Growth Requirements of Symbiont-Free and Symbiont Lambda-Bearing Paramecium octaurelia 299 for Folic Acid and Biopterin

We investigated the growth requirements of symbiont-free and symbiont lambda-bearing Paramecium octaurelia stock 299 for folic acid and biopterin in chemically defined culture medium. Symbiont-free P. octaurelia required both folic acid and biopterin for growth. In the absence of these substances growth of symbiont-free P. octaurelia failed after the first transfer, whereas symbiont lambda-bearing P. octaurelia could be maintained indefinitely in serial subculture. In the absence of folic acid and biopterin, sulfanil-amide inhibited growth of the symbiont lambda-beating protozoa. In the presence of folic acid and biopterin, the antiobiotic selectively inhibited growth of lambda symbionts but did not affect growth of the protozoa. In both cases, inhibition by sulfanilamide was reversed by addition of p-aminobenzoic acid to the medium. These results support our earlier finding that folic acid is required for growth of symbiont-free P. octaurelia 299 and that growth of the lambda-bearing strain without exogenous folate denoted synthesis of folic acid by the symbionts. In addition, it appears that the symbionts produce sufficient biopterin to meet the needs of the host protozoon for growth.     

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.     

Pteridine Requirement of the Hemoflagellate Leishmania tarentolae

SYNOPSIS. Leishmania tarentolae grown in a defined medium required both folic acid and an unconjugated pteridine. Continuous growth was obtained with folic acid at a minimal level of 0.34 ng/ml and biopterin at 1.7 ng/ml. Neopterin was about 1/100 as active as biopterin.     

The origin of unconjugated pteridines in Crithidia fasciculata

Summary By using folic acid-2-¹⁴C in the growth medium as the sole source of pteridine it was found that the kinetoplastid flagellate, Crithidia fasciculata, produced a total of 87 mg of labeled biopterin and 23 g of 2-amino-4-hydroxy-6-hydroxymethylpteridine from 2 mg of folic acid in 2 liters of medium. Eighty percent of the biopterin and 70% of the 6-hydroxymethylpteridine was isolated from the spent medium, the remainder from the cells. These results and the results whereby the total pteridine was supplied as a folic acid analog make it obvious that this flagellate does convert folate to biopterin and is incapable of de novo pteridine synthesis.Supported in part by Research grants AM 01005 and CA 02924 from the National Institutes of Health, United States Public Health Service, and by Deutsche Forschungsgemeinschaft.Dedicated to Professor C. B. Van Niel on the occasion of his 70th birthday.     

ORCHID MYCORRHIZA: VITAMIN PRODUCTION AND REQUIREMENTS BY THE SYMBIONTS

A Rhizoctonia species isolated from Cymbidium has been cultured successfully on a defined medium consisting of minerals, sugar, thiamine, and folic acid. Thiamine can be replaced by its thiazole component, which is probably produced by germinating orchids. The fungus apparently produces the pyrimidine moiety of thiamine, a compound which may enhance growth of certain orchid seedlings. Niacin is also provided by the fungus. Para-aminobenzoic acid, a constituent of folic acid, produced and released by orchid seeds, can satisfy the vitamin requirement of the fungus. These findings point to the possibility that orchids and their fungi may have coevolved with respect to vitamin requirements. The data also suggest that exchanges of vitamins or their components between orchids and endophytes are important aspects of the symbiotic relationship.     

Effect of water soluble vitamins and their analogues on growth of candida albicans. III. Para-aminobenzoic acid,nicotinic acid,inositol and their analogues

Summary By using high concentrations of vitamins in biotin basal synthetic mineral medium,Candida albicans was shown to possess a partial dependency forp-aminobenzoic acid, nicotinic acid and inositol. Sodium gantrisin [N¹-3,4-dimethyl-5-isoxazolyl sulfanilamide)] was growth inhibitory in the absence ofp-aminobenzoic acid and its effect was reversed byp-aminobenzoic acid. Similarly, pyridine-3-sulfonic acid was growth inhibitory to the organism in the absence of nicotinic acid and its effect was reversed by nicotinic acid. Additions of biotins, thiamine, vitamin B₁₂, nicotinic acid,p-amino-benzoic acid and inositol to basal synthetic medium showed clear-cut growth stimulation for each vitamin. Parallel omission type experiments on the other hand, demonstrated a masking effect of one vitamin on another. Ascorbic acid, riboflavin, calcium pantothenate choline, folic acid and thioctic acid were without effect. A new procedure for the determination of microbial vitamin requirements was outlined.     

Effect of vitamin B12 and folate on biosynthesis of methionine from homocysteine in the nematode Caenorhabditis briggsae.

(i) Omission of L-methionine from the medium resulted in an 80% population reduction. Substitution of D,L-homocysteine corrected methionine deficiency in C. briggsae in the presence of supraoptimal vitamin B12 and folic acid. (ii) An absolute vitamin B12 requirement in C. briggsae developed in the medium containing homocysteine at the second subculture. Concentration of 6 ng/ml of vitamin B12 (at 100 ng/ml of folic acid) was sufficient to support maximum growth of C. briggsae in the medium containing homocysteine. (iii) It was found that either supraoptimal folic acid (2000 ng/ml) or supraoptimal vitamin B12 (3750 ng/ml), with homocysteine, supported very little population growth of C. briggsae. However, supraoptimal folic acid and supraoptimal vitamin B12 together supported a maximum population growth. Therefore, it was concluded that both vitamin B12 and folic acid were required for the biosynthesis of methionine from homocysteine. Studies also showed that the two vitamins spared each other for population growth in the medium containing homocysteine.     

饲料中添加叶酸和VB12对中华绒螯蟹幼蟹生长、非特异性免疫和抗病力的影响

为研究叶酸和VB12协同作用对中华绒螯蟹(Eriocheir sinensis)幼蟹生长、非特异性免疫和抗病力的影响,选取初始体重为(2.570.03) g的幼蟹600只,随机分成4组,每组5个重复,每个重复30只幼蟹,分别投喂对照组(不添加叶酸和VB12),单一VB12组(0.2 mg/kg),单一叶酸组(2.3 mg/kg)和联合处理组(0.2 mg/kg VB12 +2.3 mg/kg叶酸)的饲料8周。在养殖实验结束后,先统计成活率和称重,然后从每个处理组随机选取30只幼蟹,用2108 CFU/mL的嗜水气单胞菌注射攻毒2周。实验结果表明:幼蟹的增重率、特定生长率、饲料效率和存活率在联合处理组最高,显著高于对照组(P0.05),但与单一叶酸或VB12组相比不存在显著差异(P0.05)。联合处理组的血清酚氧化酶活性显著高于对照组(P0.05),但与单一叶酸或VB12组也无显著性差异(P0.05)。同时,联合处理组的血清酸性磷酸酶、碱性磷酸酶、溶菌酶活性和血细胞总数等指标最高,其次是单一叶酸组和VB12组,而对照组最低。投喂联合处理组饲料幼蟹的肝胰腺超氧化物歧化酶活性最高,而丙二醛含量和累积死亡率最低。以上结果表明,叶酸和VB12对幼蟹的生长、生理代谢和免疫性能均可能有互补和协同作用,养殖生产中建议饲料中叶酸和VB12添加量分别为2.3 mg/kg和0.2 mg/kg。       

Efficacy of Supplementation with B Vitamins for Stroke Prevention: A Network Meta-Analysis of Randomized Controlled Trials

Background

Supplementation with B vitamins for stroke prevention has been evaluated over the years, but which combination of B vitamins is optimal for stroke prevention is unclear. We performed a network meta-analysis to assess the impact of different combinations of B vitamins on risk of stroke.

Methods

A total of 17 trials (86 393 patients) comparing 7 treatment strategies and placebo were included. A network meta-analysis combined all available direct and indirect treatment comparisons to evaluate the efficacy of B vitamin supplementation for all interventions.

Results

B vitamin supplementation was associated with reduced risk of stroke and cerebral hemorrhage. The risk of stroke was lower with folic acid plus vitamin B6 as compared with folic acid plus vitamin B12 and was lower with folic acid plus vitamin B6 plus vitamin B12 as compared with placebo or folic acid plus vitamin B12. The treatments ranked in order of efficacy for stroke, from higher to lower, were folic acid plus vitamin B6 > folic acid > folic acid plus vitamin B6 plus vitamin B12 > vitamin B6 plus vitamin B12 > niacin > vitamin B6 > placebo > folic acid plus vitamin B12.

Conclusions

B vitamin supplementation was associated with reduced risk of stroke; different B vitamins and their combined treatments had different efficacy on stroke prevention. Folic acid plus vitamin B6 might be the optimal therapy for stroke prevention. Folic acid and vitamin B6 were both valuable for stroke prevention. The efficacy of vitamin B12 remains to be studied.     

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.     

Biosynthesis of folic acid

Summary The influence of various vitamins on the biogenesis of folic acid has been studied in microorganisms requiring these as growth factors. In L. arabinosus, the folic acid synthesised was directly proportional to the availability of both riboflavin and pantothenic acid. The influence of cyanocobalamin on folic synthesis varied radically in different organisms. In case of the B₁₂/methionine auxotroph of E. coli there was an inverse relationship of vitamin B₁₂ to folic acid synthesis, while in Euglena the folic acid elaborated was in proportion to cyanocobalamin supplied. Synthesis of both folic acid and vitamin B₁₂ was depressed when thymine supply was adequate in the nutrition of E. coli 15 T ^-, a thymine auxotroph.     

Megaloblastic anemia and the requirement for folic acid in the cebus monkey (Cebus albifrons)

To study the development of folic acid deficiency, nine 3-year-old cebus monkeys (Cebus albifrons) were fed purified diets containing varying amounts of added folic acid. Monkeys fed the diet without added vitamin stopped growing and then lost weight. Macrocytic anemia and leukopenia developed, and megaloblastic changes were observed in precursors of both erythrocytes and leukocytes in the bone marrow. Urinary excretion of formiminoglutamic acid was increased significantly in these animals compared with controls. Repletion of deficient animals with injections of folic acid caused a rapid weight increase and reversed the hematological and biochemical abnormalities. It was estimated that the minimal folic acid requirement for adequate growth and normal hematological parameters was between 45 and 75 μg/kg body weight/day. To allow for needs above the minimal requirement, purified diets for cebus monkeys should be formulated to provide at least 150 μg of folic acid/kg body weight/day.     

Antagonists of Growth Inhibition of Crithidia by Allopurinol, a Guanine Analog

SYNOPSIS. Allopurinol, 4-hydroxypyrazolo[3,4- d ] pyrimidine, a relatively non-toxic xanthine-oxidase inhibitor used to treat gout, inhibited growth (ID_0–90 1.0 μg/ml) of the trypanosomatid flagellate of insects Crithidia fasciculata in minimal media in respect to biopterin, folic acid and purine. Allopurinol inhibition was antagonized by thymidine (4.0 μg/ml) when a ) biopterin (≥ 1.0 ng) was present alone; traces of folate entering from the inoculum or contamination of chemicals probably sufficed here to satisfy the folate requirement; b) biopterin (5% pure) 1.0 ng was added together with either pteroylglutamic acid (PGA) 4.0 ng or hypoxanthine 0.01 mg. With biopterin absent, full growth was permitted by PGA 10 ng + hypoxanthine 0.03 mg, and completely inhibited by the standard concentration of allopurinol, but in this instance thymidine did not release the inhibition.
Allopurinol is therefore useful for investigating pteridine function via inhibition analysis with the singularity that folate acts uniquely in trypanosomatids as precursor of biopterin, and the complication that both pteridines catalyze multiple biosyntheses. The biosynthetic step between folate and biopterin is postulated to be a site of inhibition of allopurinol in Crithidia.     

Folate and vitamin B12-related genes and risk for omphalocele

Both taking folic acid-containing vitamins around conception and consuming food fortified with folic acid have been reported to reduce omphalocele rates. Genetic factors are etiologically important in omphalocele as well; our pilot study showed a relationship with the folate metabolic enzyme gene methylenetetrahydrofolate reductase (MTHFR). We studied 169 non-aneuploid omphalocele cases and 761 unaffected, matched controls from all New York State births occurring between 1998 and 2005 to look for associations with single nucleotide polymorphisms (SNPs) known to be important in folate, vitamin B12, or choline metabolism. In the total study population, variants in the transcobalamin receptor gene (TCblR), rs2232775 (p.Q8R), and the MTHFR gene, rs1801131 (c.1298A>C), were significantly associated with omphalocele. In African-Americans, significant associations were found with SNPs in genes for the vitamin B12 transporter (TCN2) and the vitamin B12 receptor (TCblR). A SNP in the homocysteine-related gene, betaine-homocysteine S-methyltransferase (BHMT), rs3733890 (p.R239Q), was significantly associated with omphalocele in both African-Americans and Asians. Only the TCblR association in the total population remained statistically significant if Bonferroni correction was applied. The finding that transcobalamin receptor (TCblR) and transporter (TCN2) SNPs and a BHMT SNP were associated with omphalocele suggests that disruption of methylation reactions, in which folate, vitamin B12, and homocysteine play critical parts, may be a risk factor for omphalocele. Our data, if confirmed, suggest that supplements containing both folic acid and vitamin B12 may be beneficial in preventing omphaloceles.     

Carbohydrate Metabolism by the Acetic Acid Bacteria

Vitamin requirements for the growth of the acetic acid bacteria were investigated extensively on a. taxonomical viewpoint and the following new findings were pointed out. Neither Acetobacter nor Intermediate strain required vitamin for the growth.

Gluconobacter required generally pantothenic acid. And some strains belonging to it did moreover somewhat of thiamine, nicotinic acid and p-aminobenzoic acid, although there was a difference of requirements between strains even in the same species. Riboflavin, pyridoxine, vitamin B₁₂, folic acid, biotin and inositol were unnecessary for the growth of the acetic acid bacteria. A taxonomical division of the acetic acid bacteria based on the vitamin requirements agreed well with that on basis of the oxidative activities for carbohydrates.     

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.     

Nutritional requirements of Methanomicrobium mobile

A defined medium was developed for Methanomicrobium mobile BP. M. mobile required acetate for growth; the optimal concentration was 30 mM. Other requirements and their optimal concentrations included isobutyrate (0.65 mM), isovalerate (0.73 mM), and 2-methylbutyrate (1.5 mM). The appropriate branched-chain amino acids did not substitute for these branched-chain fatty acids. M. mobile required tryptophan at an optimal concentration of 24 microM. Indole substituted for tryptophan, but the possible precursor compounds shikimic acid and anthranilic acid and the degradation compound skatole did not. Vitamin requirements and their optimal concentrations included pyridoxine (0.49 microM), thiamine (0.15 microM), biotin (0.04 microM), and vitamin B12 (0.04 microM); p-aminobenzoic acid (0.18 microM) was required for optimal growth, but folic acid did not replace p-aminobenzoic acid. M. mobile required an unidentified growth factor found in ruminal fluid or extracts of Methanobacterium thermoautotrophicum for growth. M. mobile has a complex nutrition compared with that of other methanogens, but not an unusual nutrition in the context of organisms from the ruminal ecosystem.     

Nutritional requirements of Methanomicrobium mobile.

A defined medium was developed for Methanomicrobium mobile BP. M. mobile required acetate for growth; the optimal concentration was 30 mM. Other requirements and their optimal concentrations included isobutyrate (0.65 mM), isovalerate (0.73 mM), and 2-methylbutyrate (1.5 mM). The appropriate branched-chain amino acids did not substitute for these branched-chain fatty acids. M. mobile required tryptophan at an optimal concentration of 24 microM. Indole substituted for tryptophan, but the possible precursor compounds shikimic acid and anthranilic acid and the degradation compound skatole did not. Vitamin requirements and their optimal concentrations included pyridoxine (0.49 microM), thiamine (0.15 microM), biotin (0.04 microM), and vitamin B12 (0.04 microM); p-aminobenzoic acid (0.18 microM) was required for optimal growth, but folic acid did not replace p-aminobenzoic acid. M. mobile required an unidentified growth factor found in ruminal fluid or extracts of Methanobacterium thermoautotrophicum for growth. M. mobile has a complex nutrition compared with that of other methanogens, but not an unusual nutrition in the context of organisms from the ruminal ecosystem.     

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.     

Folic acid fortification: why not vitamin B12 also?

Folic acid fortification of cereal grains was introduced in many countries to prevent neural tube defect occurrence. The metabolism of folic acid and vitamin B12 intersect during the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine catalyzed by B12-dependent methioine synthase. Regeneration of tetrahydrofolate via this reaction makes it available for synthesis of nucleotide precursors. Thus either folate or vitamin B12 deficiency can result in impaired cell division and anemia. Exposure to extra folic acid through fortification may be detrimental to those with vitamin B12 deficiency. Among participants of National Health And Nutrition Examination Survey with low vitamin B12 status, high serum folate (>59 nmol/L) was associated with higher prevalence of anemia and cognitive impairment when compared with normal serum folate. We also observed an increase in the plasma concentrations of total homocysteine and methylmalonic acid (MMA), two functional indicators of vitamin B12 status, with increase in plasma folate under low vitamin B12 status. These data strongly imply that high plasma folate is associated with the exacerbation of both the biochemical and clinical status of vitamin B12 deficiency. Hence any food fortification policy that includes folic acid should also include vitamin B12.