Molecular cloning and characterization of NAD(+) dependent isocitrate dehydrogenase enzyme from Shewanella putrefaciens

Isocitrate dehydrogenase (IDH) is a fundamental enzyme for carbon metabolism in the Krebs cycle. This enzyme is required for oxidation-reduction reactions in both eukaryotic and prokaryotic cells and plays a critical role in their growth and pathogenesis. In this study, we cloned the gene encoding NAD(+) dependent isocitrate dehydrogenase from Shewanella putrefaciens. The expression of recombinant protein was induced with 0.5 mM of IPTG. His-tagged IDH overexpressed in E. coli was purified and characterized. The expressed IDH enzyme was purified in an active soluble form. The molecular weight of the enzyme was confirmed with Western blotting. High sequence homology was observed with IDH sequences of other Shewanella strains and remarkable sequence homology was found with other bacteria reported in the database

The influence of GSTM1 null genotype on susceptibility to in vitro oxidative stress

From epidemiological studies, there is some evidence that genetic variation at the glutathione S-transferase (CST) loci GSTM1 influences individual susceptibility to disease associated with oxidative stress. The aim of this study was to elucidate the role of the GSTM1 genotype in protection against oxidant chemicals by comparing the sensitivity, genotoxicity and cytotoxicity of lymphocytes to benzo(a)pyrene (BaP)- and cumene hydroperoxide (CumOOH)-induced in vitro oxidative challenge. Malondialdehyde and protein carbonyl levels, and oxidation of 2',7'-dichlorofluorescin diacetate were used as biomarkers of oxidative stress in lymphocytes. Following supplementation with BaP or CumOOH, time-dependent increases were observed in the production of all the markers after incubation for 12-48 h. However, we could not find any differences between GSTM1 null and positive genotypes. Furthermore, dose or time response experiments indicated that GSTM1-deficient cells were not more sensitive than control cells to BaP-or CumOOH-induced cell killing and micronucleus formation, although they were hypersensitive to BaP-inhibited cellular growth. The results suggest that lymphocytes from individuals with the GSTM1 null genotype are not abnormally susceptible to in vitro induced oxidant challenge, when exposed to CumOOH. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved

Inhibition of platelet function by GSTM1-null human peripheral lymphocytes exposed to benzo(a)pyrene-induced challenge

Recent epidemiological studies proposed that the glutathione S-transferase (GST) M1-null genotype may contribute to diseases associated with oxidative stress. The genetic polymorphism exhibited by the GSTM1 may be an important factor in risk toward oxidant chemicals. In this study, we investigated the effect of GSTM1-null genotype in lymphocyte and oxidative stress-dependent inhibition of platelet aggregation. To determine whether GSTM1 deficiency is a genetic determinant of cell toxicity toward oxidant chemicals, lymphocytes were incubated in vitro with low levels of benzo(a)pyrene (BaP), cumene hydroperoxide (CumOOH), or trans-stilbene oxide that do not decrease cell viability, and were assessed for oxidative damage and for the lymphocyte-dependent inhibition of platelet response. Malondialdehyde and carbonyl levels, and the oxidation of cis-parinaric acid, were used as biomarkers of oxidative stress in lymphocytes. Following stimulation by BaP or CumOOH, when peroxidation-dependent changes in these parameters were compared between the GSTM1-null genotype and the positive genotype, no significant differences were found between the two genotypes. On the other hand, preincubation of the lymphocytes with BaP or CumOOH attenuated their inhibitory action on ADP-induced platelet aggregation. However, our results indicate that lymphocytes of individuals with the GSTM1-null genotype have greater inhibitory activity on platelet function after exposure to BaP, but not CumOOH, although they are not more susceptible to in vitro oxidative stress

Transport of glutathione conjugate in erythrocytes from aged subjects and susceptibility to oxidative stress following inhibition of the glutathione S-conjugate pump

The aim of the present study was to investigate the effect of donor aging on the glutathione conjugate transport in erythrocytes and whether it plays a role in the resistance to oxidative stress of the erythrocytes of aging subjects. In our comparative study on intact erythrocytes of healthy aging and young adults, in which 2,4-dinitrophenyl-S-glutathione (DNP-SG) was used as model glutathione S-conjugate, we found that the efflux of DNP-SG remained unchanged in the aged subjects. This result suggests that the detoxification function is maintained against the chemical stress employed in erythrocytes of aging subjects. In the assay conditions used, which were optimized to obtain maximal inhibition of glutathione S-conjugate transport, our results also indicated that the susceptibility of erythrocytes to in vitro lipid peroxidation generated by cumene hydroperoxide was enhanced by pretreatment with DNP-SG inhibitors in both age groups. However, the difference in susceptibility was not a function of aging. Further, the results suggested that inhibition of glutathione S-conjugate pump may impair cellular protection of the erythrocytes against oxidative damage. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved

The investigation of GSTT1, GSTM1 and SOD polymorphism in bladder cancer patients

Glutathione S transferases (GSTT1, GSTM1, GSTP1) are enzymes that activate the detoxification of endogenous and exogenous agents. The genetic polymorphism in these genes may change the response of individuals to environmental toxicants. The genetic polymorphisms of GSTT1, GSTM1, GSTP1 have been studied extensively in the determination of individual cancer risks. Some studies showed a strong relationship between polymorphism of GSTs and superoxidedismutase enzymes. Using the polymerase chain reaction (PCR) the prevalence of genetic polymorphisms of GSTT1, GSTM1 and MnSOD (Manganese Superoxide Dismurase) was investigated in 104 cases and controls to seek any association with the risk of bladder cancer. The frequency of GSTT1 +/+ polymorphism was 65% (33/51) in the cases and 79% (42/53) in the controls. The frequency of the GSTM1 +/+ polymorphism was 33% (17/51) in the cases and 58% (31/53) in the controls. The frequency of the GSTM1 null genotype was 42% (22/53) in the controls and 68% (34/51) in the patients. The frequency of the SOD AA genotype was 36% (17/51) in the cases and 33% (19/53) in the controls. There was no association between the GSTT1 and SOD polymorphism and bladder cancer incidence. The incidence of the GSTM1 null genotype was increased in bladder cancer patients compared to controls (OR = 1.755, 95% CI = 1.119-2.751)

Glutathione-S-transferase M1 and T1 genetic polymorphisms and the risk of cataract development: A study in the Turkish population

In this study, we aimed to determine the effects of genetic polymorphisms of glutathione-S-transferase M1 (GSTM1) and glutathione-S-transferase T1 (GSTT1) on risk of developing different subtypes of age-related cataract in the Turkish population. Using a multiplex polymerase chain reaction (PCR), GSTM1 and GSTT1 gene polymorphisms were analyzed in 195 patients with age-related cataract (75 patients with cortical, 53 with nuclear, 37 with posterior subcapsular, and 30 with mixed type) and in 136 patients of an otherwise healthy control group of similar age. GSTM1 null genotype had a significant association with the development of cataract in female subjects (p <0.0029; OR, 2.98; 95% CI, 1.41-6.34). This relationship in female subjects was only in nuclear and mixed types cataract cases (p < 0.002; OR, 4.58; 95% CI, 1.67-12.78 and p < 0.03, respectively). There was also a statistically significant association between the combination of GSTM1-null and GSTT1-positive genotypes and the risk of cataract development in female subjects (p = 0.01; OR = 2.87; 95% CI = 1.25-6.69). Stratification by the subtypes revealed that this association was only in nuclear type cataract (p = 0.001; OR, 3.92; 95% CI, 1.34-11.71). GSTM1-null genotype or combination of the GSTM1-null and GSTT1-positive genotypes in females may be associated with increased risk of cataract development in the Turkish population