Thiamine antivitamins--an opportunity of therapy of fungal infections caused by Malassezia pachydermatis and Candida albicans.

Severe skin diseases and systemic fungaemia are caused by Malassezia pachydermatis and Candida albicans respectively. Antifungal therapies are less effective because of chronic character of infections and high percentage of relapses. Therefore, there is a great need to develop new strategies of antifungal therapies. We previously found that oxythiamine decreases proliferation of yeast (Saccharomyces cerevisiae), therefore we suggest that thiamine antivitamins can be considered as antifungal agents. The aim of this study was the comparison of thiamine antivitamins (oxythiamine, amprolium, thiochrome, tetrahydrothiamine and tetrahydrooxythiamine) inhibitory effect on the growth rate and energetic metabolism efficiency in non-pathogenic S. cerevisiae and two potentially pathogenic species M. pachydermatis and C. albicans. Investigated species were cultured on a Sabouraud medium supplemented with trace elements in the presence (40 mg l(-1)) or absence of each tested antivitamins to estimate their influence on growth rate, enzyme activity and kinetic parameters of pyruvate decarboxylase and malate dehydrogenase of each tested species. Oxythiamine was the only antivitamin with antifungal potential. M. pachydermatis and S. cerevisiae were the most sensitive, whereas C. albicans was the least sensitive to oxythiamine action. Oxythiamine can be considered as supportive agent in superficial mycoses treatment, especially those caused by species from the genus Malassezia.

Up-regulation of 2-oxoglutarate dehydrogenase as a stress response.

2-Oxoglutarate dehydrogenase multienzyme complex (OGDHC) operates at a metabolic cross-road, mediating Ca(2+)- and ADP-dependent signals in mitochondria. Here, we test our hypothesis that OGDHC plays a major role in the neurotransmitter metabolism and associated stress response. This possibility was assessed using succinyl phosphonate (SP), a highly specific and efficient in vivo inhibitor of OGDHC. Animals exposed to toxicants (SP, ethanol or MnCl(2)), trauma or acute hypoxia showed intrinsic up-regulation of OGDHC in brain and heart. The known mechanism of the SP action as OGDHC inhibitor pointed to the up-regulation triggered by the enzyme impairment. The animal behavior and skeletal muscle or heart performance were tested to correlate physiology with the OGDHC regulation and associated changes in the glutamate and cellular energy status. The SP-treated animals exhibited interdependent changes in the brain OGDHC activity, glutamate level and cardiac autonomic balance, suggesting the neurotransmitter role of glutamate to be involved in the changed heart performance. Energy insufficiency after OGDHC inhibition was detectable neither in animals up to 25 mg/kg SP, nor in cell culture during 24 h incubation with 0.1 mM SP. However, in animals subjected to acute ethanol intoxication SP did evoke energy deficit, decreasing muscular strength and locomotion and increasing the narcotic sleep duration. This correlated with the SP-induced decrease in NAD(P)H levels of the ethanol-exposed neurons. Thus, we show the existence of natural mechanisms to up-regulate mammalian OGDHC in response to stress, with both the glutamate neurotransmission and energy production potentially involved in the OGDHC impact on physiological performance. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Changes of activity and kinetics of certain liver and heart enzymes of hypothyroid and T3-treated rats

Thyroid diseases are one of the most common metabolic disorders in the human population. In this work, we present data concerning changes in the activity and kinetic parameters of several enzymes associated with both anabolic (glucose-6-phosphate dehydrogenase-G6PDH, EC 1.1.1.49; 6-phosphogluconate dehydrogenase-6PGDH, EC 1.1.1.44; malic enzyme-ME, EC 1.1.1.40; and isocitrate dehydrogenase-IDH, EC 1.1.1.42) and catabolic (NAD-dependent malate dehydrogenase-NAD-MDH, EC 1.1.1.37; and lactate dehydrogenase-LDH, EC 1.1.1.27) processes under conditions of hypothyroidism and T3 treatment. Hypothyroidism was induced in rats by the surgical removal of the thyroid gland. T3-treated rats were injected by T3 (0.5 mg T3/kg body weight daily during 10 days). We have found that T3 treatment caused an increase of NAD-MDH activity as well as heart hypertrophy whereas the activity of LDH increased in the direction of pyruvate reduction. Moreover, we observed increased activity of both enzymes in the liver. These results confirm earlier observation concerning the relevance of oxidative metabolism in the heart under T3 treatment. Hypothyroidism resulted in changes in the LDH activity in the heart whereas NAD-MDH activity did not change. Moreover, our data show that T3 treatment caused an increase of G6PDH, 6PGDH, and ME activities in the liver. We also observed a decrease of IDH activity in both organs, whereas hypothyroidism caused the opposite effect. This data indicate that either deficiency or excess of thyroid hormones can prove to be particularly dangerous for the physiology of the heart muscle by disturbing bioenergetic and anabolic processes (AU)

Changes of activity and kinetics of certain liver and heart enzymes of hypothyroid and T(3)-treated rats.

Thyroid diseases are one of the most common metabolic disorders in the human population. In this work, we present data concerning changes in the activity and kinetic parameters of several enzymes associated with both anabolic (glucose-6-phosphate dehydrogenase-G6PDH, EC 1.1.1.49; 6-phosphogluconate dehydrogenase-6PGDH, EC 1.1.1.44; malic enzyme-ME, EC 1.1.1.40; and isocitrate dehydrogenase-IDH, EC 1.1.1.42) and catabolic (NAD-dependent malate dehydrogenase-NAD-MDH, EC 1.1.1.37; and lactate dehydrogenase-LDH, EC 1.1.1.27) processes under conditions of hypothyroidism and T(3) treatment. Hypothyroidism was induced in rats by the surgical removal of the thyroid gland. T(3)-treated rats were injected by T(3) (0.5 mg T(3)/kg body weight daily during 10 days). We have found that T(3) treatment caused an increase of NAD-MDH activity as well as heart hypertrophy whereas the activity of LDH increased in the direction of pyruvate reduction. Moreover, we observed increased activity of both enzymes in the liver. These results confirm earlier observation concerning the relevance of oxidative metabolism in the heart under T(3) treatment. Hypothyroidism resulted in changes in the LDH activity in the heart whereas NAD-MDH activity did not change. Moreover, our data show that T(3) treatment caused an increase of G6PDH, 6PGDH, and ME activities in the liver. We also observed a decrease of IDH activity in both organs, whereas hypothyroidism caused the opposite effect. This data indicate that either deficiency or excess of thyroid hormones can prove to be particularly dangerous for the physiology of the heart muscle by disturbing bioenergetic and anabolic processes.

[2-Oxoglutarate dehydrogenase complex and its multipoint control]. / Kompleks dehydrogenazy 2-oksoglutaranowej i wieloplaszczyznowa regulacja aktywnosci kompleksu.

Enzymes control the course of biochemical reactions. The enzymes involved in bioenergetic processes play most important role in cell metabolism. One of them is 2-oxoglutarate dehydrogenase complex (OGDHC), the key regulatory enzyme of Krebs cycle. Krebs cycle integrates basic metabolic pathways of carbohydrates, fatty acids and amino acids during catabolic as well as anabolic reactions. Due to the key position of OGDHC in mitochondrial metabolism, its activity is controlled by many factors. Allosteric regulation by positive effectors (ADP, Pi, Ca2+, Mn2+) of the complex is very important. These effectors strongly enhances affinity of the first component of OGDHC to 2-oxoglutarate. Moreover there are negative effectors (ATP, NADH, succinyl-CoA) which affect all three enzymes of the complex. Regulation of biosynthesis of individual components of the complex by activation or inactivation of genes expression is very important for proper OGDHC activity too. Activity of OGDHC also depends on posttranslational modifications of its components. All of this control processes maintain OGDHC activity on adequate level and prevent the complex against its excessive action.

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.

Short-term regulation of the mammalian pyruvate dehydrogenase complex.

In this minireview the main mechanism of control of mammalian pyruvate dehydrogenase complex (PDHC) activity by phosphorylation-dephosphorylation is presented in the first place. The information recently obtained in several laboratories includes new data about isoforms of the PDH converting enzymes (kinase and phosphatase) and their action in view of short-term regulation of PDHC. Moreover, interesting influence of exogenous thiamine diphosphate (TDP) and some divalent cations, especially Mn(2+), on the kinetic parameters of PDHC saturated with endogenous tightly bound TDP, is discussed. This influence causes a shortening of the lag-phase of the catalyzed reaction and a strong decrease of the K(m) value of PDHC mainly for pyruvate. There are weighty arguments that the effects have an allosteric nature. Thus, besides reversible phosphorylation, also direct manifold increase of mammalian PDHC affinity for the substrate by cofactors seems an important aspect of its regulation.

Suicidal dephosphorylation of thiamine pyrophosphate coupled with pyruvate dehydrogenase complex.

Earlier it was noted that purified pyruvate dehydrogenase complex (PDC) produced by "Sigma" usually contains almost saturating amounts of thiamine pyrophosphate (ThPP). In this communication we present the observation that the endogenous ThPP coupled to PDC is dephosphorylated while staying at -10 degrees C, because in the enzyme preparation thiamine monophosphate and un-phosphorylated thiamine appear (HPLC determination). Under the same conditions exogenous ThPP is not dephosphorylated despite contact with the PDC preparation. This may suggest that interactions of some active groups of the enzyme with molecules of endogenous ThPP leads to break-up of the phosphoesters bonds, and destruction of the coenzyme. Decrease of PDC activity during storage is not in proportion with the degree of ThPP dephosphorylation. However the observed instability of PDC activity may be a consequence of the spontaneous process of its coenzyme autodestruction.

Effect of oxythiamin on growth rate, survival ability and pyruvate decarboxylase activity in Saccharomyces cerevisiae.

Oxythiamin is one of the antivitamin derivatives of thiamin which, after phosphorylation, can be bound to the catalytic centre of thiamin-dependent enzymes and inhibit these enzymes. In this work the influence of oxythiamin on the growth rate, survival and the activity of pyruvate decarboxylase of Saccharomyces cerevisiae (s288c) was investigated. Oxythiamin decreased both the growth rate and survival ability of yeast cells. Moreover, in three-day-old cultures on a medium with oxythiamin, an increase of pyruvate decarboxylase activity was observed. This unusual effect may be in response to the earlier inhibition of pyruvate decarboxylase. A high concentration of pyruvate in the cell extracts taken from the medium with oxythiamin was found. This accumulation of pyruvate could provide for enhanced biosynthesis of the pyruvate decarboxylase apoform and an increase of enzyme activity.