Ultrastructural localization of thiamine pyrophosphatase activity in the epitheliocytes of duodenal biopsies in peptic ulcer

Using electron microscopic studies the location of thiamine pyrophosphatase (TPPase) activity in the epithelial cells of duodenal mucosa was investigated in 39 patients with ulcer exacerbation and during the disease remission. The biopsy material was studied before and after the administration of physiological and therapeutic doses of thiamine. TPPase activity was detected on the membranes of Golgi complex, lateral cell membrane and microvilli. The possibility of intra- and extracellular localization of TPPase activity and the activity of various enzymes depending on the mechanism of thiamine absorption across the epithelial barrier is discussed.     

Thiamine pyrophosphokinase deficiency in encephalopathic children with defects in the pyruvate oxidation pathway

Thiamine pyrophosphate (TPP) is an essential cofactor of the cytosolic transketolase and of three mitochondrial enzymes involved in the oxidative decarboxylation of either pyruvate, α-ketoglutarate or branched chain amino acids. Thiamine is taken up by specific transporters into the cell and converted to the active TPP by thiamine pyrophosphokinase (TPK) in the cytosol from where it can be transported into mitochondria. Here, we report five individuals from three families presenting with variable degrees of ataxia, psychomotor retardation, progressive dystonia, and lactic acidosis. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate but normal pyruvate dehydrogenase complex activity in the presence of excess TPP. A reduced concentration of TPP was found in the muscle and blood. Mutation analysis of TPK1 uncovered three missense, one splice-site, and one frameshift mutation resulting in decreased TPK protein levels.     

Maternal influences on thiamine status of walleye Sander vitreus ova

Concentrations of the various forms of thiamine (vitamin B(1) ) were determined in walleye Sander vitreus ova from three central North American lakes. Total thiamine concentrations in ova from Lake Winnipeg S. vitreus were approximately three times greater (mean 12 nmol g(-1) ) than in those from Lakes Erie or Ontario. The percentage of thiamine in the active form (thiamine pyrophosphate, TPP) was highest in Lake Ontario ova (mean 88%) and lowest in those from Lake Winnipeg (mean 70%). Neither ova total thiamine concentration nor per cent ova thiamine as TPP showed any consistent relationships with maternal age, size, morphometric condition, somatic lipid concentrations or liver lipid concentrations. Ova total thiamine concentration, however, was negatively related to ovum size in some populations, as well as among populations, and was positively related to liver total thiamine concentration. Maternal transfer of thiamine to ova appears to be independent of female ontogenetic or conditional state in S. vitreus.     

The effect of thiamine supplementation on tumour proliferation. A metabolic control analysis study.

Thiamine deficiency frequently occurs in patients with advanced cancer and therefore thiamine supplementation is used as nutritional support. Thiamine (vitamin B1) is metabolized to thiamine pyrophosphate, the cofactor of transketolase, which is involved in ribose synthesis, necessary for cell replication. Thus, it is important to determine whether the benefits of thiamine supplementation outweigh the risks of tumor proliferation. Using oxythiamine (an irreversible inhibitor of transketolase) and metabolic control analysis (MCA) methods, we measured an in vivo tumour growth control coefficient of 0.9 for the thiamine-transketolase complex in mice with Ehrlich's ascites tumour. Thus, transketolase enzyme and thiamine clearly determine cell proliferation in the Ehrlich's ascites tumour model. This high control coefficient allows us to predict that in advanced tumours, which are commonly thiamine deficient, supplementation of thiamine could significantly increase tumour growth through transketolase activation. The effect of thiamine supplementation on tumour proliferation was demonstrated by in vivo experiments in mice with the ascites tumour. Thiamine supplementation in doses between 12.5 and 250 times the recommended dietary allowance (RDA) for mice were administered starting on day four of tumour inoculation. We observed a high stimulatory effect on tumour growth of 164% compared to controls at a thiamine dose of 25 times the RDA. This growth stimulatory effect was predicted on the basis of correction of the pre-existing level of thiamine deficiency (42%), as assayed by the cofactor/enzyme ratio. Interestingly, at very high overdoses of thiamine, approximately 2500 times the RDA, thiamine supplementation had the opposite effect and caused 10% inhibition of tumour growth. This effect was heightened, resulting in a 36% decrease, when thiamine supplementation was administered from the 7th day prior to tumour inoculation. Our results show that thiamine supplementation sufficient to correct existing thiamine deficiency stimulates tumour proliferation as predicted by MCA. The tumour inhibitory effect at high doses of thiamine is unexplained and merits further study.     

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.     

Up-regulation of vitamin B1 homeostasis genes in breast cancer

An increased carbon flux and exploitation of metabolic pathways for the rapid generation of biosynthetic precursors is a common phenotype observed in breast cancer. To support this metabolic phenotype, cancer cells adaptively regulate the expression of glycolytic enzymes and nutrient transporters. However, activity of several enzymes involved in glucose metabolism requires an adequate supply of cofactors. In particular, vitamin B1 (thiamine) is utilized as an essential cofactor for metabolic enzymes that intersect at critical junctions within the glycolytic network. Intracellular availability of thiamine is facilitated by the activity of thiamine transporters and thiamine pyrophosphokinase-1 (TPK-1). Therefore, the objective of this study was to establish if the cellular determinants regulating thiamine homeostasis differ between breast cancer and normal breast epithelia. Employing cDNA arrays of breast cancer and normal breast epithelial tissues, SLC19A2, SLC25A19 and TPK-1 were found to be significantly up-regulated. Similarly, up-regulation was also observed in breast cancer cell lines compared to human mammary epithelial cells. Thiamine transport assays and quantitation of intracellular thiamine and thiamine pyrophosphate established a significantly greater extent of thiamine transport and free thiamine levels in breast cancer cell lines compared to human mammary epithelial cells. Overall, these findings demonstrate an adaptive response by breast cancer cells to increase cellular availability of thiamine.     

Development of improved media for axenic cultivation of Acanthamoeba culbertsoni, Singh and Das 1970

Several varieties of peptone supported growth of A. culbertsoni to different extents reaching a maximum cell density of 1-2 X 10(6)/ml. Proteose peptone and tryptone also yielded good growth when combined with thiamine and vitamin B12. A combination of proteose peptone with glucose, yeast extract and salts promoted excellent growth of A. culbertsoni with cell density reaching 1-2 X 10(7) cells/ml; tryptone and one of the indigenous peptones also yielded comparable growth when substituted for proteose peptone in this medium. Casamino acids also supported good growth of amoebae and requirement of yeast extract could be met by a combination of thiamine, vitamin B12 and biotin. Bacto peptone did not support good growth of this amoeba but supplementation of peptone with casamino acids or amino acid mixture improved the growth supporting capacity of the medium. Development of several media with or without glucose will aid in cultivation of A. culbertsoni, studies on its metabolism as well as screening of potential drugs.     

A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro

Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn? Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn? Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific combination of nutrients acts synergistically in enhancing muscarinic M1 receptor responses, probably by facilitating receptor-mediated G protein activation.     

Differential interaction of cations with the thiamine and biotin transport proteins of Lactobacillus casei

Lactobacillus casei cells contain separate and specific binding proteins which mediate the cellular uptake of thiamine and biotin. In buffered salt solutions, these proteins exhibit a very high affinity for their vitamin substrate. Dissociation constants (Kd values) at pH 7.5 are 0.03 and 0.15 nM for thiamine and biotin, respectively. Optimal binding of biotin requires the presence of cations. This cation dependence is substantial since the Kd for biotin is 60-fold higher in a buffer containing 0.1 mM K-Hepes, compared with a buffer composed of 50 mM K-Hepes and 5 mM MgCl2. Measurements of Kd versus cation concentration showed that Mg2+ is 300-fold more effective than K+ in promoting biotin binding. The extent of cation dependence decreases as the pH is reduced from 7.5 to 5.0, suggesting that protons can partially fulfill the cation requirement. In contrast, binding of thiamine to the thiamine transport protein shows no dependence on the ionic composition of the medium. These results suggest that the transport protein for the anionic vitamin, biotin, contains a binding site for cations. Cotransport of both the vitamin and cation into the cell might then occur during the normal transport cycle, allowing the cellular uptake of the vitamin to occur against the membrane potential. Conversely, the cationic vitamin, thiamine, does not appear to be transported into the cell as a complex with other ions.     

Phosphate Derivatives of Thiamine and Na+ Channel in Conducting Membranes

The results show that thiamine derivatives are copurified with the specific proteins forming the Na+ channel in conducting membranes. Therefore, thiamine derivatives could well play a specific role in the molecular aspects of bioelectrogenesis , an interpretation that could help explain the neurological symptoms observed in human pathology as well as in animals experimentally rendered deficient in vitamin B1.     

Biotin-responsive basal ganglia disease-linked mutations inhibit thiamine transport via hTHTR2: biotin is not a substrate for hTHTR2

The water-soluble micronutrient thiamine is required for normal tissue growth and development in humans. Thiamine is accumulated into cells through the activity of two cell surface thiamine transporters (hTHTR1 and hTHTR2), which are differentially targeted in polarized tissues. Mutational dysfunction of hTHTR1 is associated with the clinical condition of thiamine-responsive megaloblastic anemia: the symptoms of which are alleviated by thiamine supplementation. Recently, two hTHTR2 mutants (G23V, T422A) have been discovered in clinical kindreds manifesting biotin-responsive basal ganglia disease (BBGD): the symptoms of which are alleviated by biotin administration. Why then does mutation of a specific thiamine transporter isoform precipitate a disorder correctable by exogenous biotin? To investigate the suggestion that hTHTR2 can physiologically function as a biotin transporter, we examined 1) the cell biological basis of hTHTR2 dysfunction associated with the G23V and T422A mutations and 2) the substrate specificity of hTHTR2 and these clinically relevant mutants. We show that the G23V and T422A mutants both abrogate thiamine transport activity rather than targeting of hTHTR2 to the cell surface. Furthermore, biotin accumulation was not detectable in cells overexpressing either the full length hTHTR2 or the clinically relevant hTHTR2 mutants, yet was demonstrable in the same assay using cells overexpressing the human sodium-dependent multivitamin transporter, a known biotin transporter. These results cast doubt on the most parsimonious explanation for the BBGD phenotype, namely that hTHTR2 is a physiological biotin transporter.