• Animal cells and systems
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• 제16권6호
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Biomolecules & Therapeutics
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• 제8권2호
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• pp.153-159
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• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• Journal of the Korean Society of Food Science and Nutrition
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• 제26권6호
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• pp.1181-1186
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• 1997

This study was performed to investigate the effects of Codonopsis lanceolata extract on the activities of antioxidative enzymes in carbon tetrachloride treated rats. Male Sprague-Dawley rats were fed until they reached about 110$\pm$10g body weight. Thereafter they were divided into normal group(N), carbon tetrachloride treated group(T), carbon tetrachloride and Codonopsis lanceolata water extract treated group(TW). Normal group were fed standard diet and carbon tetrachloride treated group were fed carbon tetrachloride once a week at the level of 0.12ml/100g body weight. Carbon tetrachloride and Codonopsis lanceolata water extract treated group were fed carbon tetrachloride once a week at the level of 0.12ml/100g body weight and Codonopsis lanceolata water extract at the level of 0.1ml/100g body weight once a day. The rats were sacrificed after 6weeks of feeding period. Content of hepatic cytochrome P-450 diminished by carbon tetrachloride was significantly increased by Codonopsis lanceolata water extract. Significant decrease in hepatic xanthine oxidase activity was found in rats treated with Codonopsis lanceolata water extract. The activity of superoxide dismutase was decreased by carbon tetrachloride, but it was significantly increased by Codonopsis lanceolata water exract. The activity of glutathione peroxidase increased by carbon tetrachloride was significantly decreased by Codonopsis lanceolata water extract. The activities of catalase and glutathione S-transferase were significantly influenced by Codonopsis lanceolata water extract. Contents of glutathione and lipid peroxide were increased by carbon tetrachloride, but they were significantly diminished by Codonopsis lanceolata water extract.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2635-2638
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• 2012

Background: Many studies have investigated the association between glutathione S-transferase T 1 (GSTT1) null genotype and risk of prostate cancer, but the impact of GSTT1 null genotype in Asians is still unclear owing to inconsistencies across results. Thie present meta-analysis aimed to quantify the strength of the association between GSTT1 null genotype and risk of prostate cancer. Methods: We searched the PubMed, Embase and Wangfang databases for studies of associations between the GSTT1 null genotype and risk of prostate cancer in Asians and estimated summary odds ratio (OR) with their 95% confidence interval (95% CI). Results: A total of 11 case-control studies with 3,118 subjects were included in this meta-analysis, which showed the GSTT1 null genotype to be significantly associated with increased risk of prostate cancer in Asians (random-effects OR = 1.49, 95% CI 1.15-1.92, P = 0.002), also after adjustment for heterogeneity (fixed-effects OR = 1.45, 95% CI 1.23-1.70, P < 0.001). No evidence of publication bias was observed. Conclusions: This meta-analysis of available data suggested the GSTT1 null genotype does contribute to increased risk of prostate cancer in Asians.

• BMB Reports
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• 제43권5호
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• pp.375-381
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• 2010

In this study, the cDNA library of Chang-liver cells was immunoscreened using common ADAMs antibody to obtain ADAM related genes. We found one positive clone that was confirmed as a new gene by Blast, which is an uncharacterized helical and coil protein and processes protease activity, and named protease-related protein 1 (ARP1). The submitted GenBank accession number is AY078070. Molecular characterizations of ARP1 were analyzed with appropriate bioinformatics software. To analyse its expression and function, ARP1 was subcloned into glutathione S-transferase fusion plasmid pGEX-2T and expressed by E. coli system. The in vitro expression product of ARP1 was recognized by common ADAMs antibody with western blot. Interestingly, ARP1 cleaves gelatine at pH9.5, which suggests it is an alkaline protease. Semi-quantitative RT-PCR result indicates that ARP1 mRNA is strongly transcribed in the liver and the treated Chang-liver cells.

• The Korean Journal of Mycology
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• 제30권2호
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• pp.124-130
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• 2002

The inductions of phase II enzymes, such as NAD(P)H : quinone reductase (QR), glutathione S-transferase (GST), glutathione (GSH) level and the inhibition of polyamine metabolism were tested for the chemopreventive potentials of the extracts from the soybean fermented with Agrocybe cylindracea (AC) or Phellinus igniarius (PI). The soybean fermented with AC or PI was potent inducer of QR activity in murine hepatoma Hepa1c1c7 cells. GST activities of the extracts from soybean fermented with AC or PI were higher than that of the extract from soybean not fermented with basidiomycetes. In addition, GSH levels of the extracts from soybean fermented with AC or PI were increased about 1.2 fold or 1.4 fold, respectively. In addition, proliferation of Acanthamoeba castellanii in a broth medium was inhibited by the extracts from soybean fermented with AC or PI at the both concentration of 20 and 40 mg/3 ml. These results suggest that soybean fermented with AC or PI may have chemopreventive potentials by inducing QR activity, increasing GSH and GST levels and inhibiting polyamine metabolism.

• Korean Journal of Poultry Science
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• 제46권3호
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• pp.161-171
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• 2019

Four-week-old male Korean native chicks (KNC) were assigned to 3 groups with 6 replicates (8 birds/replicate) in each group: a basal diet (CON, 100 ppm of Zn), basal diet fortified with 50 ppm of Zn with zinc oxide (ZnO), or basal diet fortified with 50 ppm of Zn with Zn-methionine (ZnM). Immediately after a 4-week-feeding trial, 6 birds per group were used to evaluate the effects of zinc supplements on antioxidant indicators and the mRNA expression of zinc transport genes. The nitrogen components, lipid peroxidation, and total antioxidant status in blood were not influenced by Zn fortified diets. However, the ZnM group showed a significant (P<0.05) increase in uric acid levels than those in the ZnO group. In the small intestine, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities, and malondialdehyde (MDA) level were unaffected by zinc supplements. The activity of glutathione S-transferase (GST) was significantly (P<0.05) enhanced by Zn-methionine supplementation. In the liver, the activity of GST was significantly (P<0.05) increased by Zn-methionine supplement without affecting SOD, GPX, and MDA levels. With respect to the mRNA expression of zinc transport genes, the ZnM group displayed a strong tendency for increases in intestinal ZnT-1 (P=0.09) and ZnT-5 (P=0.06) levels, compared to those in the CON group. Moreover, the ZnM group showed a tendency (P=0.10) for up-regulation of hepatic metallothionein mRNA as compared with the CON group. In conclusion, the Zn-fortified diet with 50 ppm of Zn-methionine helped to improve GST activity and Zn transport gene expression in the small intestine or liver of KNC.

• Archives of Pharmacal Research
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• 제27권7호
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• pp.757-762
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• 2004

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes including glutathione S-transferases (GSTs). NF-E2-related factor-2 (Nrf2) phosphorylation by protein kinase C (PKC) is a critical event for its nuclear translocation in response to oxidative stress. Previously, we have shown that peroxynitrite plays a role in activation of Nrf2 and Nrf2 binding to the antioxidant response element (ARE) via the pathway of phosphatidylinositol 3-kinase (PI3-kinase) and that nitric oxide synthase in hepatocytes is required for GSTA2 induction. In view of the importance of PKC and Pl3-kinase in Nrf2-mediated GST induction, we investigated the role of these kinases in peroxynitrite formation for GSTA2 induction by oxidative stress and determined the relationship between PKC and PI3-kinase. Although PKC activation by phorbol 12-myristate-13-acetate (PMA) did not increase the extents of constitutive and inducible GSTA2 expression, either PKC depletion by PMA or PKC inhibition by staurosporine significantly inhibited GSTA2 induction by tert-butylhydroquinone (t-SHa) a prooxidant chemical. Therefore, the basal PKC activity is req- uisite for GSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, induced GSTA2, which was not inhibited by PKC depletion, but slightly enhanced by PKC activation, suggesting that PKC promotes peroxynitrite formation for Nrf2-mediated GSTA2 induction. Treatment of cells with S-nitroso-N-acetyl-penicillamine (SNAP), an exogenous NO donor, in combination with t-BHQ may produce peroxynitrite. GSTA2 induction by SNAP ＋ t-BHQ was not decreased by PKC depletion, but rather enhanced by PKC activation, showing that the activity of PKC might be required for peroxynitrite formation. LY294002 a P13-kinase inhibitor blocked GSTA2 induction by t-BHQ, which was reversed by PMA-induced PKC activation. These results provide evidence that PKC may playa role in formation of peroxynitrite that activates Nrf2 for GSTA2 induction and that PKC may serve an activator for GSTA2 induction downstream of PI3-kinase.

• Journal of the Korean Society of Food Science and Nutrition
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• 제33권6호
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• pp.987-994
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• 2004

The purpose of this study was to investigate the effect of dietary seaweed in diabetic rats treated with streptozotocin (STZ) for 7 weeks. The rats (Sprague-Dawley male rats, 180∼200 g) were divided into 4 groups : normal rats fed control diet (C), diabetic rats fed control diet (CD), normal rats fed seaweed diet (M), and diabetic rats fed seaweed diet (MD). Diabetes was induced by single injection of streptozotocin (60 mg/kg, i.p.). Urinary levels of calcium and uric acid, and blood levels of hemoglobin, total cholesterol and low density lipoprotein (LDL)-cholesterol were not significantly different among groups. But high density lipoprotein (HDL)- cholesterol of M and MD groups were higher than that of C and CD groups. Activity of hepatic microsomal G6Pase was significantly (p<0.05) lower in C and M groups than that of CD and MD groups. Hepatic glutathione S-transferase (GST) of M, CD and MD groups were significantly lower than C group (p<0.05), glutathione peroxidase (GPX) of C, M and MD groups were higher than CD group. In conclusion, dietary seaweed may improve blood lipid profiles and GSH-related enzymes in STZ-induced diabetic rats.

• Journal of Nutrition and Health
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• 제38권1호
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• pp.20-29
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• 2005

This study is conducted to determine the effects of dietary levels of corn and tuna oils on the formation of preneoplastic lesions in die-thylnitrosamine (DEN) induced rat hepatocarcinogenesis. Weanling male Sprague-Dawley rats were fed 2.5, 5, 15, 25% (w/w) corn or tuna oils. Hepatocellular carcinogenesis was induced by DEN (200 mg/kg body weight) and two-thirds partial hepactectomy was carried out 3 weeks later and were sacrificed 8 weeks after DEN initiation. Tuna oil group showed smaller area of placental glutathione S-transferase (GST-P) positive foci than com oil group. Com oil group of 25% (w/w) showed the widest area of GST -P positive foci, and tuna oil group showed significantly smaller area of GST-P positive foci than com oil in 25% (w/w) level but had no differences between oil levels. Thio-barbituric acid reactive substances (TBARS) content was the highest in 25% (w/w) level of tuna oil group fed long chain and highly polyunsaturated fatty acids. Also serum ${\gamma}$ -glutamyltranspeptidase (GGT) activities in 25% level of tuna oil group were significantly higher than by other levels. As oil contents increased, glucose 6-phosphatase (G6Pase) seems to decrease in com oil groups but remained the same in tuna oil groups. Glutathione reductase (GR) activities were significantly higher in tuna oil group, and the higher the level of tuna oil, the higher GR activities. But Cu/Zn superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities didn't seem to be influenced by levels and kind of dietary fats. Therefore, as oil levels increased, com oil rich in n-6 fatty acids promoted carcinogenesis but tuna oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of n-3 fatty acids suppressed. Although lipid peroxidation products were elevated in 25% (w/w) tuna oil group, GST-P positive foci didn't increase. Therefore pre-neoplastic lesions might be reduced through mediation of a lipid peroxidation process in tuna oil. As fat contents of tuna oil increased, elevated GR activities may give a rise to produce more reduced glutathione in order to protect against free radical attack, and high G6Pase activities remained the same and they contributed to membrane stability. So tuna oil diet seems to protect hepatocarcinogenesis.