Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.)

Summary This study was conducted to examine the effect of biotin and thiamine concentrations on callus growth and somatic embryogenesis of date palm (Phoenix dactylifera L.). Embryogenic callus derived from offshoot tip explants was cultured on hormone-free MS medium containing biotin at 0, 0.1, 1, or 2 mg l⁻¹ combined with thiamine at 0.1, 0.5, 2, or 5 mg l⁻¹. Embryogenic callus weight, number of resultant embryos, and embryo length were significantly influenced by thiamine and biotin concentration. The optimum callus growth treatment consisted of 0.5 mg l⁻¹ thiamine and 2 mg l⁻¹ biotin. This treatment also gave the highest number of embryos. Embryo elongation was greatest at 0.5 or 2 mg l⁻¹ thiamine combined with 1 mg l⁻¹ biotin. Embryos from all treatments germinated and regenerants exhibited normal growth in soil. This study provides an insight into the importance of optimizing various culture medium components to overcome in vitro recalcitrace of date palm.     

Influence of dietary supplementation with thiamine on lead intoxication in rats

The influence of dietary supplementation with thiamine on lead (Pb) contents in blood and tissues, blood δ-aminolevulinic acid dehydratase (δ-ALAD) activity, and urinary excretion of δ-aminolevulinic acid (δ-ALA) was evaluated in male Sprague-Dawley rats. Groups of randomly selected animals were given a thiamine-deficient diet, a diet containing normal thiamine (20 mg/kg), or a thiamine-supplemented diet (50 mg/kg), along with control drinking water or water containing 100 ppm Pb, for 4 mo. Animals fed the thiamine-supplemented diet (50 mg/kg) and Pb showed decreased urinary excretion of δ-ALA and a decreased inhibition of δ-ALAD activity in blood compared to those given Pb with normal thiamine diet. The liver, kidney, and blood of rats receiving supplemental thiamine also contained significantly less Pb than the other two treatment groups given Pb-containing water. The protective effect of thiamine against Pb toxicity may be attributed to its interference with retention of the metal in body tissue, possibly resulting from the formation of excretable thiamine-lead complexes.     

A Thiamine/H+ Antiport Mechanism for Thiamine Entry into Brush Border Membrane Vesicles from Rat Small Intestine

Outwardly oriented H⁺ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of adult Wistar rats. At a gradient pH_in5:pH_out7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of the gradient. Under the same conditions, the binding component of uptake accounted for only 10–13% of intravesicular transport. At the same gradient, the K _m and J _max values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 ± 1.4 μm and 14.9 ± 3 pmol · mg⁻¹ protein · 6 sec⁻¹ respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H⁺ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H⁺ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H⁺ antiport, and imipramine. Conversely, the guanidine/H⁺ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase in thiamine transport rate. In the absence of H⁺ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to 99% at pH 9. Received: 17 July 1997/Revised: 16 September 1997     

Characteristic properties of metabolism of the yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> during the synthesis of α-ketoglutaric acid from ethanol

The study of free amino acid content in Yarrowia lipolytica cells grown on ethanol under thiamine deficiency showed that glutamate, alanine, and γ-aminobutyric acid (γ-ABA) occurred in the highest concentrations among the present 17 free amino acids. The culture liquid contained no amino acids. Analysis of the enzymes of oxidative metabolism in the yeast grown under these conditions showed that the cell-free homogenate contained substantial activity of glutamate decarboxylase, γ-ABA transaminase, and succinyl semialdehyde dehydrogenase. This result indicated the formation of succinate from glutamate in a reaction catalyzed by 4-aminobutyrate aminotransferase (γ-aminobutyrate bypass) under severe thiamine deficiency. These studies lead to the conclusion that cultivation of the yeast Y. lipolytica on ethanol under thiamine deficiency causes adaptive stress-induced metabolic changes. Increase of ammonium nitrogen consumption and excretion of α-ketoglutaric acid are indicative of physiological changes, the functioning of the γ-aminobutyrate bypass and high activity of malate dehydrogenase are manifestations of metabolic changes, and increased activities of the transamination reactions reflect the changes in nitrogen metabolism.     

Role of mitochondrial dysfunction and oxidative stress in the pathogenesis of selective neuronal loss in Wernicke’s encephalopathy

Thiamine deficiency results in Wernicke’s encephalopathy and is commonly encountered in chronic alcoholism, gastrointestinal diseases, and HIV AIDS. The earliest metabolic consequence of thiamine deficiency is a selective loss in activity of the thiamine diphosphate-dependent enzyme α-ketoglutarate dehydrogenase (α-KGDH), a rate-limiting tricarboxylic acid cycle enzyme. Thiamine deficiency is characterized neuropathologically by selective neuronal cell death in the thalamus, pons, and cerebellum. The cause of this region-selective neuronal loss is unknown, but mechanisms involving cellular energy failure, focal lactic acidosis, and NMDA receptor-mediated excitotoxicity have classically been implicated. More recently, evidence supports a role for oxidative stress. Evidence includes increased endothelial nitric oxide synthase, nitrotyrosine deposition, microglial activation, and lipid peroxidation. Reactive oxygen species production results in decreased expression of astrocytic glutamate transporters and decreased activities of α-KGDH, resulting in an amplification of cell death mechanisms in thiamine deficiency.     

The Production of Pyruvic and {alpha}-Oxoglutaric Acids by Mucor Species

Six out of the seven species of Mucor studied (M. plumbeus,M. racemosus, M. rouxii, M. hiemalis, M. ramannianus, and M.mucedo) are shown to produce pyruvic acid when grown on a mediumcontaining glucose, glutamate, and mineral salts. Mucor pusillusproduces no detectable pyruvate on this medium. The proportionof the carbohydrate metabolized which is excreted as pyruvateis greatest in species having a high thiamine requirement forgrowth. Addition of thiamine to the culture medium reduces pyruvateexcretion. However, in the species which need added thiaminefor growth, the growth requirement is almost completely satisfiedby 20 µg thiamine per litre whereas suppression of pyruvateexcretion requires about 200 µg thiamine per litre. Experimentswith M. plumbeus show that the yield of pyruvate is reducedwhen xylose is substituted for glucose and also when alanineis substituted for glutamate in the medium. -Oxoglutaric acidis produced by all seven species on the glucose/glutamate medium,the yield varying with the species. This acid is partly derivedfrom the glutamate in the medium.     

Role of vitamin-zinc interactions on in vitro zinc uptake by human erythrocytes

In vitro zinc uptake by human erythrocytes was studied under a range of zinc concentrations representing three different plasma zinc levels i.e., zinc deficient [0.35–0.61 ppm], zinc normal [0.74–1.59 ppm], and zinc excess [1.65–2.3 ppm]. Further, interactions of physiological levels of riboflavin, flavin adenine dinucleotide (FAD), nicotinic acid, nicotinamide adenine dinucleotide (NAD), thiamine, thiamine pyrophosphate (TPP), folic acid, and ascorbic acid with zinc uptakes were studied in independent experiments. In control experiments, as compared to the normal zinc state, the rate of change of zinc uptake over change in zinc levels was 1.6 times in the excess state and 0.12 times in the deficient state, indicating three distinct patterns. Under the zinc-deficient state, thiamine significantly enhanced the zinc uptakes (p<0.05), whereas ascorbic acid and riboflavin inhibited zinc uptakes (p<0.05). The percent hemolysis of the cells was also significantly lower in the presence of thiamine (p<0.05). Under normal and excess zinc states, the vitamin-zinc interactions were not significant. The results suggest that with erythrocytes as the vehicles, thiamine might be playing an enhancer role in uptake of zinc, whereas the action of ascorbic acid might be inhibitory for zinc uptakes under deficient zinc states.     

Thiamine requirements of various plant cells in suspension culture

The thiamine requirements of cells of several plant speciesin suspension culture were investigated. Omission of thiaminefrom the medium leads to a rapid and complete cessation of growthin subcultures of soybean, tobacco and rice cells. The criticallevels of thiamine content in these cells are considered tobe in the vicinity of 0.6, 0.5 and 0.2 µg per g dry weight,respectively. Soybean cells can grow satisfactorily when suppliedwith an equimolar mixture of two thiamine precursors, pyrimidineand thiazole moieties. Neither moiety alone can substitute forthiamine. On the other hand, Rula and peanut cells can be successfullysubcultured for ten passages in the absence of externally suppliedthiamine. When grown without thiamine, Rula cells had an averagethiamine content of 0.5-0.6 µg in darkness and 3.5 µgper g dry weight in the light. The relationship between thethiamine requirement and the degree of dedifferentiation incultured cells is discussed. ¹ Present address: Section of Phytochemical Research, Researchand Development Division, Eisai Co., Ltd., Kawashima, Gifu 112,Japan. (Received December 24, 1975; )     

Egg thiamine concentration affects embryo survival in Lake Erie walleye

We quantified thiamine in eggs of Lake Erie walleye to determine if differences exist between spawning stocks within the Maumee and Sandusky rivers, both of which drain into the Western Basin. In spring 2004, eggs of walleye were collected in the Maumee River at three occasions (early, peak and late of the spawning run) and in the Sandusky River during the spawning run. After collection, eggs were fertilized with a known amount of milt, incubated and embryo survival was determined at the pigmented eyed stage. Thiamine and its derivatives were analyzed using high performance liquid chromatography. Thiamine pyrophosphate (TPP) was the most abundant form of vitamin B₁ present in eggs (60–95% of the total vitamin B₁ concentration). Total thiamine concentrations in walleye eggs from the Maumee and Sandusky rivers at the peak of the spawning run averaged 6.1 ± 1.6 nmol g⁻¹ and 5.0 ± 2.9 nmol g⁻¹, respectively. Our results also indicated that Maumee River stock survival to the eyed stage embryo declined as the spawning season progressed (72%, 59% and 37% in the early, middle and late of spawning run, respectively) as well as the total thiamine and TPP concentrations. At the peak of the spawning run, survival to the eyed stage embryo did not differ significantly between stocks 59% versus 65% in the Maumee and Sandusky rivers, respectively, and thiamine concentrations were not significantly different between sites.     

Synthesis of α-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol

The ability of yeast to synthesize α-ketoglutaric acid (KGA) from ethanol has been studied. Thiamine-auxotrophic yeasts of different genera and species may be able to produce KGA; the main condition of synthesis is growth limitation by thiamine. Using a model culture, mutant Yarrowia lipolytica N 1, the principal conditions affecting KGA oversynthesis were identified. These were: thiamine concentration in medium and in cells, nitrogen and oxygen concentration in medium, and pH level. A KGA concentration of 49 g/l and a yield from ethanol consumed of 42% were achieved. Based on the results of the analysis of the activities of the key enzymes participating in ethanol metabolism and KGA synthesis, a concept of the mechanism of KGA biosynthesis by Y. lipolytica yeast is suggested and discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 June 1999     

Influence of soil temperature on niacin and thiamine in honey mesquite

Summary The influence of soil temperature was examined on niacin and thiamine concentration in honey mesquite (Prosopis glandulosa var.glandulosa) seedlings. The seedlings were grown in soil temperature regimes of 21, 27, and 32°C in a controlled environment growth room. Nodulation randomly occurred on the roots of the seedlings, necessitating separate analysis according to the occurrence of nodulation. Roots of nodulated seedlings from the 21°C soil temperature regime contained greater quantities of niacin and thiamine compared to root samples from seedlings grown in either 27 or 32°C regimes. Niacin concentration of non-nodulated seedlings was highest in samples from seedlings grown in the 27°C soil temperature regime and lowest in samples from seedlings grown in the 21°C regime. Thiamine concentration was the greatest from non-nodulated seedlings grown in the 27°C soil temperature regime, while the thiamine concentration of non-nodulated samples from the 32°C regime was the least. Optimal soil temperature for honey mesquite root growth appears to be about 27°C. At sub-optimal soil temperatures niacin might have limited ‘growth’ while at supra-optimal soil temperatures, thiamine might be a limiting factor. College of Agricultural Sciences Contribution No. T-9-164.     

Thiamine requirement of two different cultured cell lines of soybean

When supplied with 6-benzyladenine (0.5–5 mg/liter) insteadof thiamine, thiamine-requiring soybean cells (strain TU) couldgrow successively. The effect of cytokinin was much more remarkableat a relatively higher concentration of 2,4-dichlorophenoxyaceticacid (4 mg/liter) than at a lower concentration (0.5 mg/liter).Among calli initiated from soybean hypocotyls on a medium withoutthiamine, the thiamine-nonrequiring variant (strain G) was obtainedincidentally. As this cell line became green in light, it couldbe visually separated from the other necrotic tissues. StrainG cells could grow successively not only without thiamine butalso without phytohormones, auxin and cytokinin. This cell linehad relatively higher amounts of chlorophyll and thiamine, andgrew in rigid, large cell aggregates which differed from cellaggregates of the strain TU cell line. The thiamine requirementof plant cultured cells seems to be associated with the degreeof dedifferentiation of the cells rather than the kind of plant.In general, the higher the degree of redifferentiation of thecells, the higher is their thiamine level and the less theyrequire externally supplied thiamine. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )     

Effect of sucrose,inorganic salts,inositol, and thiamine on protease excretion during pineapple culture in temporary immersion bioreactors

Summary Although pineapple plants have been found to produce proteases ex vitro, most of the biotechnological investigations of this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore, we decided to modify the culture medium composition of this phase looking for an increase in protease excretion. Four independent experiments were performed to evaluate the effects of different levels of sucrose (0–350.4 mM), inorganic salts [0–200% Murashige and Skoog (MS) salt strength], inositol (0–2.20 mM), and thiamine (0–1.2μM). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. Specific protease activity, the most important indicator recorded, was highest with 262.8 mM sucrose, 100% MS salt strength, 0.3 μM thiamine and no inositol. Results shown here demonstrate that conditions adequate for propagation purposes (87.6 mM sucrose, 100% MS salt strength, 0.55 mM inositol, 0.3 μM thiamine) are not always adequate for protease excretion.     

Germination of Sporangia of Phytophthora palmivora (Butl.) Butl.

Sporangia of Phytophthora palmivora germinated by either forminggerm tubes or producing zoospores. Two distinct modes of germ-tubedevelopment have been described. Sporangia in distilled waterformed zoospores at 10–34°C with an optimum at 22°Cbut germinated by means of germ tubes at 30 and 34°C only.Zoospore formation was inhibited to varying degrees by cocoapod extract, I.0 per cent (w/v) peptone and yeast extract, 100–500µg1^-1 thiamine, and by very low concentrations of severalamino acids, carbohydrates, and inorganic salts. Germ-tube formation was encouraged at 22°C by 1'0 per cent(w/v) peptone and yeast extract, by cocoa pod extract and exudate,10mM CaCl2, 1–10 mM MgSO₄. 7H₂O, 0.5 per cent (w/v) fructose,galactose, glucose, lactose, maltose, and sucrose, by 100 ppmarginine, aspartic acid, glutarnic acid, glycine, leucine, andtryptophane, and by 100–500 µg 1^-1 thiamine.     

Inhibition of Thiamine Pyrophosphate Utilization by Thiamine or Its Monophosphate in Escherichia coli

The growth of a thiamine pyrophosphate auxotroph of Escherichi coli was inhibited by either thiamine or thiamine monophosphate, and the growth of a thiamine monophosphate auxotroph was inhibited by thiamine. The thiamine pyrophosphate-dependent oxidation of pyruvate was inhibited by thiamine with whole cells of the thiamine pyrophosphate auxotroph but not with cell extracts prepared from the same organism. In addition, the thiamine pyrophosphate uptake of the thiamine pyrophosphate auxotroph was inhibited by either thiamine or thiamine monophosphate. Although the thiamine pyrophosphate uptake of a revertant, selected for prototrophy from the thiamine monophosphate auxotroph, was inhibited by thiamine to an extent comparable to that observed with the thiamine monophosphate auxotroph, its growth was no longer inhibited by thiamine. A possible mechanism for the inhibition by thiamine and thiamine monophosphate in the utilization of thiamine pyrophosphate is discussed.     

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles

Thiamine, a water-soluble vitamin, is essential fornormal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety ofconditions. To date, however, little is known about the mechanism ofthiamine absorption in the native human small intestine. The objectiveof this study was, therefore, to characterize the mechanism of thiaminetransport across the brush-border membrane (BBM) of human smallintestine. With the use of purified BBM vesicles (BBMV) isolated fromthe jejunum of organ donors, thiamine uptake was found to be1) independent of Na⁺ but markedly stimulated byan outwardly directed H⁺ gradient (pH 5.5_in/pH7.5_out); 2) competitively inhibited by thecation transport inhibitor amiloride (inhibitor constant of 0.12 mM);3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structuralanalogs (amprolium, oxythiamine, and pyrithiamine), but not byunrelated organic cations (tetraethylammonium,N-methylnicotinamide, or choline); 5) notaffected by the addition of ATP to the inside and outside of the BBMV;6) potential insensitive; and 7) saturable as afunction of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 µM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. Carrier-mediated thiamine uptake was also found inBBMV of human ileum. These data demonstrate the existence of aNa⁺-independent, pH-dependent, amiloride-sensitive,electroneutral carrier-mediated mechanism for thiamine absorption innative human small intestinal BBMV.

     

Induction and growth characteristics of embryogenic avocado cultures

Embryogenic cultures were induced from immature avocado zygotic embryos representing different botanical races and complex hybrids. The optimum induction medium consisted of B5 major salts, MS minor salts, 0.4 mg l⁻¹ thiamine HCl, 100 mg l⁻¹ myo-inositol, 30 g l⁻¹ sucrose, 0.41 μM picloram and 8 g l⁻¹ TC agar. Somatic embryogenesis occurred directly from the explants on induction medium, and secondary embryos and proembryonic masses proliferated in liquid and on semisolid maintenance medium. Embryogenic culture maintainance was optimized in liquid, filter-sterilized MS medium, supplemented with 30–50 mg l⁻¹ sucrose, 4 mg l⁻¹ thiamine HCl and 0.41 μM picloram. Two types of embryogenic cultures were recognized: –genotypes that proliferated as proembryonic masses in the presence of auxin (PEM-type) and; –genotypes in which the heart stage and later stages of somatic embryos developed in the presence of auxin(SE-type). Embryogenic suspension cultures became increasingly disorganized over time, and this was associated with progressive loss of embryogenic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

Thiamine phosphate metabolism and possible coenzyme-independent functions of thiamine in brain.

The effect of depolarization of rat brain cortex slices on the relative distribution of thiamine among its various phosphate esters and on the efflux of thiamine was studied as a probe of possible coenzyme-independent neurophysiological functions of thiamine. Electrical pulses for 30 min increased lactate production but did not affect the levels of thiamine esters. Depolarization with 41 mM-potassium decreased thiamine diphosphate by only 3 percent (P= 0.05). Thiamine triphosphate levels (TTP) were unaffected by depolarization but doubled during incubation for 1 h in which time efflux of 40 percent of the total thiamine from the slices as unesterified thiamine occurred. Depolarization by potassium released a small but highly variable portion of the thiamine content of superfused cortex slices above the basal rate of efflux. The basal efflux was partially sodium dependent. Thiamine efflux was unaffected by acetylcholine, ouabain, or tetrodotoxin, compounds previously reported to increase thiamine efflux. The incorporation of ³²P₁ into the endogenous thiamine phosphates of cortex slices was studied. Incorporation into thiamine diphosphate reached only 20 percent of the specific activity of its precursor, ATP, after 2h of incubation while the incorporation into TTP approached equilibrium with ATP in 15-30 min indicating that the TTP pool was the most rapidly turning over of the thiamine phosphates. The data suggest that only a small portion of the TDP pool undergoes rapid turnover and serves as a precursor for TTP. The rapid turnover of TTP phosphoryl groups is consistent with specific functions for this compound related to its potential for phosphorylation reactions. An analog of TTP with the β, γ oxygen bridge replaced by a methylene group decreased TDP levels and increased thiamine when incubated with cortex slices, but did not effect thiamine monophosphate or triphosphate levels indicating inhibition of thiamine pyrophosphokinase.