Methylenetetrahydrofolate reductase gene polymorphisms and risk of acute lymphoblastic leukemia in children

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and is involved in DNA synthesis, DNA repair and DNA methylation. Genetic polymorphisms of this enzyme have been shown to impact several diseases, including cancer. Leukemias are malignancies arising from rapidly proliferating hematopoietic cells having great requirement of DNA synthesis. This case-control study was undertaken to analyze the association of the MTHFR gene polymorphisms 677 C"T and 1298 A"C and the risk of acute lymphoblastic leukemia in children. Materials and Methods: Eighty-six patients aged below 15 years with a confirmed diagnosis of acute lymphoblastic leukemia (ALL) and 99 matched controls were taken for this study. Analysis of the polymorphisms was done using the polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) method. Results: Frequency of MTHFR 677 CC and CT were 85.9% and 14.1% in the controls, and 84.9% and 15.1% in the cases. The 'T' allele frequency was 7% and 7.5% in cases and controls respectively. The frequency of MTHFR 1298 AA, AC, and CC were 28.3%, 55.6% and 16.1% for controls and 23.3%, 59.3% and 17.4% for cases respectively. The 'C' allele frequency for 1298 A→C was 43.9% and 47% respectively for controls and cases. The odds ratio (OR) for C677T was 1.08 (95% CI 0.48- 2.45, p = 0.851) and OR for A1298C was 1.29(95% CI 0.65-2.29, p = 0.46) and OR for 1298 CC was 1.31 (95% CI 0.53-3.26, p =0.56). The OR for the combined heterozygous status (677 CT and 1298 AC) was 1.94 (95% CI 0.58 -6.52, p = 0.286). Conclusion: The prevalence of 'T' allele for 677 MTHFR polymorphism was low in the population studied. There was no association between MTHFR 677 C→T and 1298 A→C gene polymorphisms and risk of ALL, which may be due to the small sample size.

Desensitization to ATP-Mg inhibition of 3-hydroxy-3-methylglutarylCoA reductase by heat treatment of microsomes

HMGCoA reductase is found to be inhibited by palmitylCoA and free CoA. The inhibition of this enzyme by ATP-Mg, but not by palmityl CoA, is lost on preincubation of microsomes at 50°C for 15 min.

Functional Mimics of Glutathione Peroxidase: Bioinspired Synthetic Antioxidants

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the biological system's ability to detoxify these reactive intermediates. Mammalian cells have elaborate antioxidant defense mechanisms to control the damaging effects of ROS. Glutathione peroxidase (GPx), a selenoenzyme, plays a key role in protecting the organism from oxidative damage by catalyzing the reduction of harmful hydroperoxides with thiol a ``catalytic triad'' with tryptophan and glutamine, which cofactors. The selenocysteine residue at the active site forms activates the selenium moiety for an efficient reduction of peroxides. After the discovery that ebselen, a synthetic organoselenium compound, mimics the catalytic activity of GPx both in vitro and in vivo, several research groups developed a number of small-molecule selenium compounds as functional mimics of GPx, either by modifying the basic structure of ebselen or by incorporating some structural features of the native enzyme. The synthetic mimics reported in the literature can be classified in three major categories: (i) cyclic selenenyl amides having a Se-N bond, (ii) diaryl diselenides, and (iii) aromatic or aliphatic monoselenides. Recent studies show that ebselen exhibits very poor GPx activity when aryl or benzylic thiols such as PhSH or BnSH are used as cosubstrates. Because the catalytic activity of each GPx mimic largely depends on the thiol cosubstrates used, the difference in the thiols causes the discrepancies observed in different studies. In this Account, we demonstrate the effect of amide and amine substituents on the GPx activity of various organoselenium compounds. The existence of strong Se ... O/N interactions in the selenenyl sulfide intermediates significantly reduces the GPx activity. These interactions facilitate an attack of thiol at selenium rather than at sulfur, leading to thiol exchange reactions that hamper the formation of catalytically active selenol. Therefore, any substituent capable of enhancing the nucleophilic attack of thiol at sulfur in the selenenyl sulfide state would enhance the antioxidant potency of organoselenium compounds. Interestingly, replacement of the sec-amide substituent by a tert-amide group leads to a weakening of Se ... 0 interactions in the selenenyl sulfide intermediates. This modification results in 10- to 20-fold enhancements in the catalytic activities. Another strategy involving the replacement of tert-amide moieties by tert-amino substituents further increases the activity by 3- to 4-fold. The most effective modification so far in benzylamine-based GPx mimics appears to be either the replacement of a tert-amino substituent by a sec-amino group or the introduction of an additional 6-methoxy group in the phenyl ring. These strategies can contribute to a remarkable enhancement in the GPx activity. In addition to enhancing catalytic activity, a change in the substituents near the selenium moiety alters the catalytic mechanisms. The mechanistic investigations of functional mimics are useful not only for understanding the complex chemistry at the active site of GPx but also for designing and synthesizing novel antioxidants and anti-inflammatory agents.

Developmental regulation of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) gene expression in rat liver

The expression of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes has been studied as a function of development in rat liver. The levels of cytochrome P-450 (b+e) mRNAs and their transcription rates are too low for detection in the 19-day old fetal liver before or after phenobarbitone treatment. However, glutathione transferase (Ya+Yc) mRNAs can be detected in the fetal liver as well as their induction after phenobarbitone treatment can be demonstrated. These mRNAs contents as well as their inducibility with phenobarbitone are lower in maternal liver than that of adult nonpregnant female rat liver. Steroid hormone administration to immature rats blocks substantially the phenobarbitone mediated induction of the two mRNA families as well as their transcription. It is suggested that steroid hormones constitute one of the factors responsible for the repression of the cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes in fetal liver.

Irreversible inactivation of 3-hydroxy-3-methylglutaryl-CoA reductase by H2O2

A concentration-dependent inactivation of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase was found on reincubation of rat liver microsomal preparations with H2O2 and at lower concentrations in the presence of KCN which inhibited the contaminating catalase. The inactivation was not affected in the presence quenchers of hydroxyl radicals and singlet oxygen and was also obtained when H2O2 was added during the reaction. HMG-CoA, but not NADPH, partially protected the enzyme from H2O2-inactivation. Even at high concentration DTT was unable to reverse this inactivation. The soluble 50 kDa-enzyme was similarly inactivated by H2O2, and the tryptic-digest of the inactivated protein indicated the presence of a disulfide-containing peptide. The results support the view that H2O2 by directly acting on the catalytic domain possibly converts an active thiol group to an inaccessible disulfide and irreversibly HMG-CoA reductase.

On the involvement of intramolecular protein disulfide in the irreversible inactivation of 3-hydroxy-3-methylglutaryl-CoA reductase by diallyl disulfide

Treatment with diallyl disulfide, a constituent of garlic oil, irreversibly inactivated microsomal and a soluble 50 kDa form of HMG-CoA reductase. No radioactivity was found to be protein-bound on treating the soluble enzyme with [35S]diallyl disulfide, indicating the absence of the mixed disulfide of the type allyl-S-S-protein. SDS-PAGE and Western blot analyses of the diallyl-disulfide-treated protein showed no traces of the dimer of the type protein-S-S-protein, but clearly indicated BME-reversible increased mobility, as expected of an intramolecular protein disulfide. The sulfhydryl groups, as measured by alkylation with iodo[2-14C]acetic acid, were found to decrease in the diallyl-disulfide-treated enzyme protein. Tryptic peptide analysis also gave support for the possible presence of disulfide-containing peptides in such a protein. It appears that diallyl disulfide inactivated HMG-CoA reductase by forming an internal protein disulfide that became inaccessible for reduction by DTT, and thereby retaining the inactive state of the enzyme.

Purification and Partial Characterization of Microsomal NADH-Cytochrome b5 Reductase from Higher Plant Catharanthus roseus

A simple three step procedure was used to purify microsomal NADH-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD+-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 μM), 2,6-dichlorophenolindophenol (Km 65 μM) and cytochrome Image via cytochrome b5 but not to menadione.

Effect of Substrate Binding Loop Mutations on the Structure, Kinetics, and Inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network. (C) 2011 IUBMB mum Life, 63(1): 30-41,2011

Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50kDa protein) to the membrane, present in the 58kDa-protein appears to be involved in mediating the inhibition by silatrane.

Glutathione peroxidase3 of Saccharomyces cerevisiae protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 mu M cadmium (Cd)-treatment, whereas an untreated gpx3 Delta strain showed a drastic reduction in overall phospholipids which was further reduced with 50 mu M Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3 Delta cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3 Delta. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3 Delta cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3 Delta cells during Cd-treatment.

Expression of the Japanese encephalitis virus NS3 and NS2b proteins as glutathione S-transferase fusions

Flaviviruses generate their structural and nonstructural proteins by proteolytic processing of a single large polyprotein precursor. These proteolytic events are brought about both by host cell signalase and a virally encoded protease. The virally encoded proteolytic activity has been shown to reside within the nonstructural protein 3 (NS3) and requires the product of the nonstructural 2b (NS2b) gene. In order to obtain sufficient quantities of pure NS2b and NS3 proteins for kinetic analysis, we have expressed both these proteins in recombinant systems as fusions to glutathione S-transferase (GST). The fusion constructs were driven by the strong bacteriophage T7 promoter. Transfection of these constructs into the African green monkey kidney cell line CV-1 previously infected with a recombinant vaccinia virus expressing the T7 RNA polymerase resulted in synthesis of the fusion proteins. Both the fusion proteins could be purified to homogeneity in a single step using a glutathione agarose affinity matrix.