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PubMed2001

Phylogenetic analyses and comparative genomics of vitamin B6 (pyridoxine) and pyridoxal phosphate biosynthesis pathways.

Vitamin B6 in its active form pyridoxal phosphate is an essential coenzyme of many diverse enzymes. Biochemistry, enzymology and genetics of de novo vitamin B6 biosynthesis have been primarily investigated in Escherichia coli. Database searches revealed that the key enzymes involved in ring closure of the aromatic pyridoxin ring (PdxA; PdxJ) are present mainly in genomes of bacteria constituting the gamma subdivision of proteobacteria. The distribution of DXS, a transketolase-like enzyme involved in vitamin B6 biosynthesis as well as in thiamine and isoprenoid biosynthesis and the distribution of vitamin B6 modifying enzymes (PdxH: oxidase; PdxK: kinase) was also analyzed. These enzymes are also present in the genomes of animals. Two recent papers (Ehrenshaft et al., 1999, Proc. Natl. Acad. Sci. USA. 96: 9374-9378; Osmani et al., 1999, J. Biol. Chem. 274: 23565-23569) show the involvement of an extremely conserved protein (a member of the UPF0019 or SNZ family) found in all three domains of life (bacteria, archaea, eukarya) in an alternative vitamin B6 biosynthesis pathway. Members of this family were previously identified as a stationary phase inducible protein in yeast, as an ethylene responsible protein in plants and in a marine sponge, as a singlet oxygen resistance protein in Cercospora nicotianae and as a cumene hydroperoxide and H2O2 inducible protein in Bacillus subtilis. In yeast, the SNZ protein interacts with another protein called SNO which also represents a member of a highly conserved protein family (called UPF0030 or SNO family). Phylogenetic trees for the DXS, PdxA, PdxJ, PdxH, PdxK, SNZ and SNO protein families are presented and possible implications of the two different vitamin B6 biosynthesis pathways in cellular metabolism are discussed. A radically different view of bacterial evolution (Gupta, 2000, Crit. Rev. Microbiol. 26: 111-131) which proposes a linear rather than a treelike evolutionary relationship between procaryotic species indicates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage. This proposal might help to explain why the PdxA/PdxJ pathway is largely restricted to this subdivision.

Bacterial Proteins↗

PubMedDate not supplied

[Biosynthesis of inulinases by Bacillus bacteria].

Biosynthesis of extracellular inulinase by bacteria Bacillus polymyxa 29, B. polymyxa 722, and B. subtilis 68 was studied. The optimal parameters for the producer growth were as follows: pH 7.0, 33-35 degrees C, the growth duration within 72 h. The presence of mineral reduced or of organic nitrogen was necessary for the enzyme biosynthesis. The inulinase biosynthesis was sharply activated in the presence of carbohydrates. B. polymyxa 722 and B. polymyxa 29 displayed the maximal activity on a starch-containing culture medium, the maximal activity of B. subtilis 68 was found in the presence of sucrose. Inulin did not induce the inulinase biosynthesis by the strains studied. The time course of bacteria growth and of the enzyme biosynthesis was studied.

Bacillus↗

PubMed2002

[Regulations and pathophysiological significance of the biosynthesis of tetrahydrobiopterin in human endothelial cells].

Tetrahydrobiopterin(BH4) serves as an essential cofactor for the biosynthesis of nitric oxide (NO). BH4 is de novo synthesized from GTP and GTP cyclohydrolase I(GCH I) is the rate-limiting enzyme in the biosynthesis of BH4. Under inflammatory conditions, it is reported that endothelial cells release large amount of BH4. In this study, we examined the regulation mechanism of the biosynthesis of BH4 in human umbilical vein endothelial cells(HUVEC). Prostacyclin and forskolin, reagents of stimulation of cAMP signaling cascade, reduced cytokine induced biosynthesis of BH4 through the inhibition of expression of GCH I mRNA. On the other hand, stimulations of NO-cGMP signaling pathway inhibited GCH I activities through the post translational modification of GCH I enzyme. Both two signaling cascade lead to vasodilation. It is suggested that the biosynthesis of BH4 can be regulated by negative feed back regulation systems between endothelium and smooth muscle cells to prevent over stimulated vasodilation.

Biopterins↗

PubMed1992

On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.

Animals↗

PubMed1976

Inhibition of prostaglandin biosynthesis by non-narcotic analgesic drugs.

The existence of a relationship between inhibition of prostaglandin biosynthesis and analgesic or anti-inflammatory activity was investigated in the case of the non-narcotic analgesics glafenine, floctafenine and clometacine, in comparison to indomethacin and acetylsalicylic acid. These compounds inhibit prostaglandin biosynthesis from arachidonic acid in a guinea-pig lung homogenate as strongly as indomethacin. On its biosynthesis in rat epididymal tissue stimulated by noradrenaline, glafenine equals indomethacin inhibitory potency, whereas floctafenine and clometacine are less active. Acetylsalicylic acid is the least active in both preparations. In vivo, prostaglandin biosynthesis induced in rat peritoneal fluid by injection of acetic acid is inhibited by the 5 drugs, ranked as follows: floctafenine greater than indomethacin greater than glafenine greater than clometacine greater than acetylsalicylic acid. The pharmacological profile of glafenine, floctafenine and clometacine is characterized by a relatively strong effect on acetic acid writhing and a relatively weak effect on carrageenin oedema, U.V. erythema and adjuvant arthritis. The inhibition of prostaglandin biosynthesis seems better correlated with their analgesic activity than with their anti-inflammatory effects. The results show that prostaglandins could play an important role in the genesis of tissulary pain in animals.

Acetates↗

PubMed1992

Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells.

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.

Aminocaproic Acid↗

PubMed1992

Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.

Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents.

Animals↗

PubMed1992

5-Lipoxygenase-activating protein is the target of a novel hybrid of two classes of leukotriene biosynthesis inhibitors.

An 18-kDa leukocyte membrane protein, termed 5-lipoxygenase-activating protein (FLAP), has recently been shown to be the target of two structurally distinct classes of leukotriene biosynthesis inhibitors. These classes of inhibitors are based on indole and quinoline structures and are represented by MK-886 and L-674,573, respectively. A novel class of hybrid structure based on the indole and quinoline classes of inhibitors, termed quindoles, has recently been developed. These compounds, exemplified by L-689,037, are potent inhibitors of leukotriene biosynthesis, both in vitro and in vivo. In the present study, we have developed and characterized a potent radioiodinated photoaffinity analogue of L-689,037, termed [125I]L-691,678. This compound was used in immunoprecipitation studies with FLAP antisera to show that the quindole series of leukotriene biosynthesis inhibitors interact directly with FLAP. In addition, we show that MK-886, L-674,573, and L-689,037 specifically compete, in a concentration-dependent manner, with both [125I]L-691,678 and [125I]L-669,083, a photoaffinity analogue of MK-886, for binding to FLAP. These results suggest that these three classes of leukotriene biosynthesis inhibitors share a common binding site on FLAP, providing further evidence that FLAP represents a suitable target for structurally diverse classes of leukotriene biosynthesis inhibitors.

5-Lipoxygenase-Activating Proteins↗

PubMed2004

Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp. strain SCRC-2738.

The 38 kb eicosapentaenoic acid (EPA) biosynthesis gene cluster of Shewanella sp. strain SCRC-2738 was cloned into the cosmid vector (pEPA). A 27 kb nucleotide sequence of the XhoI to SpeI region of pEPA showed EPA production (6.3%) in E. coli JM109. Among the nine open reading frames (ORFs) in this sequence, only five (ORFs 2 and 5-8) were essential for EPA production. High levels of production (16%-22%) were found in E. coli JM109 transformed with a multicopy pNEB vector carrying only the five essential ORFs and in that transformed with a pNEB vector that integrated ORFs 3, 5, 6, 7 and 8, and vector pSTV28 that integrated the ORF2 encoding phosphopantetheinyl transferase (PPTase). Thus, production of EPA appears to be regulated by the presence of all the biosynthesis gene products and by the ratio of PPTase to the other gene products. The temperature -EPA production relationship in E. coli strain DH5alpha varied between constructs, suggesting that it is controlled not only by EPA biosynthesis enzymes but also by other factors in vivo. There was a strict upper temperature limit for EPA biosynthesis: no EPA was synthesized at 30 degrees C in E. coli transformants carrying any gene construct for EPA biosynthesis.

Base Sequence↗

PubMed2005

[Regulatory effects of ammonium ions on the biosynthesis of meilingmycin].

Based on the effects of different ammonium sulfate concentrations on meilingmycin biosynthesis, the results show that lower concentration of ammonium ions stimulates the biosynthesis of meilingmycin, while the concentration of higher than 5mmol/L inhibits the mycelial growth and the biosynthesis of the products. However, the sugar consumption rate increases with the elevating concentration of ammonium sulfate. On this basis, six enzymes, which are greatly related to the meilingmycin biosynthesis and the glucose metabolism, were measured and analyzed during the meilingmycin fermentation process. The results suggest that glucose-6-phosphate dehydrogenase, citrate synthase, succinate dehydrogenase and fatty acid synthase are stimulated by higher concentration of ammonium ions, while valine dehydrogenase and methylmalonyl-CoA carboxyltransferase are inhibited. From the results it follows that ammonium ions favors primary metabolism, that is, the HMP passway and the TCA circle is enhanced, as well as the source of the precursors for the biosynthesis of meilingmycin is restricted, which bring about the lower production of meilingmycin.

Ammonium Sulfate↗

PubMed2005

Melanin potentiates kanamycin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

Ototoxicity is one of the well known side effects of kanamycin. The mechanism underlying the organ specificity of the side effect is not understood. Since many pharmacologic agents are known to form complexes with melanin and melanin is an abundant constituent of the inner ear, we investigated whether kanamycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. We found that kanamycin forms complexes with melanin. The amount of kanamycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the kanamycin-melanin complex formation: strong binding sites with the association constant K1 - 3 x 10(5) M(-1), and the weak binding sites with K2 - 4 x 10(3) M(-1). The number of total binding sites (n1 + n2) was calculated as about 0.64 micromol kanamycin per 1 mg melanin. We found that kanamycin induced inhibition of collagen and DNA biosynthesis (IC50 - 5 microM). Melanin at 100 microg/ml produced about 25% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to kanamycin-treated cells (5 microM) augmented the inhibitory action of kanamycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in cell growth and collagen metabolism, may be one of the targets for kanamycin-induced inhibition of these processes. As shown by Western immunoblot analysis melanin augmented kanamycin-induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2. The obtained results demonstrate that melanin potentiates the inhibitory effect of kanamycin on IGF-I receptor-dependent signaling pathway in cultured fibroblasts. The data suggest a potential mechanism for the organ specificity of kanamycin-induced hearing loss in patients which may result from melanin-induced augmentation of the inhibitory effects of kanamycin on collagen and DNA biosynthesis.

Anti-Bacterial Agents↗

PubMed1990

Inhibition by adenosine 3':5'-monophosphate of eicosanoid and platelet-activating factor biosynthesis in the mouse PT-18 mast cell.

A mouse spleen-derived mast cell line (PT-18) was employed to examine the mechanisms of adenosine 3':5'-monophosphate (cAMP)-mediated inhibition of antigen-induced lipid mediator biosynthesis. Specifically, we tested the hypothesis that increasing cAMP in mast cells inhibits lipid mediator biosynthesis by a mechanism independent of effects on histamine release (degranulation) or changes in cytosolic calcium concentration. Forskolin inhibited antigen-induced prostaglandin D2 (PGD2), leukotriene C4 (LTC4), and leukotriene B4 (LTB4) production by 30-50%. In contrast, forskolin had no inhibitory effect on antigen-induced increases in cytosolic calcium concentration, as monitored by the calcium indicator fura-2, or histamine release from the cells. The combination of the phosphodiesterase inhibitor isobutylmethylxanthine with forskolin inhibited the antigen-induced production of PGD2 and LTC4 by 90-100% and histamine release by about 60%. These responses were accompanied by a virtual abolition of the antigen-induced increase in cytosolic calcium. To test further the hypothesis that increasing cAMP can lead to inhibition of lipid mediator biosynthesis in the absence of effects on cytosolic calcium, we employed the calcium ionophores A23187 and ionomycin. Forskolin alone or in combination with isobutylmethylxanthine had no effect on ionophore-induced increases in cytosolic calcium but effectively inhibited leukotriene biosynthesis. In addition, increasing cyclic AMP led to an inhibition of ionophore-induced production of platelet-activating factor and liberation of arachidonic acid. These data suggest that a relatively modest increase in cAMP-dependent protein kinase activity in mast cells leads to inhibition of the lipase-catalyzed cleavage of arachidonic acid from membrane phospholipids in the absence of measurable effects on either histamine release or changes in cytosolic calcium concentration. This effect results in a selective inhibition of the biosynthesis of lipid mediators including LTC4, LTB4, PGD2, and platelet-activating factor.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Biosynthesis of fibronectin by rabbit aorta.

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied, fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased following a 24-h preincubation of the aortic rings, and concomitant increases in steady state mRNA for fibronectin were found. These in vitro studies documented the utility of aortic rings for the general purpose of studying protein synthesis in vascular cells and provide new information on the characteristics of fibronectin biosynthesis by aortic tissue.

Animals↗

PubMed1991

[Inhibitory effects of interferon and inhibitors of polyamine biosynthesis on clonal growth of Raji cells derived from Burkitt's lymphoma].

Interferon (IFN)-alpha, -gamma, and two inhibitors of polyamine biosynthesis (alpha-difluoromethyl ornithine; DFMO and methyl glyoxal bis-guanyl hydrazone; MGBG) inhibited the clonal growth of Raji cells. The combination-treatment using two agents, particularly IFN-alpha and DFMO represented synergistic effects. In order to investigate the mechanism of the synergism between IFNs and inhibitors of polyamine biosynthesis, the concentrations of polyamines and the levels of 2'-5' oligo-adenylate synthetase (2-5 AS) activity in the treated Raji cells were determined. Not only the inhibitors of polyamine biosynthesis, but also IFNs decreased the content of spermidine in the cells. Furthermore, the clonogenic growth-inhibition by IFN-alpha was recovered partially by exogenous spermidine. On the other hand, 2-5AS activity in the cells was elevated to 57 times by the treatment with interferon-alpha and further more, the combinations of IFN and the inhibitors of polyamine biosynthesis induced higher activities of 2-5AS as compared with those by each agent. From these results, it was suggested that the synergistic effects of IFNs in combinations with inhibitors of polyamine biosynthesis on the inhibition of clonal growth of Raji cells were closely associated with the decrease of intracellular polyamine level and the increase of 2-5AS activity in the treated cells.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol.

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.

Animals↗

PubMed1977

[Effect of some organosilicon compounds (silatranes) on the biosynthesis of collagen in cartilagenous tissue of chick embryo].

Effect of some organosilicon compounds from the class of silatranes on the biosynthesis of collagen in cartilagenous tissue of chick embryos in vitro was studied. The criteria for assessing the intensity of collagen biosynthesis was the formation of peptide bond 14C-hydroxyproline from 14C-proline of incubation medium. All the compounds studied (methylsilatrane, ethoxysilatrane and chloromethylsilatrane) stimulated total protein biosynthesis and collagen biosynthesis in cartilagenous tissue of chick embryos. The most pronounced stimulation of the biosynthesis of collagen was observed at 3-10(-3) M concentration of silatranes within 180 min. of incubation (14C-proline was added to the incubation medium 60 min after the beginning of the incubation). The activity of partly purified collagen prolyl-hydroxylase in the presence of silatranes was also studied.

Animals↗

PubMed1991

Stimulation of nerve growth factor biosynthesis in developing rat brain by reserpine: steroids as potential mediators.

The stimulation of beta-adrenergic receptors by isoproterenol increases nerve growth factor (NGF) biosynthesis in C6 rat glioma cells, suggesting that norepinephrine may regulate NGF biosynthesis in vivo. We have tested this hypothesis in 21-day-old rats by depleting catecholamine stores with reserpine. Northern blot analysis of NGF mRNA, in combination with a two-site enzyme immunoassay for NGF, showed that depletion of catecholamines was associated with a 3-fold increase in NGF mRNA, which was followed by a significant increase in the NGF content of cerebral cortex. The increase in NGF mRNA was most marked 9 hr after reserpine administration (2 mg/kg, subcutaneously) and was no longer apparent 24 hr after drug administration, when brain monoamine stores were still depleted. Moreover, the lowest dose of reserpine that significantly increased NGF mRNA levels induced only a small change in the content of cortical catecholamines. These results suggest that reserpine mediates the increase in NGF production by a mechanism other than monoamine depletion. Because reserpine increases plasma glucocorticoid concentrations through the pituitary-adrenal axis, we investigated whether adrenal steroids could be responsible for the induction of NGF biosynthesis. The effect of reserpine on NGF biosynthesis was abolished in adrenalectomized rats. Moreover, dexamethasone, a synthetic glucocorticoid, given at a dose of 0.5 mg/kg, subcutaneously, increased the amount of NGF mRNA and NGF in cerebral cortex. NGF biosynthesis in the central nervous system may, thus, be regulated by adrenocortical hormonal secretion.

Adrenal Cortex Hormones↗

PubMed1990

Role of substrate depletion in the inhibition of thymidylate biosynthesis by the dihydrofolate reductase inhibitor trimetrexate in cultured hepatoma cells.

The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH2H4PteGlun) from 2.0-0.2 microM, respectively. This is accompanied by an increase in H2PteGlun from 1.2 microM in control cultures to 4.7 microM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH2H4PteGlun in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH2H4PteGlun in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH2H4PteGlun) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H2PteGlun for thymidylate synthase Ki = 5.0 microM) suggest that H2PteGlun accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.

Carcinoma, Hepatocellular↗