Peculiarities of carbohydrate metabolism in the rat liver due to the limited accessibility of thiamine

It was found that the progressive development of vitamin B1 deficiency in rats caused by varying doses of oxythiamine results in a sharp decrease of the free NAD/NADH ratio in the liver after injection of a high dose of the antivitamin. The value of this ratio was calculated from the intracellular concentrations of pyruvate, lactate and Keq for lactate dehydrogenase, whose activity remained practically unchanged throughout the experiments. The increase of the cAMP level in the liver caused by corresponding doses of oxythiamine was concomitant with a marked activation of cAMP-dependent processes.     

Antivitamin activity of O-acyloxythiamine disulfides

B1-antivitamin activity of symmetrical oxythiamine disulphide esters with succinic and o-phthalic acids has been studied in experiments on albino mice. It is shown that O-acyloxythiamine disulphides exert more profound and prolonged inhibitory effect on the transketolase activity in the animal body in comparison with the known antagonist of vitamin B1, oxythiamine, and the initial oxythiamine disulphide.     

Transketolase inhibitors based on disulfide derivatives of oxythiamine with branched hydrocarbon chains

The experiments on mice have shown that oxythiamine disulphide derivatives with the branched hydrocarbon chains are less toxic in the organism as compared to oxythiamine and corresponding disulphides with the unbranched hydrocarbon chains and also induce a more pronounced inhibition of transketolase in the liver and other tissues. It is found that under the effect of the above substances the recovery of enzymic activity is slower than in the case with the oxythiamine application.     

Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.     

Antivitamin activity of oxythiamine disulfide nicotinate]

The B1-antivitamin activity of oxythiamine disulphide nicotinate has been determined in experiments on albino mice and it is shown that in the liver this derivative exerts the equal action while in the blood and heart--a more profound and prolonged inhibitory action on the transketolase activity in comparison with oxythiamine disulphide. Like the initial compound oxythiamine disulphide nicotinate does not penetrate through hemato-encephalic barrier and does not inhibit the brain transketolase.     

Comparative antivitamin activity of oxythiamine disulfide and its monosulfoxide

The B1-antivitamin activity of oxythiamine disulphide monosulphoxide has been determined in experiments on albino mice. It is shown that this derivative is less toxic and exerts a more profound and prolonged inhibitory action on the pyruvate dehydrogenase and transketolase activity in the animal body in comparison with initial oxythiamine disulphide.     

Effect of indomethacin on cyclic nucleotide metabolism and vascular reactivity in rats

The content and metabolism of cyclic nucleotides in the aorta as well as contractility were studied in rats given different doses of indometacin. High doses of the drug (5 mg/kg) favored an abrupt decrease in cAMP and cGMP levels in the aorta and did not essentially affect vascular response to electric stimulation. Low doses of indometacin (2 mg/kg), particularly when coupled with salt load, resulted in the decreased cAMP/cGMP ratio and in the potentiation of vasoconstrictor responses. It has been demonstrated that the reduced proportion of cAMP in cyclic nucleotide metabolism is related to the activation effect of indometacin on cAMP-phosphodiesterase activity. The role of the prostaglandin-cyclic nucleotide system in the vascular tone control is discussed.     

Biosynthesis of mitochondrial protein in rat liver under conditions of vitamin B 1 deficiency following oxythiamine injection

The incorporation of labelled precursors into mitochondrial proteins of liver under different duration of oxythiamine (antivitamin B1) effect was studied in the whole organism and in a cell-free system. After 24 hrs following the injection, oxythiamine at a dose of 400 mg/kg of body weight increases the mitochondrial protein synthesis in vivo without changing the protein-synthesizing capacity of isolated mitochondria. After 72 hrs following the injection of the same dose of preparation, a sharp increase in the rate of protein label incorporation into the mitochondria was observed. The protein synthesis in mitochondria in the whole body studies also showed an increase. It is assumed that oxythiamine enhances the inductive synthesis of mitochondrial thiamine phosphate-dependent enzymes or activates the syntheses of other enzymic systems, capable of increasing the utilization of alpha-keto acids accumulated under conditions of thiamine deficiency.     

Certain characteristics of the rat liver transketolase

Modification of the method for transketolase purification in the rat liver was used to reveal the existence of two molecular forms of this enzyme. One of the forms does not react to development of avitaminosis in animals caused by oxythiamine in different doses. The method is developed for isolation of the basic transketolase form in the rat liver with the 50-80% yield of activity. Km values of two sites for coenzyme binding on protein do not depend on the extent of holoenzyme reconstruction from apoenzyme.     

Production of acetylcholine in rat brain following thiamine deprivation and treatment with thiamine antagonists

Abstract— The effects of thiamine deprivation and of treatment with the thiamine antagonists, oxythiamine and pyrithiamine, on the storage and synthesis of acetylcholine were studied in rats. Rats treated with pyrithiamine always developed ataxia and convulsions, and they died in an average of 36 ± 5.0 hr after onset of convulsions. Injections of sublethal doses of eserine after onset of convulsions had no effect or shortened survival time. If injections were started before the onset of convulsions, the survival time was increased to 56 ± 3.3 hr. The content of total acetylcholine-like compounds, measured by bioassay, in the brain was decreased in all three types of thiamine deficiency. On the other hand, the amount of parenterally administered [¹⁴C]pyruvate converted to [¹⁴C]acetylcholine in vivo was affected only by treatment with pyrithiamine. The increase found was probably due to an increased permeability of the blood-brain barrier to the pyruvate. Conversion of [¹⁴C]pyruvate to [¹⁴C]acetylcholine in vitro was decreased significantly in homogenates of brains from both oxythiamine and pyrithiamine-treated animals.     

Regulation of ion uptake in membrane vesicles from rat brain by thiamine compounds

We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude homogenate with thiamine or pyrithiamine increases chloride uptake while oxythiamine has the reverse effect. Thiamine and oxythiamine also affect 22Na+ and 86Rb+ uptake in the same way as for 36Cl- but to a lesser extent. Thiamine-dependent 36Cl- uptake is activated by sodium bicarbonate (10 mM) and partially inhibited by bumetanide (0.1 mM) and 2,4-dinitrophenol (0.1 mM). Preincubation with thiamine increases the thiamine triphosphate content of the vesicles. The hypothesis that TTP is the activator of a particular chloride uptake mechanism is discussed.     

Effect of thiamine deficiency and excess on activity of NA+, K+, Mg2+-ATPase in plasma membranes of rat liver

The activity of Na+, K+, Mg2+-ATPase in plasma membranes of the rat liver was studied as affected by thiamine, oxythiamine and food B1-avitaminosis. It is shown that the ATPase activity of the liver plasma membranes is inhibited only in case of modelling the alimentary thiamine deficiency.     

Formation of the skeletal system of white rat and golden hamster embryos during experimental hypovitaminosis B1

In pregnant females B1-hypovitaminosis was induced by injecting various doses of oxythiamine--a specific antimetabolite for B1 vitamin. The rat and hamster embryos were respectively treated on the 20th and 15th days of development after the technique suggested by Dauson-Dyban with staining the osseous anlages of the skeletons with alizarine red. The results of the investigations performed in 193 skeletons of the rat embryos and in 196 skeletons of the golden hamster embryos revealed a progressive decrease, as the dose of oxythiamine increased, in length of ossification anlages of the extremity bones. However, susceptibility to lesions in various bones of the extremity and skull skeletons was not similar under conditions of progressive oxythiamine-induced B1-hypovitaminosis and depended on time of their anlage formations.     

Zinc(II), cadmium(II) and mercury(II) complexes of the vitamin B1 antagonist oxythiamine

The reaction of oxythiamine chloride hydrochloride (HOxTCl x HCl) with ZnCl2, CdCl2 and HgCl2 in ethanol yielded the complexes [ZnCl3(HOxT)], [CdCl3(HOxT)] and [HgCl3(HOxT)]. In water, the reaction with CdCl2 afforded [CdCl2(OxT)], but reaction with ZnCl2 or HgCl2 yielded unidentified products. The four new complexes were characterized by mass spectrometry and IR spectroscopy in the solid state and by 1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopy in hexadeuterated dimethylsulfoxide (DMSO-d6), and three were also studied by X-ray diffractometry. In [ZnCl3(HOxT)] and [HgCl3(HOxT)] the oxythiamine ligand is bound to the metal via N(1') and adopts the V conformation exhibited by thiamine in biological contexts. The infrared (IR) spectrum of [CdCl3(HOxT)] suggests a similar coordination mode. In [CdCl2(OxT)] each OxT zwitterion coordinates to one Cd(II) ion via its N(1') atom and to another via its N(3') and O atoms, giving rise to a polymeric chain along the x-axis. The coordination number of the metal is made up to six by Cdc...Cl interactions, two of which link the polymeric chains in pairs. This seems to be the first metal complex containing the oxythiamine ligand as a zwitterion, with the N(3')-H/O(4'alpha)-H group deprotonated. Neither HOxTCl nor its zinc(II) complex showed any significant activity in vitro against HeLa cells.     

Effect of agents for the chemical prevention of radiation sickness on the cyclic nucleotide content in the bone marrow of mice

The effect of different chemical compounds on the cAMP/cGMP ratio in the bone marrow of mice and radioresistance of animals has been studied. It has been shown that all compounds possessing radioprotective properties give rise to the cAMP/cGMP ratio in the bone marrow of mice. No changes in cAMP and cGMP level were noted after the administration of nonradioprotective substances. The maximal radioprotective effect coincide in time with the largest increase of the cAMP/cGMP ratio. The injection of radioprotectors at different doses demonstrate clearly that only at radioprotective doses the increase in the cAMP/cGMP ratio takes place. The administration of some substances 2, 15 and 60 min after the irradiation of mice shows that the radioprotective effect, though modest, was evident only in one case of elevated cAMP/cGMP ratio (the injection of 2-Mercaptoethylamine 2 min after the irradiation). Evident radioprotective effect occurs at the cAMP/cGMP ratio of about 170-200%; the ratio of about 130-140% corresponds to small radioprotection.     

Effect of GLP-1 on lipid metabolism in human adipocytes.

We have studied the effect of several doses of GLP-1, compared to that of insulin and glucagons, on lipogenesis, lipolysis and cAMP cellular content, in human adipocytes isolated from normal subjects. In human adipocytes, GLP-1 exerts a dual action, depending upon the dose, on lipid metabolism, being lipogenic at low concentrations of the peptide (ED50, 10(-12) M), and lipolytic only at doses 10-100 times higher (ED50, 10(-10) M); both effects are time- and GLP-1 concentration-dependent. The GLP-1 lipogenic effect is equal in magnitude to that of equimolar amounts of insulin; both hormones apparently act synergically, and their respective action is abolished by glucagon. The lipolytic effect of GLP-1 is comparable to that of glucagon, apparently additive to it, and the stimulated value induced by either one is neutralized by the presence of insulin. In the absence of IBMX, GLP-1, at 10(-13) and 10(-12) M, only lipogenic doses, does not modify the cellular content of cAMP, while from 10(-11) M to 10(-9) M, also lipolytic concentrations, it has an increasing effect; in the presence of IBMX, GLP-1 at already 10(-12) M increased the cellular cAMP content. In human adipocytes, GLP-1 shows glucagon- and also insulin-like effects on lipid metabolism, suggesting the possibility of GLP-1 activating two distinct receptors, one of them similar or equal to the pancreatic one, accounting cAMP as a second messenger only for the lipolytic action of the peptide.     

Lymphocyte activation: the dualistic effect of cAMP

The effects of exogenously added cyclic nucleotides on DNA synthesis have been investigated in human peripheral blood lymphocytes stimulated with phytohemagglutinin (PHA). At low doses of PHA the addition of exogenous cAMP resulted in an inhibition of DNA synthesis. At optimal or supraoptimal doses of PHA the addition of cAMP, db-cAMP, or 8-Br-cGMP resulted in enhancement of DNA synthesis. Measurement of cell associated cAMP and cGMP levels in lymphocytes exposed to PHA with or without exogenously added cAMP revealed a gradual increase in cAMP levels and a fluctuating decline in cGMP levels.     

The effect of hexose monophosphate shunt inhibitors on adenohypophyseal and ceruloplasmin reactions to oestrogen.

Oestradiol benzoate, as an aqueous microcrystal suspension, was administered i.m. to rats in doses of 1 mg twice a week; it induced adenohypophyseal hyperplasia and an increase of the thyroxine-binding capacity of the adenohypophyseal proteins in vitro and raised the blood ceruloplasmin level. The simultaneous administration of a hexose monophosphate shunt inhibitor--6-aminonicotinamide (200 microgram/rat/day in food) or oxythiamine (8 mg/rat/day in food)--did not modify the reaction of the adenohypophysis; the hexose monophosphate shunt thus probably does not play a significant role in the adenohypophyseal reaction to oestrogens. By themselves, both inhibitors raised the blood ceruloplasmin level and their effect summated with that of oestradiol. The mechanism of action of the inhibitors is not known, but a nonspecific stress effect leading to an increase in the ceruloplasmin level as an "acute phase protein" is considered to be the most likely.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.     

Effect of water soluble vitamins and their analogues on growth of Candida albicans II. Vitamin B12, thiamine,oxythiamine, neopyrithiamine,substituted pyrimidines and thiazoles

Summary By employing wide ranges in vitamin concentrations in biotin basal mineral synthetic medium, it was demonstrated that vitamin B₁₂ markedly stimulated the growth ofCandida albicans, the organism showing a partial dependency upon this vitamin. Growth inhibition by 5-fluorouracil was reversed non-competitively by vitamin B₁₂, suggesting that B₁₂ has a role in nucleic acid biosynthesis of the organism. Thiamine was growth stimulatory, the organism being partially dependent upon this vitamin as well. Neopyrithiamine and oxythiamine were growth inhibitory in thiamine-free biotin basal mineral synthetic medium although the halves of each inhibitor compound were non-inhibitory. Neopyrithiamine inhibition was reversed by intact thiamine but not by pyrimidine thiamine or thiazole thiamine; while oxythiamine inhibition was reversed by thiamine and pyrimidine thiamine but not by thiazole thiamine, the inference being drawn that oxythiamine selectively blocks utilization of pyrimidine thiamine. Twenty-seven different substituted pyrimidines, thiazoles and related thiamine compounds were all utilizable byC. albicans in thiamine-free basal synthetic mineral medium, the organism presumably synthesizing thiamine when presented with the constituent parts of these thiamine analogues. Substitution of sulfur of the thiazole ring with oxygen, as in -methyloxazolium, failed to produce an inhibitory compound forC. albicans. Acetylthiamine, allithiamine, cocarboxylase, tetrahydrothiamine and dihydrothiamine were equally as growth stimulatory as thiamine.