• BMB Reports
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• v.31 no.3
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• pp.287-295
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• 1998

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-Ieucine, L-isoleucine, and L-valine. The sulfonylurea-resistant ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS3 was used to transform Escherichia coli strain XL1-Blue, and the mutant tobacco ALS (mALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-mALS was purified in a single step on a glutathione-Sepharose column. ALS activities of 0.9-2.5 ${\mu}mol/min/mg$ protein were observed in the GST-mALS, and the Km values for pyruvate, FAD, and TPP were 10.8-24.1, $(1.9-8.9){\times}10^{-3}$, and 0.14-0.38 mM, respectively. The purified GST-mALS was resistant to both the sulfonylurea and the triazolopyrimidine herbicides, and lost its sensitivity to end products, L-valine and L-leucine. For comparision, the tobacco wild-type recombinant ALS fused with GST, GST-wALS, was also characterized with respect to its pyruvate and cofactor bindings. These results suggest that the purified mutant recombinant tobacco ALS was functionally active, that the mutations resulting in herbicide resistance has affected pyruvate and cofactor bindings," and that the two classes of herbicides interact at a common site on the plant ALS.

• Horticultural Science & Technology
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• v.29 no.6
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• pp.623-632
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• 2011

To increase the anti-carcinogens phenylethylisothiocyanate (PEITC), myrosinase (MYR), and glutathione S-transferase (GST), genes related to PEITC pathway were isolated and the gene expressions were regulated by Agrobacterium transformation. Isolated cDNAs, MYR, and GST genes were 1,647 bp and 624 bp, respectively, and the protein expression was confirmed through pET system. Thereafter, we constructed a sense-oriented over-expressing myrosinase (pBMY) and RNAi down-regulated GST (pJJGST) binary vectors for the Chinese cabbage transformation. After the transformation, thirteen over-expressing transgenic Chinese cabbage plants (IMS) with pBMY and five down-regulated ones (IGA) with pJJGST were selected by PCR analysis. Selected $T_0$ transgenic plants were generated to $T_1$ plants by self-pollination. Based on the Southern blot analysis on these $T_1$ transgenic plants, 1-4 copies of T-DNA were transferred to Chinese cabbage genome. Thereafter, RNA expression level of myrosinase gene or GST gene was analyzed through real-time RT PCR of IMS, IGA, and non-transgenic inbred lines. In case of IMS lines, myrosinase gene was increased 1.03-4.25 fold and, in IGA lines, GST gene was decreased by 26.42-42.22 fold compared to non-transgenic ones, respectively. Analysis of PEITC concentrations using GC-MS it showed that some IMS lines and some IGA lines increased concentrations of PEITC up to 4.86 fold and up to 3.89 fold respectively compared to wild type. Finally in this study IMS 1, 3, 5, 12, and 15 and IGA 1, 2, and 4 were selected as developed transgenic lines with increasing quantities of anti-carcinogen PEITC.

• Animal cells and systems
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• v.12 no.3
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• pp.149-155
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• 2008

We have isolated and characterized Agrius convolvuli cDNA encoding a c-type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino-terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21(DE3) pLysS cells for pGEX 4T-1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T-1 vector, which contained the glutathione S-transferase(GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS-PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion-Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti-Agrius lysozyme.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2075-2081
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• 2014

The association between glutathione-S-transferase polymorphisms (GSTM1, GSTT1 and GSTP1) and risk of acute leukemia in Asians remains controversial. This study was therefore designed to evaluate the precise association in 23 studies identified by a search of PubMed and several other databases, up to December 2013. Using random or fixed effects models odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated. Heterogeneity across studies was assessed, and funnel plots were constructed to test for publication bias. The meta-analysis showed positive associations between GST polymorphisms (GSTM1 and GSTT1 but not GSTP1) and acute leukemia risk [(OR=1.47, 95% CI 1.18-1.83); (OR=1.32, 95% CI 1.07-1.62); (OR=1.01, 95% CI 0.84-1.23), respectively] and heterogeneity between the studies. The results suggested that the GSTM1 null genotype and GSTT1null genotype, but not the GSTP1 polymorphism, might be a potential risk factors for acute leukemia. Further well-designed studies are needed to confirm our findings.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.10
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• pp.5037-5042
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• 2012

Background: Tobacco contains agents which generate various potent DNA adducts that can cause gene mutations. Production of DNA adducts may be neutralized by glutathione S transferase (GST) along with other phase I and phase II enzyme systems. The existence of null type of GST among the population increases the susceptibility to various disorders and diseases. The present study focuses on the impact of high tobacco usage and possible null type mutation in GST loci. Methods: Genotypes of GST were detected by multiplex polymerase chain reaction in unrelated 504 volunteers of high tobacco using natives of Gujarat. Allelic frequencies were calculated using Statistical Package for Social Studies-16 software. Hardy Weinberg Equilibrium (HWE) was calculated using Chi square test. Two sided Fisher's significance test was used to compare allelic frequencies of different populations. Results: The frequency of homozygous null genotype of GSTM1 and GSTT1 were 20% (95% CI 16.7-23.9) and 35.5% (95% CI 31.4-39.9) respectively. The GSTM1 and GSTT1 null allele frequency distribution in the Gujarat population was significantly deviating from HWE. GSTT1 null frequency of Gujaratians was significantly higher and different to all reported low tobacco using Indian ethnics, while GSTM1 was not differing significantly. Conclusion: Tobacco usage significantly influences the rate of mutation and frequency of GSTT1 and M1 null types among the habituates. The rate of mutation in GSTT1 loci was an undeviating response to the dose of tobacco usage among the population. This mutational impact of tobacco on GSTT1 postulates the possible gene - environment interaction and selection of null genotype among the subjects to prone them under susceptible status for various cancers and even worst to cure the population with GSTT1 dependent drugs.

• YAKHAK HOEJI
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• v.44 no.4
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• pp.300-307
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• 2000

Expression of xenobiotic-metabolizing enzymes can be altered by xenobiotics, which represents changes in the production of reactive metabolic intermediates as well as toxicities in tissues. Metabolic intermediates derived from xenobiotics are considered to produce the reactive oxygen species including drug free radicals and hydroxyl free radicals, which would be ultimately responsible for drug-induced toxicities. The effects of 1,2-benzothiazine anti-inflammatory agents on the expression of xenobiotic-metabolizing enzymes including major cytochrome P450s, microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) were studied in the liver with the aim of providing the part of information on potential production of reactive metabolites and hepatotoxicity by the agents. The synthetic compounds 24, 36 and 39 exhibited anti-inflammatory effects in rats as assessed by the Randall-Selitto method. The anti-inflammatory effect was detected as early as at 30 min after gavaging the agents with the ED5O being noted at 80 mg/kg, which was comparable to that of ibuprofen. Treatment of rats with each compound (100 mg/kg, 3d) resulted in no significant induction in the immunochemically-detectable cytochromes P45O 1A1/2, P450 2B1/2, P45O 2 Cl1 and P45O 2El. Changes in the mEN expression were also minimal, as evidenced by both Western blot and Northern blot analyses. Hepatic GST expression was slightly increased by the agents: GST Ya protein and mRNA expression was ～1.5-fold increased after treatment with compounds 24 and 39, whereas GST Yb1/2 and Yc1/2 mRNA levels were elevated 2- to 3-fold. In summary the effects of the synthetic 1,2-benzothiazines on the expression of major P45O, mEH and G57 were not significant, providing evidence that metabolic activation of the agents, potential drug interaction and hepatotoxicity would be minimal.

• Fisheries and Aquatic Sciences
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• v.4 no.2
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• pp.88-92
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• 2001

To examine effects of cadmium on the respiratory burst of kidney phagocytes and antioxidant defense in liver, juvenile red seabream Pagrus major were fed a cadmium-incorporated diet $(1g\;CdC1_2/kg\;diet)$. The respiratory burst activity measured by chemiluminescence (CL) was significantly reduced by oral intake of cadmium. Lipid peroxidation in liver expressed as thiobarbituric acid reactive substances (TBARS) was significantly higher in the fish fed a cadmium-incorporated diet than that of the fish fed a control diet both on Day 3 and Day 9. Liver Glutathione S-transferase (GST) activitiy was significantly increased both on Day 3 and Day 9 by feeding a cadmium-incorporated diet, when compared with the controls. From the present results, it can be concluded that oral intake of cadmium in red seabream is associated with marked reduction of respiratory burst capacity of kidney phagocytes which can elevate susceptibility of fish against infecting pathogens. Cadmium administration also elicits significant increment of lipid peroxidation in liver, and fish try to detoxify cadmium by increasing GST activity.

• Toxicological Research
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• v.8 no.1
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• pp.131-137
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• 1992

Sprague-Dawley rats aged six weeks divided into four groups and group 1, 2, 3 and 4 of rats were given an intrapertioneal injection of diethylnitrosamine at 200 mg/kg body weight. Group 4 was Control. Two weeks after beginning of the experiment, group 1 of rats were begun to feed on water containing 0.05% phenobarbital sodium as a promoter for six weeks, and CP-2 were intrapertioneally given to rats of group 2(20mg/kg) and group 3(1mg/kg). Three weeks after beginning of the experiment, partial hepatectomy was performed in all rats. Preneoplastic foci were identified histopathologically by glutathione S-transferase placental form (GST-P) activity. In the Immunohistochemical quantitative analysis of carcinogen-induced foci, it was concluded that CP-2 was not carcinogen.

• The Plant Pathology Journal
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• v.29 no.1
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• pp.110-115
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• 2013

Theobroxide, a novel compound isolated from a fungus Lasiodiplodia theobromae, stimulates potato tuber formation and induces flowering of morning glory by initiating the jasmonic acid synthesis pathway. To elucidate the effect of theobroxide on pathogen resistance in plants, Nicotiana benthamiana plants treated with theobroxide were immediately infiltrated with Pseudomonas syringae pv. tabaci. Exogenous application of theobroxide inhibited development of lesion symptoms, and growth of the bacterial cells was significantly retarded. Semiquantitative RT-PCRs using the primers of 18 defense-related genes were performed to investigate the molecular mechanisms of resistance. Among the genes, the theobroxide treatment increased the expression of patho-genesis-related protein 1a (PR1a), pathogenesis-related protein 1b (PR1b), glutathione S-transferase (GST), allen oxide cyclase (AOC), and lipoxyganase (LOX). All these data strongly indicate that theobroxide treatment inhibits disease development by faster induction of defense responses, which can be possible by the induction of defense-related genes including PR1a, PR1b, and GST triggered by the elevated jasmonic acid.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.36 no.6
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• pp.573-577
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• 2003

Effects of 4-nonylphenol (4-NP) on mixed function oxygenase (MFO) in the kidneies of rockfish (Sebastes schlegeli) were investigated. The cytochrome P45O (CYP) contents and NADPH cytochrome P450 reductase, NADH cytochrome b5 reductase and 7-ethoxyredorufin-O-deethylase (EROD) activities of microsome, glutathione S-transferase (GST) activity of cytosol in rockfish exposed to 4-NP for 7 days using an intraperitoneal injection (25 mg/kg) were quantitatively determined. The GST activity of rockfish exposed to 4-NP were higher, up to 5.2 times higher, than those in the control fish. The activities of NADPH-and NADH-dependent reductases were inhibited. On the other hand, CYP contents and EROD activity of the 4-NP exposed fish demonstrated neither an increasing or decreasing trend.