Study of the restoration and balance of thiamine in the tissues of albino mice in alcoholic intoxication

Acute inhalative alcoholic intoxication has been studied for its effect on the influx and level of free and bound thiamine in tissues of white mice. It is established that acute inhalative ethanol intoxication increases 35S-thiamine incorporation in tissues and decreases the level of endogenic free and bound thiamine. The results obtained permit a conclusion on intensification of the thiamine renewal in tissues with its sufficient influx from outside as affected by the ethanol narcosis.     

Interaction of functionally coupled vitamins in the distribution and metabolism of [14C]nicotinic acid in tissues and blood cells

Leucocytes adsorb by two orders of magnitude more labeled nicotinic acid ([14C]Na) than erythrocytes (as calculated on a per cell basis). The dynamics of binding of labeled vitamin by leucocytes is biphasic with the formation of predominantly [14C]nicotinic coenzymes already at very short time intervals after their injection to rats. Simultaneous injections of thiamine, riboflavin, lipoate and pantotenate increased the level of total labeled nicotinate metabolites in the blood and leucocytes 2.1- and 4.1-fold, respectively. The metabolism of subcutaneously injected [14C]NA was predominantly localized in the digestive system with a markedly pronounced two-phase dynamics of changes of the level of total labeled metabolites in the liver and small intestine concomitant with their secretion together with digestive juices. The functionally coupled vitamins injected simultaneously sharply increased the incorporation of the total label into liver tissues (up to 45% of the injected dose against 33% in the control) and the increase in the level of [14C]pyridine nucleotides. Similar effects were observed upon accumulation of labeled metabolites of [14C]NA in small intestine membranes. The increase in the maximal accumulation of nicotinate under effects of other group B vitamins in brain, heart and spleen tissues correlated with the dynamics, of their accumulation in the blood. In the postmaximal period in cardiac muscle and brain tissues, the second increase in the [14C]NA binding correlated with the dynamics of its accumulation in the digestive system.(ABSTRACT TRUNCATED AT 250 WORDS)     

New aspects in the study of the mechanism of intestinal absorption of thiamine in rats

Intestinal absorption of thiamine was studied in rats using such metabolic inhibitors as ouabain, sodium azide and theophylline. These substances changed certain biochemical parameters of the mucosa in the small intestine affecting the level of thiamine transport across the intestinal epithelium. It was found that the intestinal transport of thiamine at lower concentration was an active process depending on the activity of Na, K, Mg-ATPase in the intestinal microvilli, and on the activity of mitochondrial processes. The used metabolic inhibitors increased the intestinal diffusion of thiamine at its higher concentration suggesting that these inhibitors changed membrane permeability by affecting enterocyte homeostasis. At both studied concentrations thiamine was rapidly absorbed from the digestive tract reaching the state of saturation which suggested a carrier character of this transport.     

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.     

Relation of nutrition to health in aging persons; a four-year follow-up of a study in San Mateo County

A follow-up study of 577 San Mateo County residents over 50 years of age who were originally studied in 1948 was carried out. Three hundred fifty still were available for reevaluation. Mortality studies showed a higher death rate in males than in females, in persons of the lower economic levels, and in those with systolic blood pressure of more than 180 mm. of mercury. Correlations between factors studied and morbidity were not conclusive, but suggested relationships between low economic status and digestive system disease; low hemoglobin and high incidence of respiratory disease; high caloric intake and digestive system disease; low thiamine intake and nervous system disease; low ascorbic acid intake and diseases of the circulatory and digestive systems.     

Molecular mechanisms of thiamine utilization

Thiamine is required for all tissues and is found in high concentrations in skeletal muscle, heart, liver, kidneys and brain. A state of severe depletion is seen in patients on a strict thiamine-deficient diet in 18 days, but the most common cause of thiamine deficiency in affluent countries is alcoholism. Thiamine diphosphate is the active form of thiamine, and it serves as a cofactor for several enzymes involved primarily in carbohydrate catabolism. The enzymes are important in the biosynthesis of a number of cell constituents, including neurotransmitters, and for the production of reducing equivalents used in oxidant stress defenses and in biosyntheses and for synthesis of pentoses used as nucleic acid precursors. Because of the latter fact, thiamine utilization is increased in tumor cells. Thiamine uptake by the small intestines and by cells within various organs is mediated by a saturable, high affinity transport system. Alcohol affects thiamine uptake and other aspects of thiamine utilization, and these effects may contribute to the prevalence of thiamine deficiency in alcoholics. The major manifestations of thiamine deficiency in humans involve the cardiovascular (wet beriberi) and nervous (dry beriberi, or neuropathy and/or Wernicke-Korsakoff syndrome) systems. A number of inborn errors of metabolism have been described in which clinical improvements can be documented following administration of pharmacological doses of thiamine, such as thiamine-responsive megaloblastic anemia. Substantial efforts are being made to understand the genetic and biochemical determinants of inter-individual differences in susceptibility to development of thiamine deficiency-related disorders and of the differential vulnerabilities of tissues and cell types to thiamine deficiency.     

Study on the effect of thiamine on the metabolism of yeast by intrinsic fluorescence.

Thiamine in living human bodies exists mainly as diphosphate, which works as a co-enzyme of the sugar metabolism system (active vitamin B1). Thiamine deficiency brings many clinically significant problems, such as dysphoria, quadriplegia and dyspepsia. Intrinsic fluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex systems such as biological cells and tissues. Cellular fluorescence provides a sensitive index of the functional state of a living cell (1). Different amounts of thiamine were added to culture medium and the fluorescence of tryptophan and NADH from yeast was determined. When the thiamine concentration was greater than 0-0.16 microg/mL, the intensity of tryptophan fluorescence increased linearly, whereas the NADH fluorescence decreased. When the thiamine concentration was above 0.24 microg/mL, the fluorescence of tryptophan and NADH was almost unchanged. We concluded that low thiamine concentration in culture medium had a large effect on the growth of Saccharomyces cerevisiae and possible reasons are discussed.     

Thiamine Triphosphate and Membrane-Associated Thiamine Phosphatases in the Electric Organ of Electrophorus electricus

The main electric organ of Electrophorus electricus is particularly rich in thiamine triphosphate, which represents 87% of the total thiamine content in this tissue. The thiamine pyrophosphate concentration, however, is very low in the eel electric organ and skeletal muscle as compared with other eel or rat tissues. Furthermore, electroplax membranes contain a whole set of enzymes responsible for the dephosphorylation of thiamine tri-, pyro- and monophosphate. Thiamine triphosphatase has a pH optimum of 6.8 and is dependent on Mg2+. The real substrate of the enzyme is probably a 1:1 complex of Mg2+ and thiamine triphosphate. Thiamine pyrophosphatase is activated by Ca2+. The apparent Km for thiamine triphosphate and Vmax are found to be, respectively, 1.76 mM and 5.95 nmol/mg of protein/min. Thiamine triphosphatase activity is inhibited at physiological K+ concentrations (up to 90 mM) and increasing Na+ concentrations (50% inhibition at 300 mM). ZnCl2 (10 mM) inhibits 90% of the enzyme activity. ATP and ITP are also strongly inhibitory. No significant effect of neurotoxins is seen. Membrane-associated thiamine triphosphatase is affected differently by proteolytic enzymes and is partially inactivated by pretreatment with phospholipase C and neuraminidase. The physiological significance of thiamine triphosphatase is discussed in relation to a specific role of thiamine in the nervous system.     

消化系统的真菌感染

消化系统真菌感染是指真菌感染食管、胃肠、肝胆、胰或消化系统的其他组织或器官,该文对消化系统真菌感染的病原菌、临床表现、病理特征和治疗措施进行综述。     

Cytokinin activation of thiamine biosynthesis in tobacco callus cultures

Bioassays of tissue extracts show that high (500-1000 μg/liter) kinetin concentrations which permit growth of tobacco callus cultures on media without added thiamine activate the biosynthesis of this vitamin by the tissues. Although the tissue concentration of thiamine may fall appreciably, it is maintained at a level adequate for survival and slow growth of the cultures, and there is a large net increase in total thiamine content per culture with time. In the second and subsequent passages of tissue on a thiamine free medium, growth is obtained only when high kinetin concentrations are maintained. Effective inhibition of growth by antithiamines suggests that thiamine is utilized by the high-kinetin tissue.

In the presence of low (30-100 μg/liter) kinetin concentrations, which would be optimal for growth in the presence of thiamine, growth only occurs early in the first passage of tissue from a medium with the vitamin to one without it. The thiamine concentration in the tissues falls to low levels, and no net biosynthesis is apparent. The tissues turn dark and die after 2 to 3 weeks. In contrast with this, in the absence of both added thiamine and kinetin no appreciable growth occurs, but the tissues keep their normal appearance, retain their thiamine content, and may stay alive for several weeks.

     

Circatidal variation in epithelial cell proliferation in the mussel digestive gland and stomach

Epithelial cell renewal in mussel (Mytilus galloprovincialis, Lmk) digestive gland and stomach was investigated by bromodeoxyuridine (BrdU) immunohistochemistry. Mussels were exposed to 4 mg BrdU/l seawater continuously. Starting at 6 h after treatment, samples were collected every 2 h for 2 days and BrdU labelling was estimated by direct counting at the light microscope, with values being noted per thousand BrdU-positive cells. BrdU-positive reaction was observed in the nuclei of digestive, basophilic, duct and stomach cells, and in haemocytes. Cell renewal in digestive diverticula was synchronised following a circatidal pattern: BrdU labelling increased during low tide and decreased during high tide. Clearcut mitotic figures were identified in digestive cells, thereby confirming that mature cell types proliferate, in agreement with results from immunohistochemistry for proliferating cell nuclear antigen and BrdU. Epithelial cell renewal in the stomach also appeared to be synchronised.This investigation was funded by the Basque Government (GVPI95-36 and GVP99-1) and by a grant to Consolidated Research Groups (UPV/EHU)     

Tissue-specific and inducible Cre-mediated recombination in the gut epithelium

We generated two complementary systems for Cre-mediated recombination of target genes in the mouse digestive epithelium and tested them with a Cre-reporter mouse strain. Cre was expressed under the control of a 9 kb regulatory region of the murine villin gene (vil-Cre). Genetic recombination was initiated at embryonic day (E) 9 in the visceral endoderm, and by E12.5 in the entire intestinal epithelium, but not in other tissues. Cre expression was maintained throughout adulthood. Furthermore, transgenic mice bearing a tamoxifen-dependent Cre recombinase (vil-Cre-ERT2) expressed under the control of the villin promoter were created to perform targeted spatiotemporally controlled somatic recombination. After tamoxifen treatment, recombination was detectable throughout the digestive epithelium. The recombined locus persisted for 60 days after tamoxifen administration, despite rapid intestinal cell renewal, indicating that epithelial progenitor cells had been targeted. The villin-Cre and villin-Cre-ERT2 mice provide valuable tools for studies of cell lineage allocation and gene function in the developing and adult intestine.     

Cloning of the human thiamine transporter, a member of the folate transporter family.

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.     

Development of the alt Mutant of Pisum sativum L

The alt (albina-terminalis) mutant of Pisum sativum L. germinates normally, produces several nodes, and then above a sharp transition produces 2 to 3 bleached nodes, ceases growth, and eventually dies. Green nodes have normal chlorophyll content, absorption spectra, photosynthetic rates, and ultrastructure. In bleaching tissues, the chloroplasts degenerate rapidly, followed by extensive disruption and loss of the remaining cytoplasm and organelles. Application of tissue extracts of normal genotypes of pea, corn, and bean stimulates apical development of alt. The resulting tissues have essentially normal structure and function. Application of thiamine, thiamine monophosphate, and thiamine pyrophosphate also stimulate normal apical development at concentrations of 1 micromolar and above. Partial characterization of the stimulus from pea seed extracts is consistent with thiamine as the active factor.     

Thiamine status in humans and content of phosphorylated thiamine derivatives in biopsies and cultured cells

Background

Thiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines.

Methodology and Principal Findings

Thiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines.

Conclusions and Significance

The high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.     

The effect of thiamine and its metabolites on the activity of tissue and purified lactate dehydrogenase

It is shown that thiamine and its metabolites effect lactate dehydrogenase activity and lactate content in the tissues. Thiochrome and thiamine phosphate increase the lactate level in the liver and small intestine. The given effect correlates with the inhibition of the tissue and purified lactate dehydrogenase by thiochrome.     

EFFECTS OF CALCIUM AND MAGNESIUM DEFICIENCY ON THIAMINE DISTRIBUTION IN RAT BRAIN AND LIVER

Wistar rats were divided into 4 groups and the following different synthetic diets were given for 1 month: group 1, calcium- and magnesium-deficient; group 2, calcium-deficient, magnesium-sufficient; group 3, calcium-sufficient, magnesium-deficient; group 4, calcium-sufficient, magnesium-sufficient (normal diet). After 1 month on these dietary regimens, the rats were killed. In calcium-deficiency (groups 1 and 2), thiamine concentration in synaptosomal and myelin-membrane fractions in the brain had decreased and the ratio of free thiamine to total thiamine and non-protein bound thiamine amount had increased in the brain. In magnesium-deficiency (groups 1 and 3), thiamine concentration in the liver and activities of thiamine-dependent enzymes in liver had decreased. These results indicate that calcium plays a role in binding thiamine in nerve membrane structures, which have a specific role in the conduction process of nervous tissues. In contrast, magnesium has little effect on thiamine in nervous tissues but may play an important role in thiamine-dependent enzyme systems in the liver.     

Effects of Dietary Pb on Accumulation,Histopathology, and Digestive Enzyme Activities in the Digestive System of Tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-μg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.     

Accumulation of thiamine and its metabolites in rat liver cells and mitochondria

Penetration of thiamine and its metabolites through the liver mitochondria and blood cells of white rats has been studied. It is shown that the catabolic forms of thiamine, thiochrome and 4-methyl-5 oxyethylthiasole penetrate through the mitochondria membranes at a larger extent than thiamine and its phosphoric esters. An increase in concentration of thiamine and its metabolites in the incubation medium from 0.1 mM to 3.2 mM leads to intensification of this process. The larger permeability of thiochrome and 4-methyl-5 oxyethylthiasole through biological membranes permits explaining the principles of catabolic thiamine forms removal from the tissues and organism.     

Thiamine pyrophosphate biosynthesis and transport in the nematode Caenorhabditis elegans

Thiamine (vitamin B1) is required in the diet of animals, and thiamine deficiency leads to diseases such as beri-beri and the Wernicke-Korsakoff syndrome. Dietary thiamine (vitamin B1) consists mainly of thiamine pyrophosphate (TPP), which is transformed into thiamine by gastrointestinal phosphatases before absorption. It is believed that TPP itself cannot be transported across plasma membranes in significant amounts. We have identified a partial loss-of-function mutation in the Caenorhabditis elegans gene (tpk-1) that encodes thiamine pyrophosphokinase, which forms TPP from thiamine at the expense of ATP inside cells. The mutation slows physiological rhythms and the phenotype it produces can be rescued by TPP but not thiamine supplementation. tpk-1 functions cell nonautonomously, as the expression of wild-type tpk-1 in one tissue can rescue the function of other tissues that express only mutant tpk-1. These observations indicate that, in contrast to expectation from previous evidence, TPP can be transported across cell membranes. We also find that thiamine supplementation partially rescues the phenotype of partial loss-of-function mutants of the Na/K ATPase, providing genetic evidence that thiamine absorption, and/or redistribution from the absorbing cells, requires the full activity of this enzyme.