Digested bacterial cell powder (DBCP) as a source of reduced-form folates for pigs: use of a trimethoprim-resistant strain and the bioavailability of folates in DBCP.

The production of digested bacterial cell powder (DBCP) as a source of reduced-form folates for pigs was studied. Trimethoprim-resistant mutants of Brevibacterium lactofermentum ATCC 13869 accumulated a significantly higher amount of the reduced form of folate in the cells than the wild-type strain. DBCPs were prepared from the resistant mutant strain and the wild-type strain. The utilization of the reduced-form of folate in DBCP was evaluated by measuring the plasma folate level after orally administering DBCP to G?ttingen minipigs. The folates in both DBCPs proved to have equally high bioavailability in the pigs.     

Folates in plants: biosynthesis, distribution, and enhancement

Folates are crucial intermediates for a set of reactions that involve the transfer of single-carbon units (C1 metabolism). They are directly involved in the synthesis of nucleic acids, methionine, pantothenate, glycine and serine, and indirectly, through S-adenosyl methionine, in all methylation reactions. Humans cannot synthesize folates de novo. In these organisms, folate deficiency has severe effects on health and affects large population groups around the world. Because plants are the main source of dietary folates, there are great concerns to select plant food having high concentrations of folates or to engineer their folate metabolism to increase the initial amount. All these attempts rely on what we know about the metabolism of folates. During these last 10 years, the complex pathway leading to the synthesis of folates has been deciphered. Our knowledge about folate synthesis and distribution during plant growth and development also increased substantially. However, important aspects of folate metabolism remain unclear, such as catabolism, transport and regulation of the homeostasis. The aim of this review was to summarize our recent findings, to describe the few attempts reported in the literature to engineer folate level in plants, and to discuss potential strategies that could be used for enhancement.     

Distribution of Folates in Ochromonas malhamensis

SYNOPSIS. Ochromonas malhamensis contains folate derivatives active for Streptococcus faecalis, Pediococcus cerevisiae and Lactobacillus casei. These activities increase about 6-, 4- and 3-fold, respectively, on treatment with chicken liver conjugase, establishing the presence of polyglutamyl folates in the organism. The nature of the folate derivatives as well as their functional groups has been ascertained by chromatography on DEAE cellulose columns and assay of the activities of the eluted fractions, before and after the conjugase treatment, using differential microbiologic assay technic. The folate complex has been resolved into 7 fractions corresponding to N¹⁰-formyltetrahydrofolic acid, N⁵-formyltetrahydrofolic acid, unsubstituted tetrahydrofolic acid, and 4 polyglutamyl folates. The implications of these findings are indicated.     

Folates of rat tissue: Bioassay of tissue folypolyglutamates and a relationship of liver folypolyglutamates to nutritional folate sufficiency

The folate content of young rat tissues extracted into boiling ascorbate was assayed by Lactobactillus casei both without and after treatment by a folate-free preparation of conjugase. The total folate content of various tissues was: liver, 8.9 μg/g; kidney, 2.6; adrenal, 2.6; bone marrow, 2.4; spleen, 0.9; erythrocytes, 0.8; small intestinal mucosa, 0.7; small intestinal smooth muscle, 0.8; heart, 0.6; brain, 0.4, and skeletal muscle, 0.1 μg/g tissue. For most tissues, with the exception of muscle and kidney, approximately 80% of the total folates assayed as longer chain length folylpolyglutamates.When liver folates were analyzed from rats fed folate-supplemented, control and folate-deficient diets, a relationship was found between folate nutrition and distribution of folylpolyglutamates. The proportion of total folates in the form of longer chain length folylpolyglutamates was greatest in the livers of folate-deficient rats and least in the livers of folate-supplemented rats.     

叶酸在植物体内功能的研究进展

叶酸(folates)是一类水溶性B族维生素, 包括四氢叶酸(THF)及其衍生物, 是植物体中参与C1转移反应的重要辅酶。其在嘌呤、胸苷酸、DNA、氨基酸和蛋白质的生物合成以及甲基循环中发挥重要作用。近年来, 人们对叶酸在植物体内的功能又有了新发现。例如, 叶酸可通过结合核糖开关实现基因表达调控; 叶酸在苯丙氨酸转化为酪氨酸过程中可作为电子供体; 光裂合酶和植物隐花色素的叶酸辅酶可捕获光能; 此外, 叶酸还参与叶绿素的生物合成以及种子抗氧化胁迫等过程。该文详细综述了上述新发现, 并对植物体内叶酸功能的主要研究方向进行了展望。     

Folates and Folic Acid: From Fundamental Research Toward Sustainable Health

Folates are of paramount importance in one-carbon metabolism of most organisms. Plants and microorganisms are able to synthesize folates de novo, making them the main dietary source for humans and animals, which are dependent on food or feed supplies for folates. Folate deficiency is an increasing problem in the developing, as well as in the developed regions of the world, affecting millions of people. Different strategies, such as food fortification and folic acid supplementation, remain far from accessible for the poor rural populations in developing countries. Increasing knowledge concerning folate biosynthesis, transport and catabolism does not only deepen our insight on the regulation of folate metabolism but also provides the keys towards folate enhancement through metabolic engineering in bacteria, as well as in plants. Recently, promising results were obtained using such an approach, but further fundamental research is a prerequisite to develop a practicable solution to fight folate deficiency. In parallel, progress in the development and improvement of folate analysis has been made. Here, we provide the state-of-the-art of folate biosynthesis, catabolism, and salvage. Finally, we report on progress in folate biofortification and discuss the agroeconomical aspect of biofortified crop plants.     

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms. Stable cell lines were created that express cDNAs for the mitochondrial and cytosolic isoforms of human FPGS under control of a doxycycline-inducible promoter in the AUXB1 cell line. AUXB1 are devoid of endogenous FPGS activity due to a premature translational stop at codon 432 in the fpgs gene. Loss of folates was not measurable from these doxycycline-induced cells or from parental CHO cells over the course of three CHO cell generations. Likewise, there was no detectable transfer of folate polyglutamates either from the cytosol to mitochondria, or from mitochondria to the cytosol. The cell line expressing cytosolic FPGS required exogenous glycine but not thymidine or purine, whereas cells expressing the mitochondrial isoform required exogenous thymidine and purine but not glycine for optimal growth and survival. We concluded that mitochondrial FPGS is required because folate polyglutamates are not substrates for transport across the mitochondrial membrane in either direction and that polyglutamation not only traps folates in the cytosol, but also in the mitochondrial matrix.     

The potential of bifidobacteria as a source of natural folate

Aims: To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate‐free and complex folate‐containing media. Methods and Results: HPLC was used to analyse deconjugated folates extracted from bacterial biomass. Most strains had a total folate content above 4000 μg per 100 g dry matter (DM). The highest value of 9295 μg per 100 g DM was found in Bifidobacterium catenulatum ATCC 27539 and the lowest in Bifidobacterium animalis ssp. animalis ATCC 25527 containing 220 μg per 100 g DM. Ten strains grew in a synthetic folate‐free medium (FFM), showing folate autotrophy and suggesting folate auxotrophy of the remaining nine. In the autotrophic strains, a consistently higher folate level was found in FFM as compared to a more complex folate‐containing medium, suggesting reduced requirements for folates in the presence of growth factors otherwise requiring folates for synthesis. The contents of total folate, 5‐CH₃‐H₄folate and H₄folate were strain dependent. 5‐CH₃‐H₄folate dominated in most strains. Conclusions: Our results show that bifidobacteria folate content and composition is dynamic, is strain specific and depends on the medium. Suitable selection of the growth conditions can result in high levels of folate per cell unit biomass. Significance and Impact of the Study: This suggests that certain bifidobacteria may contribute to the folate intake, either directly in foods, such as fermented dairy products, or in the intestine as folate‐trophic probiotics or part of the natural microbiota.     

Genetic toxicology of 1,2-dibromo-3-chloropropane (DBCP)

1,2-Dibromo-3-chloropropane (DBCP) is a nematocide, which has been used extensively as a soil fumigant in agriculture. Since sterility was found among male workers involved in the manufacture of DBCP, great concern has been focused on the genetic hazards of DBCP. DBCP gave positive results in many tests such as microbial, in vitro cytogenetics, and Drosophila studies. In mammalian test systems, DBCP caused chromosomal aberrations in the bone marrow cells and dominant-lethal mutations in germ cells in rats. In mice, there were no signs of DBCP-induced heritable mutation in germ cells, although point mutations were detected in somatic cells. The occurrence of Y-chromosomal non-disjunction was indicated in DBCP-exposed male workers by an increased number of sperm containing 2 Y-chromosomes.     

Growth properties of a folA null mutant of Escherichia coli K12.

In Escherichia coli, dihydrofolate reductase is required for both the de novo synthesis of tetrahydrofolate and the recycling of dihydrofolate produced during the synthesis of thymidylate. The coding region of the dihydrofolate reductase gene, folA, was replaced with a kanamycin resistance determinant. Unlike earlier deletions, this mutation did not disrupt flanking genes. When the mutation was transferred into a wild-type strain and a thymidine-(thy) requiring strain, the resulting strains were viable but slow growing on rich medium. Both synthesized less folate than their parents, as judged by the incorporation of radioactive para-aminobenzoic acid. The derivative of the wild-type strain did not grow on any defined minimal media tested. In contrast, the derivative of the thy-requiring strain grew slowly on minimal medium with thy but exhibited auxotrophies on some combinations of supplements. These results suggest that when folates are limited, they can be distributed appropriately to folate-dependent biosynthetic reactions only under some conditions.     

Analysis and characterization of the folates in the nonmethanogenic archaebacteria.

A detailed analysis of the folate coenzymes in the nonmethanogenic archaebacteria has been performed. By using the Lactobacillus casei microbiological assay for folates, the levels of folates in Sulfolobus solfataricus and Sulfolobus acidocaldarius were found to be 3.7 and 8.3 ng/g (dry weight) of cells, respectively, compared with 88,000 and 28,000 ng/g (dry weight) of cells in Halobacterium halobium and Halobacterium strain GN-1, respectively. The levels of folates found in the Sulfolobus spp. were approximately 100 times less than those found in the typical eubacterium, whereas the levels in the halobacteria were approximately 10 times higher. The folate in Sulfolobus solfataricus was shown to consist of only 5-formyltetrahydropteroylglutamate, and the folate in Halobacterium strain GN-1 was shown to consist of only pteroyldiglutamate. The low folate levels in the Sulfolobus spp. are the same as those found in the methanogenic bacteria, suggesting that another C1 carrier may function in these cells.     

Total Synthesis of Acetate from CO2: Methyltetrahydrofolate, an Intermediate, and a Procedure for Separation of the Folates

Whole-cell preparations of Clostridium thermoaceticum were exposed to a short pulse of (14)CO(2) under conditions in which double-labeled acetate was synthesized. Radioactive methyltetrahydrofolate monoglutamate, diglutamate, and triglutamates were isolated from extracts of the cells. The radioactivity was found to be exclusively in the five methyl position. The specific activities of the methyltetrahydrofolate derivatives were very high and were in accord with the proposal that methyltetrahydrofolates are the precursors of the methyl of acetate. A new method of separation of folates employing QAE-Sephadex chromatography and a linear gradient with triethylammonium bicarbonate is presented which completely resolves the common folate monoglutamates and, upon freeze-drying, yields salt-free preparations.     

Folate metabolism in plants: an Arabidopsis homolog of the mammalian mitochondrial folate transporter mediates folate import into chloroplasts

The distribution of folates in plant cells suggests a complex traffic of the vitamin between the organelles and the cytosol. The Arabidopsis thaliana protein AtFOLT1 encoded by the At5g66380 gene is the closest homolog of the mitochondrial folate transporters (MFTs) characterized in mammalian cells. AtFOLT1 belongs to the mitochondrial carrier family, but GFP-tagging experiments and Western blot analyses indicated that it is targeted to the envelope of chloroplasts. By using the glycine auxotroph Chinese hamster ovary glyB cell line, which lacks a functional MFT and is deficient in folates transport into mitochondria, we showed by complementation that AtFOLT1 functions as a folate transporter in a hamster background. Indeed, stable transfectants bearing the AtFOLT1 cDNA have enhanced levels of folates in mitochondria and can support growth in glycine-free medium. Also, the expression of AtFOLT1 in Escherichia coli allows bacterial cells to uptake exogenous folate. Disruption of the AtFOLT1 gene in Arabidopsis does not lead to phenotypic alterations in folate-sufficient or folate-deficient plants. Also, the atfolt1 null mutant contains wild-type levels of folates in chloroplasts and preserves the enzymatic capacity to catalyze folate-dependent reactions in this subcellular compartment. These findings suggest strongly that, despite many common features shared by chloroplasts and mitochondria from mammals regarding folate metabolism, the folate import mechanisms in these organelles are not equivalent: folate uptake by mammalian mitochondria is mediated by a unique transporter, whereas there are alternative routes for folate import into chloroplasts.     

DNA damage and cell death induced by 1,2-dibromo-3-chloropropane (DBCP) and structural analogs in monolayer culture of rat hepatocytes: 3-aminobenzamide inhibits the toxicity of DBCP

1,2-Dibromo-3-chloropropane (DBCP) and a number of halogenated propane analogs induced DNA damage in rat hepatocytes in vitro measured by an automated alkaline elution method. Short-term (2 hrs) cytotoxic effects of DBCP were not observed until the DBCP concentration exceeded 1 mM. The short-term cytotoxicity of all the DBCP analogs occurred in the same concentration range. Significant membrane damage, measured as cell detachment, was observed after extended exposure to lower concentrations of DBCP (100 M) for 20 hrs. The relative, delayed cytotoxic effect of DBCP and analogs correlated with their ability to cause DNA damage. In general, the halogenated propanes with more bromines relative to chlorines were the more potent compounds. Propane analogs lacking the third halogen had little cytotoxic activity. The addition of the proposed specific poly(ADP-ribosyl)transferase inhibitor 3-aminobenzamide (3-ABA) protected against DBCP-induced cytotoxic effects and NAD⁺ depletion. However, 3-ABA also reduced DBCP-induced DNA damage, DBCP metabolic loss, and the formation of water soluble and covalently bound DBCP metabolites. Thus, 3-ABA may block DBCP-induced cell death by decreasing the formation of reactive DBCP-metabolites.Abbreviations 3-ABA 3-aminobenzamide - 3-AB acid 3-aminobenzoic acid - Asc ascorbate - BSA bovine serum albumin - DBCP, 1,2-diB-3-CP 1,2-dibromo-3-chloropropane - DMSO dimethylsulfoxide - DPPD N,N-diphenyl-p-phenylenediamine - GSH glutathione - Hoechst 33258 [2(2-(4-hydroxy-phenyl)-6-benzimidazole-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride)] - 1,2,3-triBP 1,2,3-tribromopropane - 1,3-diB-2-CP 1,3-dibromo-2-chloropropane - 1,3-diC-2-BP 1,3-dichloro-2-bromopropane - 1,2,3-triCP 1,2,3-trichloropropane - 1,2-diBP 1,2-dibromopropane     

A mutation inactivating the mitochondrial inner membrane folate transporter creates a glycine requirement for survival of chinese hamster cells

A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284-1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.     

Identification of poly-gamma-glutamyl chain lengths in folates of Bacillus subtilis.

Bacillus subtilis strains 168 met ile leu and 23 thy contain folates which differ from one another in the number of glutamyl residues. The folate species were identified by reductive cleavage to the corresponding p-aminobenzoylglutamyl poly-gamma-glutamates and chromatography on diethylaminoethyl-cellulose. Pteroyltriglutamate is the predominant folate type, accounting for 86 to 88% of the total. Pteroyltetraglutamate is the only other type present in appreciable quantities, accounting for 5 to 6% of the total folates. Pteroyldiglutamate and pteroylpentaglutamate are present in small amounts, accounting for 1 to 3% and 1% of the total folates, respectively. Strain 168 met ile leu contains a very small amount of pteroylmonoglutamate (less than 0.5% of the total folates), but the other strain contains none.     

Mutagenicity of 1,2-dibromo-3-chloropropane (DBCP) in the mouse spot test

The spot test with 1,2-dibromo-3-chloropropane (DBCP) was carried out using male PW and female C57BL/6 mice. DBCP induced recessive colour spots in offspring with a significantly high frequency of 2.9%, showing that this chemical is mutagenic for somatic cells of mice in vivo.     

Subcellular localization of gamma-glutamyl carboxypeptidase and of folates.

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [3H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions. The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85--90% folate polyglutamates. Injected [3H] folic acid was initially concentrated in the microsomal fraction, as measured by 3h cpm per ng folate. Initially, injected [3H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total 3H cpm determined to be [3H] folate penta- and hexaglutamates. At 24 h, the conversion of [3H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates. Injected [3H] folic acid after 2h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.     

Evaluation of the Protective and Therapeutic Properties of DBCP for Control of Root-Knot Nematode on Tomato

Twelve soil drenches over a period of 30 days with DBCP concentrations of 40 μg/ml did not completely prevent infection of tomato plants by root-knot nematode juveniles. Repeated DBCP drenches of 40 μg/ml halted gall development during the drenches, but 10 days after drenching was discontinued galls were apparent. DBCP drenches at 200 μg/ml prevented tomato root development, and 40 μg/ml slowed it. Ten μg/ml increased the height of root-knot-infected plants, but not their top weights. Treated plants were lanky. Protective drenches of 2.5 to 40 μg/ml of DBCP decreased nematode populations and increased fruitfulness. DBCP as a therapeutant reduced the incidence of galling on new roots and halted increases in gall size on previously infected roots but did not improve fruitfulness or plant size significantly.     

Controlled modulation of folate polyglutamyl tail length by metabolic engineering of Lactococcus lactis

The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates >90% of the produced folate intracellularly, predominantly in the polyglutamyl form. Approximately 10% of the produced folate is released into the environment. Overexpression of folC in L. lactis led to an increase in the length of the polyglutamyl tail from the predominant 4, 5, and 6 glutamate residues in wild-type cells to a maximum of 12 glutamate residues in the folate synthetase overproducer and resulted in a complete retention of folate in the cells. Overexpression of folKE, encoding the bifunctional protein 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and GTP-cyclohydrolase I, resulted in reduction of the average polyglutamyl tail length, leading to enhanced excretion of folate. By simultaneous overexpression of folKE and folC, encoding the enzyme folate synthetase or polyglutamyl folate synthetase, the average polyglutamyl tail length was increased, again resulting in normal wild-type distribution of folate. The production of bioavailable monoglutamyl folate and almost complete release of folate from the bacterium was achieved by expressing the gene for gamma-glutamyl hydrolase from human or rat origin. These engineering studies clearly establish the role of the polyglutamyl tail length in intracellular retention of the folate produced. Also, the potential application of engineered food microbes producing folates with different tail lengths is discussed.