Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Summary The specific binding activity to [¹⁴C]thiamine was found to be located in the plasma membrane ofSaccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant ofSaccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

Active transport of [32P]thiamine diphosphate inEscherichia coli

Summary Active uptake of [³²P]thiamine diphosphate byE. coli was analyzed using an improved method of gel filtration chromatography. The radioactive coenzyme was accumulated without dephosphorylation. From this result it was concluded that thiamine kinase is not involved in the membrane transport of thiamine inE. coli.We are indebted to Miss M. Abe for her technical assistance.     

Active transport of dimethialium inSaccharomyces cerevisiae

Summary Dimethialium, a derivative of thiamine which has a methyl group in place of hydroxyethyl group at the 5-position of the thiazole moiety, was found to be accumulated in nonproliferating cells ofSaccharomyces cerevisiae by the same transport mechanism for thiamine. The results strongly support the supposition that thiamine as well as dimethialium can be transported and accumulated without obligatory phosphorylation in yeast cells, since dimethialium is not phosphorylated by yeast thiamine pyrophosphokinase.We wish to thank the late Dr S. Yurugi, Takeda Research Laboratories, for a generous gift of dimethialium.     

Interaction of basic dyes with the thiamine transport system inSaccharomyces cerevisiae

Summary Basic dyes such as methylene blue and triphenyltetrazolium chloride were found to inhibit thiamine transport inSaccharomyces cerevisiae. Conversely, the reduction of methylene blue and triphenyltetrazolium chloride by yeast cells was inhibited by thiamine. A thiamine transport mutant ofSaccharomyces cerevisiae showed decreased utilization of these dyes. From the results, the possibility that the uptake of basic dyes may proceed via a membrane-bound thiamine-binding protein in the thiamine transport system of the yeast is discussed.This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan.     

Differential effects of opiates on the incorporation of [14C] thiamine in the central nervous system of the rat

Summary Opiate agonist (morphine), pure antagonist (naloxone), mixed agonist-antagonist (nalorphine) and analgesically inactive enantiomorph (dextrorphan) produced differential stereoselective effects on the incorporation of [¹⁴C] thiamine in the central nervous system of the rats. The possible role of thiamine in opiate effects and its implications are discussed.To whom reprint requests should be addressed. The authors thank Dr. S. J. Mulé, Assistant Commissioner and Director, ODAS Testing and Research Laboratory, for his kind interest and support and Mrs J. Vardy, Mr A. Kramer and Mr V. Aschettino for assistance.     

Synthesis of L-[3-2H,18O]glycerol and its incorporation into the 4-methyl-5-hydroxyethyl thiazole moiety of thiamine byEscherichia coli

Summary L-[3-²H,¹⁸O]glycerol was prepared and fed toEscherichia coli in order to determine the origin of the oxygen atom in the biosynthesized thiazole moiety of thiamine. Measurement by GC-MS of the isotope incorporation into the thiazole from this substrate confirmed that the 2 hydrogens and the oxygen on the C-3 carbon of glycerol are incorporated directly into the thiazole.     

Mg2+-ATPase defective mutant ofEscherichia coli and thiamine transport

Summary Mg²⁺-ATPase deficient mutant ofEscherichia coli showed an evident dependency of thiamine uptake on the oxidative metabolism of glucose, whereas the parent strain did not. In both cells, this uptake was completely inhibited by H⁺ conductors.Acknowledgment. We are indebted to Miss M. Abe for her excellent technical assistance.     

Sodium deoxycholate promotes the absorption of heparin administered orally, probably by acting on gastrointestinal mucosa, in rats

Sodium deoxycholate (DOC), selected as a promoter of gastrointestinal absorption of heparin, was administered orally to rats, followed, at increasing intervals, by heparin. Maximal plasma clearing activity (PC) was obtained with a 60-min interval, though PC was still elicited after 24 h, suggesting that DOC acts on the gastrointestinal mucosa. Inhibition of blood coagulation was also observed after oral heparin. The suggestion that DOC increases heparin absorption is supported by increased plasma levels of heparin. No signs of several gastrointestinal damage were seen.     

Sodium deoxycholate promotes the absorption of heparin administered orally,probably by acting on gastrointestinal mucosa,in rats

Summary Sodium deoxycholate (DOC), selected as a promoter of gastrointestinal absorption of heparin, was administered orally to rats, followed, at increasing intervals, by heparin. Maximal plasma clearing activity (PC) was obtained with a 60-min interval, though PC was still elicited after 24 h, suggesting that DOC acts on the gastrointestinal mucosa. Inhibition of blood coagulation was also observed after oral heparin. The suggestion that DOC increases heparin absorptions is supported by increased plasma levels of heparin. No signs of several gastrointestinal damage were seen.     

Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals

In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate. Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins. Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates. Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E. coli) or withering (A. thaliana). In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized. This enzyme was found only in mammals. In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases. The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling. A decreased content may contribute to the symptoms observed during thiamine deficiency.Received 7 March 2003; received after revision 11 April 2003; accepted 14 April 2003     

Thiamine pyrophosphate: An essential cofactor for the α-oxidation in mammals – implications for thiamine deficiencies?

The identification of 2-hydroxyphytanoyl-CoA lyase (2-HPCL), a thiamine pyrophosphate (TPP)-dependent peroxisomal enzyme involved in the α-oxidation of phytanic acid and of 2-hydroxy straight chain fatty acids, pointed towards a role of TPP in these processes. Until then, TPP had not been implicated in mammalian peroxisomal metabolism. The effect of thiamine deficiency on 2-HPCL and α-oxidation has not been studied, nor have possible adverse effects of deficient α-oxidation been considered in the pathogenesis of diseases associated with thiamine shortage, such as thiamine-responsive megaloblastic anemia (TRMA). Experiments with cultured cells and animal models showed that α-oxidation is controlled by the thiamine status of the cell/tissue/organism, and suggested that some pathological consequences of thiamine starvation could be related to impaired α-oxidation. Whereas accumulation of phytanic acid and/or 2-hydroxyfatty acids or their α-oxidation intermediates in TRMA patients given a normal supply of thiamine is unlikely, this may not be true when malnourished. Received 23 December 2005; received after revision 10 April 2006; accepted 28 April 2006     

Possible role of intestinal alkaline phosphatase activity in thiamine transport

Summary Thiamine deficiency caused a marked decrease of intestinal alkaline phosphatase (al-Pase) activity, but had no effect on the Ca⁺⁺-ATPase activity and Ca⁺⁺-absorption in rats. The al-Pase activity was significantly decreased 1 h after oral administration of ethanol at 0.5 and 2.5 g/kg. In contrast, Mg⁺⁺-, Ca⁺⁺- and (Na⁺+K⁺)-ATPase activities did not change after the administration of ethanol. These findings show that the al-Pase activity, unlike the Ca⁺⁺-ATPase activity, is not related to Ca⁺⁺-absorption. A possible role of al-Pase activity in the active transport of thiamine in the intestine was discussed.     

Mg2+-HCO-3-ATPase and carbonic anhydrase in rat intestinal mucosa

Mg2+-dependent and HCO-3-stimulated ATPase activity was highest in the brush border (microvilli) of rat duodenal mucosa compared with that of the other gastrointestinal mucosa. This ATPase may be useful to neutralize the gastric acid in the duodenal lumen. Carbonic anhydrase seems to accomplish a subsidiary role in the above reaction.     

Thiamine-binding activity of Saccharomyces cerevisiae plasma membrane

The specific binding activity to [14C]thiamine was found to be located in hte plasma membrane of Saccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant of Saccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

Pharmakologische Untersuchungen bei Thiaminmangel I. Änderungen des Katecholamingehalts im Gewebe

Summary (1) The increased tissue catecholamine level of the thiamine deficient rat was shown in the atrium, the ventricle, the brain cortex and in the spleen but not in the brain stem and the adrenal gland. (2) The increased response to tyramine of the thiamine deficient heart is most probably due to the high catecholamine level caused by thiamine deficiency. (3) The increased catecholamine content in the thiamine deficient rat does not result from the increased pyruvic acid.

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Herrn Professor Dr.F. Th. von Brücke zum 60. Geburtstag im Januar 1968 gewidmet.     

Trefoil factors

Trefoil Factor 1 (TFF1), the first member of the trefoil factor family, is normally expressed in the stomach mucosa. Ectopic expression is also observed in various human pathological conditions, notably in numerous carcinomas and gastrointestinal acute inflammatory disorders. In vivo experimental data using TFF1-deficient mice highlight the pleiotropic functions of TFF1: (i) it is a gastric tumor suppressor gene involved in gastric ontogenesis and homeostasis; (ii) it protects gut mucosa from aggression; (iii) it participates in folding secreted proteins inside the endoplasmic reticulum. At the cellular level, it limits cell proliferation and apoptosis, and favors cell differentiation. Collectively, these data suggest that TFF1 may provide an alternative pharmacological tool for the prevention and treatment of human gastrointestinal diseases.     

Effect of lipophilic cations on thiamine transport system in isolated rat hepatocytes

The effect of lipophilic cations such as triphenylmethylphosphonium, tetraphenylphosphonium and tetraphenylarsonium in addition to dibenzyldimethylammonium on thiamine transport in isolated rat hepatocytes was studied. Lipophilic cations at the concentration 10 microM almost completely inhibited thiamine uptake. Kinetic studies showed that these compounds were competitive inhibitors with a very high affinity. These results suggest that lipophilic cations in addition to quaternary ammonium compounds also share a common binding site for thiamine in isolated rat hepatocytes.     

Inhibition of thiamine transport in baker's yeast by methylene blue

Summary Methylene blue was found to inhibit thiamine transport competitively (K_i=0.63 M) in baker's yeast. The dye was also effective in abolishing the growth inhibition ofSaccharomyces cerevisiae by pyrithiamine which is known to be taken up by a common transport system for thiamine in yeast cells. A possible mechanism for the inhibition by methylene blue of the thiamine transport system in baker's yeast is discussed.     

Occurrence of thiaminase II in Saccharomyces cerevisiae

It was found that cell-free extracts of Saccharomyces cerevisiae contain thiaminase II which hydrolyzes thiamine and thiamine analogs. The possible involvement of this enzyme and thiamine-synthesizing enzymes in thiamine production from thiamine antagonists is discussed.     

Thiamine triphosphate metabolism and its turnover in the rat liver

Summary When rats were injected with a thiamine disulfide derivative, the content of thiamine triphosphate (TTP) in the liver doubled within 3 h after a preceeding rise in thiamine pyrophosphate; it then returned to the basal level within the next 3h, indicating a net increase of TTP in vivo and its rapid turnover.