Nutritional requirements of Plasmodium falciparum in culture. III. Further observations on essential nutrients and antimetabolites

In a semi-defined minimal medium for cultivation of Plasmodium falciparum, ribose, mannose, fructose, galactose, and maltose could not replace glucose. Hypoxanthine was the preferred purine source for the parasite over adenine, guanine, inosine, adenosine and guanosine although all supported growth equally. Inhibitors of nucleoside uptake had low potency in killing the parasites but depressed incorporation of [3H]adenosine more than [3H]hypoxanthine. Glutamate could not be replaced by 5-oxoproline, indicating that the gamma-glutamyl transferase pathway for amino acid uptake is probably not found in this organism. Adenine, nicotinamide, and orotic acid could not supplement glutamine-deficient medium. The pyridoxine antagonists isoniazid and 4-deoxypyridoxine were reversed by amino acid supplementation, suggesting that transaminases may be targets of these drugs. Orotic acid, but not glutathione or its amino acid components, partially reversed the effects of 8-methylamino-8-desmethyl riboflavin. Thus, the flavin enzyme, dihydroorotic acid dehydrogenase, but not glutathione reductase, appears to be a target of this riboflavin antagonist. Five biotin antagonists had no significant activity. The choline antagonist 2-(tert-butylamino)ethanol and thiamin uptake inhibitors had nonspecific inhibitory effects, which were not reversed by the respective target vitamin. Buthionine sulfoximine and methionine sulfoximine, inhibitors of glutathione synthesis, had significant oxygen-dependent toxicity. Six sulfonamides showed marked variation in potency and efficacy. Sulfathiazole and sulfadoxine were reversed differentially by p-aminobenzoic acid, folic acid, and folinic acid. Folinic acid was more effective than folic acid at reversing the toxicity of the dihydrofolate reductase inhibitors aminopterin and pyrimethamine; p-amino-benzoic acid had no effect.     

THE INFLUENCE OF EXCESS METHIONINE ON THE FREE AMINO ACIDS OF BRAIN AND LIVER OF THE WEANLING RAT*

Abstract— —High circulating levels of l -methionine produced by inclusion in the diet or parenteral injection of the amino acid caused alterations in the free amino acid pattern of liver and brain tissues. Acute effects following l -methionine injection were more pronounced than those following long term feeding where adaptation played a role. The net effect following parenteral injection was to increase the total free amino acids of liver while decreasing those of brain. Individually, hepatic levels of aspartic acid, threonine, serine, glutamine, glutamic acid, glycine, and alanine were depressed while levels of taurine, cystathionine, methionine, lysine, and ornithine were markedly elevated. Brain levels of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, and γ-aminobutyric acid were markedly depressed and increased levels of cystathionine, methionine, lysine, and glutamine were observed. A generalized aminoaciduria occurred shortly after excessive methionine intake. Disruption of the free amino acid pools was of two kinds. The first depended on the continued presence of excess l -methionine, the second did not.     

Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2.

The enzyme serine transhydroxymethylase (EC 2.1.2.1; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible both for the synthesis of glycine from serine and production of the 5,10-methylenetetrahydrofolate necessary as a methyl donor for methionine synthesis. Two mutants selected for alteration in serine transhydroxymethylase regulation also have phenotypes characteristic of metK (methionine regulatory) mutants, including ethionine, norleucine, and alpha-methylmethionine resistance and reduced levels of S-adenosylmethionine synthetase (EC 2.5.1.6; adenosine 5'-triphosphate:L-methionine S-adenosyltransferase) activity. Because this suggested the existence of a common regulatory component, the regulation of serine transhydroxymethylase was examined in other methionine regulatory mutants (metK and metJ mutants). Normally, serine transhydroxymethylase levels are repressed three- to sixfold in cells grown in the presence of serine, glycine, methionine, adenine, guanine, and thymine. This does not occur in metK and metJ mutants; thus, these mutations do affect the regulation of both serine transhydroxymethylase and the methionine biosynthetic enzymes. Lesions in the metK gene have been reported to reduce S-adenosylmethionine synthetase levels. To determine whether the metK gene actually encodes for S-adenosylmethionine synthetase, a mutant was characterized in which this enzyme has a 26-fold increased apparent Km for methionine. This mutation causes a phenotype associated with metK mutants and is cotransducible with the serA locus at the same frequency as metK lesions. Thus, the affect of metK mutations on the regulation of glycine and methionine synthesis in Salmonella typhimurium appears to be due to either an altered S-adenosylmethionine synthetase or altered S-adenosylmethionine pools.     

In Vitro Susceptibility of Acanthamoeba Culbertsoni to Inhibitors of Folate Biosynthesis1

ABSTRACT. The effects of different sulphonamides, dihydrofolate reductase inhibitors and other inhibitors of folate metabolism on growth of Acanthamoeba culbertsoni in a chemically defined medium are reported. Among the sulphonamides, sulphamethoxazole and sulphadiazine were most effective followed by sulphanilamide and sulphaguanidine. Inhibition by each sulphonamide was reversed by p-aminobenzoic acid as well as folic acid. 7-Methylguanosine, a pteridine synthesis-inhibitor, did not inhibit multiplication of A. culbertsoni. Among the dihydrofolate reductase inhibitors, pyrimethamine blocked the amoebic growth at 100 μg/ml, while trimethoprim and cycloguanil palmoate failed to cause significant inhibition of growth even at 250 μg/ml. Metoprine inhibited amoebic growth completely at 50 μg/ml. Methotrexate and a thymidylate synthetase inhibitor 5-fluorouracil inhibited growth strongly, with IC₅₀ values (the concentration of the drug which causes 50% inhibition of the growth at 72 h) of 1.97 and 2.45 μg/ml, respectively. Inhibition by methotrexate, metoprine or 5-fluorouracil could not be reversed by folic acid, folinic acid, thymidine, or folinic acid plus thymidine. the results indicate unusual features in A. culbertsoni folate metabolism.     

Evaluation of amino acids as turfgrass nematicides

Laboratory experiments revealed that DL-methionine, sodium methionate, potassium methionate, and methionine hydroxyl analog at rates of 224 and 448 kg amino acid/ha reduced the number of Belonolaimus longicaudatus mixed life-stages and Meloidogyne incognita J2 in soil, whereas L-threonine and lysine were not effective in reducing the number of either nematode. Futhermore, greenhouse experiments demonstrated that DL-methionine, sodium methionate, potassium methionate, and methionine hydroxyl analog were equally effective against B. longicaudatus at rates of 112, 224, and 448 kg amino acid/ha, and the highest rate (448 kg amino acid/ha) of all amino acids was more effective in reducing the number of B. longicaudatus than the lower rate. However, phytotoxicity was observed on creeping bentgrass (Agrostis palustris) treated with 448 kg amino acid/ha of methionine hydroxyl analog and DL methionine. In addition, in one of two field experiments on bermudagrass (Cynodon dactylon × C. transvaalensis) turf percentage green cover was increased and the number of B. longicaudatus was reduced by 224 kg amino acid/ha of DL-methionine and potassium methionate compared to untreated controls in one of two trials.     

Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

Vitamin C or Vitamin B6 supplementation prevent the oxidative stress and decrease of prostacyclin generation in homocysteinemic rats

We hypothesize that homocysteinemia causes oxidative stress, decreases the aortic ability to generate prostacyclin and that antioxidants have a protective role. Four groups of eight rats each were fed for 8 weeks the control diet (group A), control diet with folic acid omitted and excess methionine (Me) added to drinking water (group B), diet B + 500 mg/kg of Vitamin C (group C) or diet B + 60 mg/kg Vitamin B6 (group D). The three groups of rats fed folic acid deficient (FD) diets (groups B, C and D) were homocysteinemic as indicated by the significant increase in their serum homocysteine (HC) concentration. Rats fed diet B had oxidative stress as indicated by an increase in serum thiobarbituric acid reactive substances (TBARS) and advanced oxidation protein products (AOPP) and urinary isoprostanes and had a decreased ability of their aortas to generate prostacyclin. Homocysteinemic rats fed a FD diet + Vitamin C (group C) or Vitamin B6 (group D) also had high levels of serum homocysteine but the oxidative stress markers and the ability of their aortas to generate prostacyclin returned to normal. This indicates that the homocysteinemic effect is through an oxidative mechanism and that Vitamin C as a free radical scavenger prevents these effects. Serum Vitamin C and liver glutathione concentrations significantly increased in rats fed excess Vitamin B6 compared to the control or FD rats. This may explain why Vitamin B6 has an antioxidative effect.     

Nutritional Requirements of Plasmodium falciparum in Culture. III. Further Observations on Essential Nutrients and Antimetabolites1

In a semi-defined minimal medium for cultivation of Plasmodium falciparum, ribose, mannose, fructose, galactose, and maltose could not replace glucose. Hypoxanthine was the preferred purine source for the parasite over adenine, guanine, inosine, adenosine and guanosine although all supported growth equally. Inhibitors of nucleoside uptake had low potency in killing the parasites but depressed incorporation of [³H]adenosine more than [³H]hypoxanthine. Glutamate could not be replaced by 5-oxoproline, indicating that the γ-glutamyl transferase pathway for amino acid uptake is probably not found in this organism. Adenine, nicotinamide, and orotic acid could not supplement glutamine-deficient medium. The pyridoxine antagonists isoniazid and 4-deoxypyridoxine were reversed by amino acid supplementation, suggesting that transaminases may be targets of these drugs. Orotic acid, but not glutathione or its amino acid components, partially reversed the effects of 8-methylamino-8-desmethyl riboflavin. Thus, the flavin enzyme, dihydroorotic acid dehydrogenase, but not glutathione reductase, appears to be a target of this riboflavin antagonist. Five biotin antagonists had no significant activity. The choline antagonist 2-(tert-butylamino)ethanol and thiamin uptake inhibitors had nonspecific inhibitory effects, which were not reversed by the respective target vitamin. Buthionine sulfoximine and methionine sulfoximine, inhibitors of glutathione synthesis, had significant oxygen-dependent toxicity. Six sulfonamides showed marked variation in potency and efficacy. Sulfathiazole and sulfadoxine were reversed differentially by p-aminobenzoic acid, folic acid, and folinic acid. Folinic acid was more effective than folic acid at reversing the toxicity of the dihydrofolate reductase inhibitors aminopterin and pyrimethamine; p-aminobenzoic acid had no effect.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine.

The effects of media vitamin B₁₂(CNB₁₂), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH₄folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mm methionine elevates the amount of B₁₂ methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μm. Conversely, excess 100 μm folic acid increases the amount of B₁₂ holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 mm methionine. These increases in B₁₂ methyltransferase promoted by 100 μm media folate and 10 mm methionine are inhibited by cycloheximide. 5-MeH₄folate will support growth and induce methyltransferase synthesis more efficiently than folic acid.Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 μm CNB₁₂, 0.1 mm homocysteine, and 100 μm folic acid or 10 μm dl-5-MeH₄folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB₁₂ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mm methionine-containing to methionine-free media is dependent upon the B₁₂ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μm CNB₁₂ to stabilize and convert apo- to holomethyltransferase, and 100 μm folate (or 10 μm dl-5-MeH₄folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B₁₂ enzyme content over the level already induced by 100 μm folate in 0.1 mm methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH₄folate fail to induce elevated amounts of B₁₂ methyltransferase in this mutant. Excess 10 mm medium methionine does, however, elevate its B₁₂ enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B₁₂ enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B₁₂ methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media.     

Folic acid and the methylation of homocysteine by Bacillus subtilis

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C₁ transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B₁₂. 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes.     

The Nutrition of Paramecium aurelia, Stock 299

SYNOPSIS. Paramecium aurelia , stock 299 (symbiote-free) was cultivated in a synthetic medium consisting of amino acids, vitamins, purine and pyrimidine derivatives, fatty acids, stigmasterol, sodium acetate and salts. The medium supported the continued growth of this stock in serial subculture. Populations up to 17,000 organisms/ml were obtained in 9 or 10 days in the medium supplemented with a phospholipid. Synthetic 1-oleoyl-2-stearoyl-dl-phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and cephalin were comparable in growth-promoting activity. The nutritional need for each of the components of the medium was examined. The following were determined to be essential nutrilites for P. aurelia : arginine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine, folic acid, nicotinamide, calcium pantothenate, pyridoxal, riboflavin, thiamine, DL-6-thioctic acid, guanosine, uridine (or cytidine), oleic acid, stigmasterol, calcium and magnesium. Serine replaced glycine for growth in the presence of thymidine. In the absence of thymidine, comparatively high levels of folic acid were required for optimal growth. Sodium acetate did not replace DL-6-thioctic acid. Populations were reduced in the absence of the non-essential amino acids, alanine, asparagine, aspartic acid and glutamic acid. These were restored to optimal levels by the addition of sodium acetate to the medium. Pyruvate was about as effective as acetate in this respect; glucose and certain other carbohydrates were not.     

Lack of D-amino-acid oxidase activity causes a specific renal aminoaciduria in the mouse

Thin-layer chromatography and amino acid analysis showed that urine of mutant ddY/DAO- mice lacking D-amino-acid oxidase activity contained more serine, proline, alanine and methionine than that of normal ddY/DAO+ mice. Among these four, an increase in alanine was conspicuous. However, the urinary levels of 11 other amino acids and glucose were not different between the ddY/DAO- and ddY/DAO+ mice. Amino acid analysis showed that the plasma levels of serine, proline and methionine were not elevated in the ddY/DAO- mice, though a slight increase in alanine was observed. Genetic crosses showed that aminoaciduria and lack of D-amino-acid oxidase activity were concomitantly transmitted as a set through generations. These results indicated that the lack of enzyme activity caused a specific renal aminoaciduria. Whether this enzyme merely diminishes the D-amino acid load presented for reabsorption, or actually participates catalytically in the reabsorption process, remains undetermined.     

Effects of dietary protein and amino acid levels on the expression of selected cationic amino acid transporters and serum amino acid concentration in growing pigs

The absorption of lysine is facilitated by leucine, but there is no information regarding the effect of crude protein, lysine and leucine levels on the expression of cationic amino acid transporters in pigs. Therefore, an experiment was conducted with 20 pigs (14.9 +/- 0.62 kg initial body weight) to evaluate the effect of two protein levels, and the content of lysine, threonine, methionine and leucine in low crude protein diets on the expression of b(0,+) and CAT-1 mRNA in jejunum, Longissimus dorsi and Semitendinosus muscles and serum concentration of amino acids. Treatments were as follows: (i) wheat-soybean meal diet, 20% crude protein (Control); (ii) wheat diet deficient in lysine, threonine and methionine (Basal diet); (iii) Basal diet plus 0.70% L-lysine, 0.27% L-threonine, 0.10% DL-methionine (Diet LTM); (iv) Diet LTM plus 0.80% L-leucine (Diet LTM + Leu). Despite the Basal diet, all diets were formulated to meet the requirements of lysine, threonine and methionine; Diet LTM + Leu supplied 60% excess of leucine. The addition of lysine, threonine and methionine in Diet LTM increased the expression of b(0,+) in jejunum and CAT-1 in the Semitendinosus and Longissiums muscles and decreased CAT-1 in jejunum; the serum concentration of lysine was also increased (p < 0.01). Further addition of L-leucine (Diet LTM + Leu) decreased the b(0,+) expression in jejunum and CAT-1 in the Longissimus dorsi muscle (p < 0.05), increased the serum concentration ofleucine and arginine and decreased the concentration of isoleucine (p < 0.05). Pigs fed the Control diet expressed less b(0,+) in jejunum, and CAT-1 in the Semitendinosus and Longissiums muscles expressed more CAT-1 in jejunum (p < 0.05) and had lower serum concentration ofisoleucine, leucine and valine (p < 0.05), but higher lysine concentrations (p < 0.01) than those fed Diet LTM. These results indicated that both, the level and the source of dietary amino acids, affect the expression of cationic amino acid transporters in pigs fed wheat-based diets.     

Regulation of serine transhydroxymethylase activity in Salmonella typhimurium

The regulation of serine transhydroxymethylase (EC 2.1.2.1.; l-serine:tetrahydrofolic-5,10-hydroxymethyltransferase) has been investigated in Salmonella typhimurium LT2. Our results indicate that limitation of a methionine auxotroph for methionine does not cause derepression of this enzyme as reported for Escherichia coli. However, a sixfold decrease in specific activity was observed when S. typhimurium cells were grown in glucose minimal medium supplemented with serine, glycine, methionine, adenine, guanine, and thymine. None of these compounds added to the growth medium individually produced more than a 42% reduction of wild-type enzyme activity. This enhanced repression by the combination of compounds suggests a form of cumulative repression of this enzyme. Growth of serine and thymine auxotrophs, with the respective requirement of each limiting, did not result in increased enzyme activity. However, growth of a purine auxotroph with a limiting amount of either guanine or inosine resulted in a five- to sevenfold increase in enzyme activity. A second condition causing significant derepression (fourfold increase) above the levels observed with cells grown in minimal medium was the addition of 0.5 mug of trimethoprim per ml, an inhibitor of the dihydrofolate reductase activity. (A partial report on this work was presented at 1974 meeting of the American Society for Microbiology.)     

The effect of different dietary levels of DL-methionine and DL-hydroxy analogue on the antioxidant status of young turkeys infected with the haemorrhagic enteritis virus

Background

The results of experiments involving broiler chickens and turkeys indicate that increased dietary methionine (Met) levels may improve the antioxidant protection of tissues in fast-growing birds. This is an important consideration since viral infections induce oxidative stress. The aim of this study was to verify the hypothesis that turkey diets with increased Met content can suppress oxidation processes induced by infection caused by the haemorrhagic enteritis virus (HEV), and that the noted effect is determined by the chemical form of this amino acid: DL-methionine (DLM) or DL-hydroxy analogue of Met (MHA).

Results

Dietary Met content above 40% higher than the level recommended by the NRC (1994) intensified lipid peroxidation in the small intestine, leading to an increase in malondialdehyde (MDA) and lipid peroxide (LOOH) levels, but it also stimulated antioxidant mechanisms in the blood and liver of turkeys infected with HEV. In comparison with DLM, MHA contributed to more severe symptoms of oxidative stress, such as elevated MDA levels in the intestines, and a decrease in glutathione peroxidase (GPx) activity and ferric-reducing ability of plasma (FRAP).

Conclusions

In HEV-infected turkeys, diets with increased Met content did not exert a clear antioxidant effect, which was noted in uninfected birds. The prooxidant activity of Met observed in the small intestinal wall was suppressed in the blood and liver of turkeys, most likely due to intensified synthesis of uric acid and glutathione. In comparison with MHA, DLM had a more beneficial influence on the analysed parameters of the redox status in the small intestine, blood and liver of turkeys.

     

The combined effects of FUdR addition and methionine depletion on the X-chromosome fragile site.

The fragile site on the X chromosome [fra(X)] associated with mental retardation is not normally seen in lymphocytes cultured in media containing folic acid or lacking methionine. The requirement for methionine has been taken to mean that amino acid metabolism or one-carbon transfer via S-adenosyl-methionine is most important in fra(X) expression. The inhibitory effect of folic acid can be overcome, as we have shown, by the addition of 5'-fluorodeoxyuridine (FUdR) to the culture medium, suggesting that depletion of dTMP available for DNA synthesis is most important in expression. We tested the combined effects of FUdR addition and methionine depletion on the fra(X). We found expression of the fra(X), indicating that methionine per se is not necessary for expression. Our work supports the suggestion that expression of the fra(X) is due to specific limitation of the dTMP pool available for DNA synthesis.     

Methionine and serine synergistically suppress hyperhomocysteinemia induced by choline deficiency, but not by guanidinoacetic acid, in rats fed a low casein diet

The effects of dietary supplementation with 0.5% methionine, 2.5% serine, or both on hyperhomocysteinemia induced by deprivation of dietary choline or by dietary addition of 0.5% guanidinoacetic acid (GAA) were investigated in rats fed a 10% casein diet. Hyperhomocysteinemia induced by choline deprivation was not suppressed by methionine alone and was only partially suppressed by serine alone, whereas it was completely suppressed by a combination of methionine and serine, suggesting a synergistic effect of methionine and serine. Fatty liver was also completely prevented by the combination of methionine and serine. Compared with methionine alone, the combination of methionine and serine decreased hepatic S-adenosylhomocysteine and homocysteine concentrations and increased hepatic betaine and serine concentrations and betaine-homocysteine S-methyltransferase activity. GAA-induced hyperhomocysteinemia was partially suppressed by methionine alone, but no interacting effect of methionine and serine was detected. In contrast, GAA-induced fatty liver was completely prevented by the combination of methionine and serine. These results indicate that a combination of methionine and serine is effective in suppressing both hyperhomocysteinemia and fatty liver induced by choline deprivation, and that methionine alone is effective in suppressing GAA-induced hyperhomocysteinemia partially.     

Synergistic effect of folic acid and vitamin B12 in ameliorating arsenic-induced oxidative damage in pancreatic tissue of rat

The efficacies of two nutritional factors, folic acid and vitamin B12, were assessed in this study against arsenic-induced islet cellular toxicity. Rats were divided into four groups consisting of five rats in each group: Group A, control; Group B, arsenic-treated; Group C, arsenic+folic acid; and Group D, arsenic+folic acid+vitamin B12. The dose of arsenic, folic acid and vitamin B12, respectively, was 3 mg, 36 microg and 0.63 microg kg(-1) body weight day(-1) for 30 days. Results showed that, compared to control group, there was a significant increase in the levels of nitric oxide (NO), malondialdehyde (MDA) and hydroxyl radical (OH-) formation in the pancreatic tissue of arsenic-treated rats, while the activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and cellular content of antioxidant glutathione (GSH) were low in these animals. The serum level of tumor necrosis factor-alpha (TNF-alpha) and IL-6 was significantly high in these animals. Light microscopic examination showed a marked fall in the number of islet cells. Concomitant administration of either folic acid or folic acid and vitamin B12 with arsenic significantly restored all these parameters. Although folic acid alone could not restore the normal level of TNF-alpha and IL-6, combined folic acid and vitamin B12 could restore it. Folic acid and vitamin B12 combined also could recover islet cell count. These results suggest that folic acid+vitamin B12 are capable of reducing arsenic-induced cellular oxidative and inflammatory toxic changes. Thus, supplement with vitamin B12+folic acid may be predicted as a possible nutritional management strategy against arsenic-induced toxicity.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.