterations in coupling GABA- and benzodiazepine receptors that might be linked to changes in conformantial state of this membrane-bound complex and could partially explain diabetes-induced impairments of GABAergic transmission evaluated in the present study. Our study suggests that nicotinamide and especially GABA play an important role in improving the functioning of brain GABA-benzodiazepine complex impaired in diabetes through specific ligand-mediated mechanism and can be useful in the management of diabetes-associated brain failures.

[Interaction of GABA-ergic component of NAD with benzodiazepine receptors during epileptogenesis]. / Uchastie GAMK-eergicheskogo komponenta vo vzaimodelstvii NAD s benzodiazepinovymi retseptorami pri 'epileptogeneze

Nitrozepam has been shown to decrease specific binding of [14C]NAD by the synaptic mem-branes approximately by 40%. Alongside with the latter GABA in the concentration of 10(-5) M potentiates the effect observed. Blocking in vitro of GABA receptors by bicuculin removes both the tranquilizer and GABA effect. While simulating epileptogenesis in animals the nitrazepam effect on [14C]NAD specific binding sites was somewhat more expressed in comparison with the norm. The interaction of NAD reception systems with benzodiazepine under the pathology is capable to be mediated not only by increasing GABA-ergic transfer, but it can occur place without GABA-ergic component of GABA-benzodiazepine receptor complex.

[Effect of withdrawal of phenazepam and nicotinamide on the status of the system of reception of benzodiazepines and NAD]. / Vliianie otmeny fenazepama i nikotinamida na sostoianie sistem retseptsii benzodiazepinov i NAD.

Comparative investigations in reception of NAD, benzodiazepines and GABA by synaptic membranes under a single and multiple administration of phenazepam and nicotinamide to animals as well as after withdrawal of long-term administration of these drugs have been carried out. The both drugs activate GABA-inhibiting system of synaptic membranes. Both the sections of specific binding of benzodiazepines and of NAD take part in realization of "cancellation" syndrome. Chronic administration of phenazepam and nicotinamide results in the change of coupling in GABA-benzodiazepine receptor complex. After the withdrawal of chronic administration of nicotinamide the function of GABA-benzodiazepine receptor complex is normalized more quickly.

[Participation of a diazepam-binding inhibitor in the interaction between NAD and GABA-benzodiazepine receptor complex in synaptic membranes]. / Uchastie diazepamsviazyvaiushchego ingibitora vo vzaimodeistvii NAD s GAMK-benzodiazepineovym retseptornym kompleksom sinapticheskikh membran.

Diazepam-binding inhibitor isolated from synaptic membranes exerts a pronounced inhibitory effect on the specific benzodiazepine-receptor binding of 3H-flunitrazepam and simultaneously leads to an increase of synaptosomal uptake of 14C-GAMA. At the same time the inhibitor also depresses the specific binding of 14C-NAD by synaptic membranes, but displaying a greater effect. In both cases the inhibition was competent. Whether the isolated neuropeptide may act as an intermediary in the interaction with the reception system of NAD with GABA-benzodiazepine-receptor complex.

[Participation of benzodiazepine receptors in the mechanism of action of nicotinamide in nerve cells]. / Uchastie benzodiazepinovykh retseptorov v mekhanizme deistviia nikotinamida v nervnoi kletke.

Bicucculine being introduced to rats has induced an increase of [14C]GABA capture with synaptosomes and simultaneous decrease of GABA and NAD level in the brain. The decrease of the inhibiting effect of GABA is accompanied by the increase of specific binding of [3H]flunitrazepam with benzodiazepine receptors at the expense of the increase of binding capacity from 0.33 to 0.45 g/mol per 100 mg of protein. Under these conditions the dissociation constant remains unchanged. Such an activation of benzodiazepine receptors was observed under the lack of NAD in the organism (model of PP-hypovitaminosis). Introduction of the surplus doses of nicotinamide neutralizes the convulsant effect on benzodiazepine receptors.

[The effect of NAD on binding and liberation of (14)C-GABA during convulsant administration]. / Vliianie NAD na zakhvat i vysvobozhdenie (14)C-GAMK pri vvedenii konvul'santa.

Administration of corazol into animals led to a decrease in content of NAD and gamma-aminobutyric acid (GABA) in brain. Under these conditions, binding of 14C-GABA was increased and its liberation was inhibited in the synaptosomes of the brain cortex. Additional administration of nicotinamide, accompanied by considerable increase in content of NAD and GABA, caused a decrease in accumulation of exogenous GABA in the synaptosomes and removed the effects produced by the convulsant agent. Kinetics of 14C-GABA binding in the presence of NAD demonstrated that the more effective inhibition of the binding occurred in the animals treated with the convulsant drug. NAD appears to affect the GABA-ergic transmission at the postsynaptic level.

[Interaction of NAD with the GABA-ergic system of the rat brain]. / Vzaimodeistvie NAD s GAMK-ergicheskoi sistemi golovnogo mozga krys.

After administration of corazole content of gamma-aminobutyric acid (GABA) was increased in the rat brain within 2 min and 10 min by 28% and 80%, respectively. Content of the GABA was distinctly decreased in the prespastic phase. During this period specific binding of 3H-muscimol to the GABA receptors was decreased. NAD at concentrations 10(-6) M and 10(-7) M activated the GABA receptors and inhibited binding of 14C-GABA to the synaptosomes of both intact rats and the animals treated with the convulsant agent. NAD appears to cause an effect as an inhibitory neurotransmitter at the postsynaptic level.

[The functional characteristics of the inhibitory mediator systems in the brain synaptosomes of PP vitamin-deficient rats]. / Osobennosti funktsionirovaniia tormoznykh mediatornykh sistem sinaptosom golovnogo mozga u PP-gipovitaminoznykh krys.

A decrease of the NAD and serotonin level in the brain of rats with PP hypovitaminosis is shown. NAD in concentration of 10(-6) M in vitro exerts a less pronounced inhibiting influence on the neuronal uptake of [14C]serotonin and [14C]GABA by brain synaptosomes of rats with PP hypovitaminosis. GABA content under such conditions increases as compared with the control and correlates with changes in the [14C]GABA uptake system.

[Effect of benzodiazepines on NAD binding to synaptic membrane in the rat brain]. / Vliianie benzodiazepinov na sviazyvanie NAD sinapticheskimi membranami golovnogo mozga krys.

The receptor protein solubilized from synaptic membranes specifically binds [14C] NAD (dissociation constant--0.75 microM, capacity of binding sites--0.0125 nmol of metaid per 1 mg of protein). All the studied benzodiazepines (phenazepam, nitrazepam, clonazepam, flunitrazepam) are able to displace [14C] NAD from its receptor sites, the mixed type of inhibition being manifested. An inhibition constant for flunitrazepam, a ligand of benzodiazepine receptors, equals 10 microM. GABA promotes an inhibiting effect of benzodiazepines. It is supposed that neurotropic action of NAD is realized through the GABA-benzodiazepine complex of neuronal membranes.

[Solubilization and various properties of NAD-binding receptor protein from rat brain synaptic membranes]. / Soliubilizatsiia i izuchenie nekotorykh svoistv NAD-sviazyvaiushchego retseptornogo belka sinapticheskikh membran mozga krys.

The NAD-binding receptor protein has been solubilized from the synaptic membranes of the rat brain by different detergents. Digitanin proved to be the most effective detergent which exerted no action on the specific binding of [14C]NAD with the solubilized receptor protein. Kinetic parameters of the soluble ligand-receptor complex were studied. The affinity of the solubilized receptor protein to NAD did not change as compared to the protein of native membranes. The specific binding of [14C]NAD was saturated at Kd = 0.53 microM, Bmax = 0.011 nmol per 1 mg of protein. The molecular weight of the soluble NAD-receptor complex determined under native conditions was equal to 115 kDa.

[Solubilization of the central benzodiazepine receptor and study of its interaction with nicotinamide]. / Soliubilizatsiia tsentral'nogo retseptora benzodiazepinov i izuchenie ego vzaimodeistviia s nikotinamidom.

It was shown that nicotinamide and NAD inhibit the specific binding of [3H]flunitrazepam to benzdiazepine receptors without causing a direct influence of gamma-aminobutyric acid (GABA) receptors. The GABA-benzdiazepine complex was separated by solubilization with 0.5% lubrol PX. The solubilized preparations contain the binding sites for [3H]flunitrazepam alone (Kd = 5.9 nm). The Kd value for the membrane-bound benzdiazepine receptor is 2.9 nM. NAD inhibited the specific binding of [3H]flunitrazepam to the solubilized membrane preparation when used at concentrations by several orders of magnitude lower than that of nicotinamide. Using gel filtration on Sepharose 6B-CL, the molecular mass of the soluble benzdiazepine receptor protein was determined.

[Properties and biosynthesis of acetyl-CoA-carboxylase from chicken liver with administration of nicotinic acid during stimulation of lipogenesis]. / Svoistva i biosintez atsetil-CoA-karboksilazy pecheni tsypliat pri vvedenii nikotinovoi kisloty na fone stimuliatsii lipogeneza.

Acetyl-CoA-carboxylase is isolated and purified to a homogeneous state from the chicken liver with alimentary lipogenesis stimulation. Under the action of nicotinic acid in vivo the specific enzyme activity is shown to decrease considerably followed by some variations in its properties. According to the results obtained during ultracentrifugation and PAAG electrophoresis nicotinic acid causes partial enzyme deaggregation with simultaneous increase of its phosphorylation. The latter is accompanied by a rise in the content of phosphate labile to alkali on acetyl-CoA-carboxylase subunits. Nicotinic acid in vivo has practically no effect on acetyl-CoA-carboxylase synthesis and decay rate. Its inhibiting action is induced by stimulation of enzyme phosphorylation.