Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned cDNA for leukotriene B4 12-hydroxydehydrogenase (LTB4 12-HD)/15-ketoprostaglandin 13-reductase (PGR) from guinea-pig liver. LTB4 12-HD catalyzes the conversion of LTB4 into 12-keto-LTB4 in the presence of NADP+, and plays an important role in inactivating LTB4. The cDNA contained an ORF of 987 bp that encodes a protein of 329 amino-acid residues with a 78% identity with porcine LTB4 12-HD. The amino acids in the putative NAD+/NADP+ binding domain are well conserved among the pig, guinea-pig, human, rat, and rabbit enzymes. The guinea-pig LTB4 12-HD (gpLTB4 12-HD) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli, which exhibited similar enzyme activities to porcine LTB4 12-HD. We examined the 15-ketoprostaglandin 13-reductase (PGR) activity of recombinant gpLTB4 12-HD, and confirmed that the Kcat of the PGR activity is higher than that of LTB4 12-HD activity by 200-fold. Northern and Western blot analyses revealed that gpLTB4 12-HD/PGR is widely expressed in guinea-pig tissues such as liver, kidney, small intestine, spleen, and stomach. We carried out immunohistochemical analyses of this enzyme in various guinea-pig tissues. Epithelial cells of calyx and collecting tubules in kidney, epithelial cells of airway, alveoli, epithelial cells in small intestine and stomach, and hepatocytes were found to express the enzyme. These findings will lead to the identification of the unrevealed roles of PGs and LTs in these tissues.
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PMID:Immunohistochemical localization of guinea-pig leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase. 1173 4

The etiology of acute myeloid leukemia (AML) is largely unknown. Biologic and epidemiologic data implicate exogenous toxicants, including cytotoxic drugs, benzene, radiation, and cigarette smoking. Allelic variation in genes encoding enzymes such as NADP(H) quinone oxidoreductase (NQO1) and glutathione S-transferase T1 (GSTT1) that metabolize environmental toxicants predispose to subtypes of AML, including therapy-related AML. We assayed NRAS oncogene mutation and FLT3 internal tandem duplication in 447 AML patients with an abnormal karyotype treated in Medical Research Council (MRC) AML clinical trials. Functional allelic variant frequencies in genes encoding carcinogen-metabolizing enzymes GSTT1, GSTM1, CYP1A1, CYP2D6, CYP2C19, SULT1A1, and NQO1 were previously determined for this cohort. FLT3 internal tandem duplication (ITD) frequency was 17%, and NRAS mutation 12% for the entire cohort. The 2 mutations were found together in only 4 patients. No association was found between enzyme allelic variant frequencies and the presence of FLT3 ITD for the entire cohort or within cytogenetic subgroups. CYP1A1*2B (Val) high-inducibility variant allele was overrepresented in patients with NRAS mutation compared with no mutation, for (1) the entire AML cohort (n = 8/53 vs 26/371; odds ratio [OR] = 2.36; 95% confidence interval [CI] 1.01-5.53) and (2) the poor-risk karyotype group (n = 6/14 vs 4/89; OR = 15.94; 95% CI 3.71-68.52) comprising patients with partial/complete deletion of chromosome 5 or 7, or abnormalities of chromosome 3. The CYP1A1*2B allele may predispose to the development of these subgroups of AML by augmented phase 1 metabolism to highly reactive intermediates of CYP1A1 substrates, including polycyclic aromatic hydrocarbons, or by generation of oxidative stress as a metabolic by-product.
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PMID:CYP1A1*2B (Val) allele is overrepresented in a subgroup of acute myeloid leukemia patients with poor-risk karyotype associated with NRAS mutation, but not associated with FLT3 internal tandem duplication. 1246 38

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.
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PMID:Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase. 1459 57

The in vitro metabolism of 3,4-dihydro-6-hydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran (CR-6), a potent lipid peroxidation inhibitor and scavenger of nitric oxide and peroxynitrite species that is currently in phase II trials for antitumoral therapy, has been investigated in rat liver microsomes in the presence of NADP(H). Five major metabolites were identified by comparison with authentic standards, namely, the quinone 2-(3'-hydroxy-3'-methylbutyl-5-methoxy-1,4-benzoquinone (2a) and its ring-closed spiro form oxaspiro[4.5]-2,2-dimethyl-8-methoxy-dec-8-ene-7,10-dione (2b), the hydroquinone 2-(3'-hydroxy-3'-methylbutyl)-5-methoxyhydroquinone (3), the hydroxylated metabolite 3,4-dihydro-4,6-dihydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran (4), and the catechol 3,4-dihydro-6,7-dihydroxy-2,2-dimethyl-1(2H)-benzopyran (5). When the incubations were carried out in the presence of GSH, the HPLC peaks corresponding to the quinone metabolites 2a/b were absent and two novel products were formed showing MS fragmentation patterns consistent with the structure of GSH conjugates of quinone 2a. The time dependence on the formation of metabolites 2a,b and 3 was measured in incubations induced with phenobarbital (PB), dexamethasone, and beta-naphthoflavone (betaNF). For the dexamethasone-induced microsomes, the amount of hydroquinone 3 decreased from minute 10 to minute 30 while that of 2a,b increased in a complementary manner. Similar effects were observed for the incubations carried out using PB- and betaNF-induced microsomes. On the other hand, CR-6 inhibited 7-ethoxyresorufin O-dealkylation activity (IC(50) = 25 microM) in incubations with betaNF-induced microsomes. Likewise, addition of pentoxyresorufin to the incubations of CR-6 with PB-induced microsomes showed a time-dependent inhibition (IC(50)= 75 microM) of the dealkylation activity. These results are in agreement with the putative generation of reactive metabolites from CR-6 that could deactivate P450 1A and P450 2B, respectively. When these incubations were carried out in the presence of 10 mM GSH, the inhibition of P450 2B could be partially prevented. Finally, preincubation of CR-6 with liver microsomes from PB-induced rats resulted in a strong increase in microsomal glutathione S-transferase (mGST) activity (up to a maximum of approximately 5-fold). When the preincubation was carried out in the presence of 10 mM GSH, the activation of mGST was blocked. Overall, these results suggest that CR-6 undergoes in vitro biotransformation indicative of the involvement of thiol-reactive metabolites.
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PMID:In vitro biotransformation of 3,4-dihydro-6-hydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran (CR-6), a potent lipid peroxidation inhibitor and nitric oxide scavenger, in rat liver microsomes. 1525 15

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), which catalyzes the conversion of 2-C-methyl-D-erythritol cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP), is an essential enzyme of the non-mevalonate (2-C-methyl-D-erythritol-4-phosphate (MEP)) pathway for isoprenoid biosynthesis. The terminal steps of the MEP pathway are still not fully understood, although this pathway is necessary for survival in various organisms such as cyanobacteria, plastids of algae and higher plants, and the apicoplast of human malaria parasites. To determine the efficient redox partner for thermophilic cyanobacterial GcpE, We have expressed the gcpE and petF genes in Escherichia coli and studied the protein-protein interaction of GcpE protein with ferredoxin I (PetF) from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. Recombinant GcpE protein was purified by an N-terminal His(6) tag and reconstituted as a [4Fe-4S](2+) metalloprotein. GcpE was shown to interact strongly with PetF via the bacterial two-hybrid system designed to detect protein-protein interactions. Moreover, a direct protein-protein interaction between PetF and GcpE was confirmed in an in vitro glutathione S-transferase (GST) pull-down assay. To investigate electron transfer activity from PetF to GcpE, we also constructed a NADPH-dependent reducing shuttle system with purified recombinant ferredoxin-NADP(+) oxidoreductase (PetH) and PetF. The result demonstrated that PetF has the ability to transfer electrons to GcpE. Thus, the combined data provide the first evidence that GcpE is a ferredoxin-dependent enzyme in T. elongatus BP-1.
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PMID:Cyanobacterial non-mevalonate pathway: (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase interacts with ferredoxin in Thermosynechococcus elongatus BP-1. 1579 53

Inappropriate exposure of neonatal sheep to estrogen during critical developmental periods inhibits or retards endometrial gland morphogenesis and reduces uterine growth. Studies were conducted to identify mechanisms mediating estrogen disruption of neonatal ovine uterine development by analysis of candidate growth factor systems and using suppression subtraction hybridization (SSH). In study 1, sheep were exposed either to corn oil as a control or to estradiol valerate (EV) from birth to Postnatal Day (PND) 14, which ablated endometrial gland development. Estradiol valerate decreased uterine FGF7 (fibroblast growth factor 7) and MET (hepatocyte growth factor receptor) expression and increased INHBA (inhibin betaA). The SSH identified a number of genes responsive to EV, which included GSTM3 (glutathione S-transferase), IDH1 (cytosolic NADP-isocitrate dehydrogenase), PECI (peroxisomal D(3),D(2)-enoyl-coenzyme A isomerase), OAS1 (2',5'-oligoadenylate 40/46-kDa synthetase), IGFBP3 (insulin-like growth factor-binding protein-3), TEGT (testis-enhanced gene transcript), CXCL10 (interferon-gamma-inducible protein 10), and IGLV (immunoglobulin V). These mRNAs were expressed predominantly in the endometrial epithelia (GSTM3, IDH1, PEC1, OAS1, and TEGT), stroma (IGFBP3), or immune cells (CXCL10 and IGLV). In study 2, effects of estrogen exposure on uterine gene expression were determined during three different critical developmental periods (PNDs 0-14, 14- 28, and 42-56). Estrogen exposure decreased expression of the SSH-identified genes, particularly those from PNDs 0-14. These studies suggest that estrogen disruption of postnatal uterine development involves period-specific effects on expression of genes predominantly in the endometrial epithelium. The SSH-identified, estrogen-disrupted genes represent new candidate regulators of postnatal endometrial adenogenesis.
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PMID:Estrogen disruption of neonatal ovine uterine development: effects on gene expression assessed by suppression subtraction hybridization. 1597 82

NADP-malic enzymes (NADP-ME) are isozymes in plants. To clarify the diversity and function of NADP-ME isozymes in rice, we produced two active GST-fused NADP-ME proteins, NADP-ME2 and NADP-ME3 in Escherichia coli, and the fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column. After enzymatic cleavage of the GST tag, final yields were 1.4 mg/g wet cell weight (wcw) for NADP-ME2 and 3.5 mg/g wcw for NADP-ME3, respectively, and the molecular weights of NADP-ME2 and NADP-ME3 were about 65 and 62 kDa, respectively. The optimum pH is 7.3 for NADP-ME2 and 7.7 for NADP-ME3. The Km values for malate of NADP-ME2 and NADP-ME3 were 2.6 and 3.1 mM, whereas the Km values for NADP were 79 and 93 microM, respectively. The Kcat values of NADP-ME2 and NADP-ME3 for malate were about 91.7 and 96.7 s-1, respectively, and the Kcat values for NADP about 88.3 and 98.3 s-1, respectively. These results suggest that the two rice isozymes of NADP-ME in vitro have similar kinetic parameter.
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PMID:Expression, purification, and characterization of two NADP-malic enzymes of rice (Oryza sativa L.) in Escherichia coli. 1629 Jan 76

The levels of the enzymes, glutathione S-transferase, catalase, NAD(P)H-cytochrome c reductases, and DT-diaphorase were determined and compared in the tissues of three invertebrates commonly used in monitoring environmental quality: a freshwater mussel, Dreissena polymorpha, the earthworm Allolobophora chlorotica and the fourth instar of Chironomus riparius. It was found that the activities of GST, catalase, and NAD(P)-cytochrome c reductases were comparable in A. chlorotica and C. riparius, whereas comparatively a higher GST and a lower catalase activity was determined in the mussel tissues. DT-diaphorase was not detectable in A. chlorotica and the C. riparius larvae tissues, whereas this enzyme is present in the gills and the rest of soft mussel tissues (soft mussel tissues minus gills). It is suggested that the relatively low catalase activity observed in the tissues of the latter organism might be compensated by the presence of the antixidant role of DT-diaphorase. In addition, the inducibility of DT-diaphorase in D. polymorpha, by butylated hydroxyanisole (BHA) and lead (Pb) was investigated. Despite the bioaccumulation of both BHA (5.2+/-0.14 microgg(-1) wet weight) and Pb (233.7+/-0.95 mgkg(-1) dry weight) in the soft mussel tissues, the mussel DT-diaphorase was not induced. Although the activity of NADPH-cytochrome c (P-450) reductase was also not affected by these reagents, its activity was 2-fold higher in the gills than the rest of soft mussel tissues.
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PMID:Comparison of key enzymes in the zebra mussel, Dreissena polymorpha, the earthworm Allolobophora chlorotica and Chironomus riparius larvae. 1705 54

Stress plays a potential role in the onset and exacerbation of depression. Chronic restraint stress in rats, and psychosocial stress in humans, is implicated in the pathophysiology of mood and anxiety disorders. Oxidative damage is an established outcome of restraint stress, which has been suggested to induce many damaging processes contributing to the pathology of stress-induced depression. However, the modulatory role of clinically effective antidepressants, such as fluoxetine, in attenuating oxidative stress has not been well characterized. Therefore, the current study was designed to investigate the antioxidant effects of chronic treatment with fluoxetine in animals submitted to restraint stress. The antioxidant potential of the antidepressant fluoxetine was compared with that of turmeric, used as a standard since it integrates both antioxidant and antidepressant properties. Chronic fluoxetine administration to stressed animals for 21 days prevented restraint stress-induced oxidative damage with an efficacy similar to that of turmeric, as evidenced by significant enhancement of key endogenous antioxidant defense components, comprising the free-radical scavenging enzymes, superoxide:superoxide oxidoreductase (EC 1.15.1.1), hydrogen-peroxide:hydrogen-peroxide oxidoreductase (EC 1.11.1.6), glutathione S-transferase (EC 2.5.1.18) and glutathione:NADP(+)oxidoreductase (EC 1.8.1.7), as well as non-enzymatic antioxidants, GSH, glucose and uric acid, which were severely depleted by restraint stress in animals receiving no treatment. Oxidative stress markers, (S)-lactate:NAD(+) oxidoreductase activity (EC 1.1.1.27), malondialdehyde levels (lipid peroxidation product) and protein carbonyl content were also significantly decreased following fluoxetine treatment. Both these drugs when given alone to non-stressed animals did not alter basal levels of antioxidant defense components and oxidative stress markers significantly. Our findings suggest that the therapeutic efficacy of fluoxetine may be mediated, at least partially, via reversal of oxidative damage as demonstrated by protective enhancement of antioxidant status following a stress-induced decline. In addition, this study demonstrates important implications for pharmacological interventions targeting cellular antioxidants as a promising strategy for protecting against oxidative insults in stress-induced depression.
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PMID:Antioxidant potential of fluoxetine in comparison to Curcuma longa in restraint-stressed rats. 1761 Aug 75

Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Delta(l)-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley.
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PMID:Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage. 1956 Oct 48


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