Characterization of the first tetrameric transcription factor of the GntR superfamily with allosteric regulation from the bacterial pathogen Agrobacterium fabrum.

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

Folic acid, but not folate, regulates different stages of neurogenesis in the ventral hippocampus of adult female rats.

Folate is an important regulator of hippocampal neurogenesis, and folic acid is needed prenatally to reduce the risk of neural tube defects. Both high levels of folic acid and low levels of folate can be harmful to health because low levels of folate have been linked to several diseases while high folic acid supplements can mask a vitamin B12 deficiency. Depressed patients exhibit folate deficiencies, lower levels of hippocampal neurogenesis, elevated levels of homocysteine and elevated levels of the stress hormone, cortisol, which may be inter-related. In the present study, we were interested in whether different doses of natural folate or synthetic folic acid diets can influence neurogenesis in the hippocampus, levels of plasma homocysteine and serum corticosterone in adult female rats. Adult female Sprague-Dawley rats underwent dietary interventions for 29 days. Animals were randomly assigned to six different dietary groups: folate deficient + succinylsulphathiazole (SST), low 5-methyltetrahydrofolate (5-MTHF), low 5-MTHF + (SST), high 5-MTHF + SST, low folic acid and high folic acid. SST was added to a subset of the 5-MTHF diets to eliminate folic acid production in the gut. Before and after dietary treatment, blood samples were collected for corticosterone and homocysteine analysis, and brain tissue was collected for neurogenesis analysis. High folic acid and low 5-MTHF without SST increased the number of immature neurones (doublecortin-expressing cells) within the ventral hippocampus compared to folate deficient controls. Low 5-MTHF without SST significantly increased the number of immature neurones compared to low and high 5-MTHF + SST, indicating that SST interfered with elevations in neurogenesis. Low folic acid and high 5-MTHF + SST reduced plasma homocysteine levels compared to controls, although there was no significant effect of diet on serum corticosterone levels. In addition, low folic acid and high 5-MTHF + SST reduced the number of mature new neurones in the ventral hippocampus (bromodeoxyuridine/NeuN-positive cells) compared to folate deficient controls. Overall, folic acid dose-dependently influenced neurogenesis with low levels decreasing but high levels increasing neurogenesis in the ventral hippocampus, suggesting that this region, which is important for regulating stress, is particularly sensitive to folic acid in diets. Furthermore, the addition of SST negated the effects of 5-MTHF to increase neurogenesis in the ventral hippocampus.

Metabolic Intermediates in Tumorigenesis and Progression.

Traditional antitumor drugs inhibit the proliferation and metastasis of tumour cells by restraining the replication and expression of DNA. These drugs are usually highly cytotoxic. They kill tumour cells while also cause damage to normal cells at the same time, especially the hematopoietic cells that divide vigorously. Patients are exposed to other serious situations such as a severe infection caused by a decrease in the number of white blood cells. Energy metabolism is an essential process for the survival of all cells, but differs greatly between normal cells and tumour cells in metabolic pathways and metabolic intermediates. Whether this difference could be used as new therapeutic target while reducing damage to normal tissues is the topic of this paper. In this paper, we introduce five major metabolic intermediates in detail, including acetyl-CoA, SAM, FAD, NAD+ and THF. Their contents and functions in tumour cells and normal cells are significantly different. And the possible regulatory mechanisms that lead to these differences are proposed carefully. It is hoped that the key enzymes in these regulatory pathways could be used as new targets for tumour therapy.

Folates and S-adenosylmethionine for major depressive disorder.

Interest in nonpharmaceutical supplements for treating major depressive disorder (MDD) has increased significantly, both among patients and among clinicians during the past decades. Despite the large array of antidepressants (ADs) available, many patients continue to experience relatively modest response and remission rates, in addition to a burden of side effects that can hinder treatment compliance and acceptability. In this article, we review the literature on folates and S-adenosylmethionine (SAMe), 2 natural compounds linked in the 1-carbon cycle metabolic pathway, for which substantial evidence supports their involvement in mood disorders. Background information, efficacy data, proposed mechanisms of action, and side effects are reviewed. Based on existing data, supplementation with SAMe, as well as with various formulations of folates, appears to be efficacious and well tolerated in reducing depressive symptoms. Compared with other forms of folates, 5-methyltetrahydrofolate (L-methylfolate or 5-MTHF) may represent a preferable treatment option for MDD given its greater bioavailability in patients with a genetic polymorphism, and the lower risk of specific side effects associated with folic acid. Although further randomized controlled trials in this area appear warranted, SAMe and L-methylfolate may represent a useful addition to the AD armamentarium.

L-methylfolate: a vitamin for your monoamines.

Synthesis of the monoamine neurotransmitters serotonin, dopamine, and norepinephrine is regulated by L-methylfolate, a derivate of the vitamin folate.

Evaluation of an ex vivo model implication for carrier-mediated retinal drug delivery.

PURPOSE: The purpose of this study was to evaluate the implication of an ex vivo model for carrier-mediated retinal drug delivery using an Ussing chamber system. METHODS: Dutch Belted Pigmented rabbits weighing 2-2.5 kg were used in these studies. Excised posterior segment tissues (RPE-choroid-sclera and sclera), mounted on the Ussing chamber, were used as an ex vivo model. Transport studies were carried out across sclera and RPE-choroid-sclera (RCS) tissue preparations in the sclera to retina (S --> R) and retina to sclera (R --> S) directions for 3 hr at 37 degrees C. The model was validated by permeability studies with paracellular and transcellular markers ([(3)H]mannitol and [(14)C]diazepam, respectively), tissue viability studies (bioelectrical and biochemical assays), and tissue histology and electron microscopy studies. Functional presence of a carrier-mediated transport system for folic acid (folate receptor alpha) was investigated on the basolateral side of the rabbit retina. RESULTS: Results from bioelectrical, biochemical, and histological evaluation of tissue provide evidence that the RCS tissue preparation remains viable during the period of transport study. Permeability values of [(3)H]mannitol across sclera were 4.18 +/- 1.09 x 10(- 5) cm/s (R --> S) and 4.11 +/- 1.09 x 10(- 5) cm/s (S --> R) and across RCS were 1.77 +/- 0.31 x 10(- 5) cm/s (S --> R) and 1.60 +/- 0.19 x 10(- 5) cm/s (R --> S). Permeability values of [(14)C]diazepam across sclera were 2.37 +/- 0.38 x 10(- 5) cm/s (R --> S) and 2.70 +/- 0.70 x 10(- 5) cm/s (S --> R) and across RCS were 3.12 +/- 0.12 x 10(- 5) cm/s (R --> S) and 2.77 +/- 0.25 x 10(- 5)cm/s (S --> R). The rate of [(3)H]folic acid transport across RCS was found to be significantly higher in the S -->R direction (16.34 +/- 0.94 fmoles min(-1) cm(-2)) as compared with R --> S direction (9.38 +/- 1.44 fmoles min(-1) cm(-2)) and nearly 10-fold higher across sclera as compared with RCS in both directions. Transport of [(3)H]folic acid was found to be pH and temperature dependent and was inhibited by 44.5%, 35.1%, and 50.3% in the presence of unlabeled folic acid, 5-methyltetrahydrofolate (MTF), and Methotrexate (MTX). CONCLUSIONS: The RCS tissue preparation mounted on the Ussing chamber system, an ex vivo model, can be a useful tool for identification and characterization of carrier-mediated systems present on RPE (a major barrier for retinal drug delivery) and to study carrier-mediated retinal drug delivery via prodrug derivatization.

Modulation of potassium evoked secretory function in rat cerebellar slices measured by real time monitoring: evidence of a possible role for methylfolate in cerebral tissue.

The real time dynamics of K+ evoked neurosecretion in cerebellar slices has been monitored electrochemically. In the presence of 5-methyltetrahydrofolate a statistically significant diminution in secretory response occurs. Agonists to probe the pharmacological basis for this indicate it is not due to voltage sensitive Ca2+ channel blockade, nor does it show any similarity of effect with kainate, whose receptor is a putative binding site for 5-methyltetrahydrofolate. The method is fully validated, although no account is taken of individual molecular species. High performance liquid chromatography combined with off line microbiological assay could only detect 5-methyltetrahydrofolate in cerebrospinal fluid. We therefore discuss our findings in relation to possible cerebral roles for cerebrospinal fluid 5-methyltetrahydrofolate in the context of both membrane and transmitter related interactions.

Correction of the DNA synthesis defect in vitamin B12 deficiency by tetrahydrofolate: evidence in favour of the methyl-folate trap hypothesis as the cause of megaloblastic anaemia in vitamin B12 deficiency.

The critical disturbance of folate metabolism caused by vitamin B12 deficiency which results in megaloblastic anaemia remains controversial. Vitamin B12 is required in the methionine synthase reaction in which homocysteine is converted to methionine and methyl tetrahydrofolate (methyl THF) to THF. The 'methyl-folate trap' hypothesis suggested that failure of demethylation of methyl THF with consequent deficiency of folate co-enzymes derived from THF is the crucial lesion caused by vitamin B12 deficiency. A more recent theory suggested that reduced supply of methionine leads to reduced availability of 'activated formate' and hence of formyl THF and it is this defect that results in failure of folate co-enzyme synthesis. The present results, based on deoxyuridine suppression tests on 103 cases of megaloblastic anaemia, show that THF itself is equally capable of correcting the failure of thymidylate synthesis in vitamin B12 deficiency as in folate deficiency. Although not as effective as formyl THF in correcting the dU blocking test in vitamin B12 deficiency, this is equally so for the correction of the test by THF compared with formyl THF in folate deficiency. The results therefore favour the theory that it is in the supply of THF and not of 'active formate' or formyl THF that vitamin B12 plays a critical role in folate metabolism.

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase.

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.

Substrate activity of synthetic formyl phosphate in the reaction catalyzed by formyltetrahydrofolate synthetase.

Formyl phosphate, a putative enzyme-bound intermediate in the reaction catalyzed by formyltetrahydrofolate synthetase (EC 6.3.4.3), was synthesized from formyl fluoride and inorganic phosphate [Jaenicke, L. v., & Koch, J. (1963) Justus Liebigs Ann. Chem. 663, 50-58], and the product was characterized by 31P, 1H, and 13C nuclear magnetic resonance (NMR). Measurement of hydrolysis rates by 31P NMR indicates that formyl phosphate is particularly labile, with a half-life of 48 min in a buffered neutral solution at 20 degrees C. At pH 7, hydrolysis occurs with P-O bond cleavage, as demonstrated by 18O incorporation from H2(18)O into Pi, while at pH 1 and pH 13 hydrolysis occurs with C-O bond cleavage. The substrate activity of formyl phosphate was tested in the reaction catalyzed by formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum. Formyl phosphate supports the reaction in both the forward and reverse directions. Thus, N10-formyltetrahydrofolate is produced from tetrahydrofolate and formyl phosphate in a reaction mixture that contains enzyme, Mg(II), and ADP, and ATP is produced from formyl phosphate and ADP with enzyme, Mg(II), and tetrahydrofolate present. The requirements for ADP and for tetrahydrofolate as cofactors in these reactions are consistent with previous steady-state kinetic and isotope exchange studies, which demonstrated that all substrate subsites must be occupied prior to catalysis. The k cat values for both the forward and reverse directions, with formyl phosphate as the substrate, are much lower than those for the normal forward and reverse reactions. Kinetic analysis of the formyl phosphate supported reactions indicates that the low steady-state rates observed for the synthetic intermediate are most likely due to the sequential nature of the normal reaction.

Altered methionine metabolism and transmethylation in cancer.

Methionine metabolism and transmethylation are frequently altered in cancer cells. The alteration is often expressed as an inability of the cancer cells to grow when methionine is replaced by homocysteine in the culture medium, a condition that allows the growth of normal cells. This metabolic defect is termed methionine dependence. Methionine dependence may reflect an overall imbalance in transmethylation which results in the overmethylation of some substances and undermethylation of others within cancer cells. Many carcinogens affect various stages of methionine/transmethylation metabolism. The ultimate effect of the alteration of methionine/transmethylation metabolism may be the disruption of the regulation of genes involved in the oncogenic process. The known protective effect of methionine against cancer may be due to prevention of altered methionine/transmethylation metabolism or compensation of the altered metabolism.

Ontogenetic development of a 5-methyltetrahydrofolate requiring enzyme in rats and mice.

Developmental characteristics of 5-methyltetrahydrofolate requiring N-methyltransferase (5 MT-NMT) have been studied in rats and mice and compared to the development pattern of marker enzymes. In rat and mouse brain, the 5 MT-NMT activity expressed in units per mg protein, was found to decrease rapidly during the first five days after birth and then more slowly, whereas two other enzymes, dopa-decarboxylase and histamine N-methyltransferase increased gradually during maturation. In kidney, the ontogenetic development of 5 MT-NMT was first characterized by an increasing activity from birth until a different age, depending on the species, and then followed by an abrupt decline. In contrast to this, histamine N-methyltransferase activity in mouse kidney was about 60 times higher at maturity than at birth. When the enzyme activities are expressed in units per g tissue, the changes in the course of the development of 5 MT-NMT were less apparent, but in any way differed markedly from those of dopa-decarboxylase and histamine N-methyltransferase. The results suggest that 5 MT-NMT does not behave as a synaptosomal or perhaps even as a neuronal enzyme. Although its physiological role remains to be elucidated, it must have a more general function in the cellular economy than merely N-methylating biogenic amines.