• Bulletin of the Korean Chemical Society
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• 제16권5호
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• pp.401-406
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• 1995

The P. fragi lipase overexpressed in E. coli as a fusion protein of 57 kilodalton (kDa) has been purified through glutathione-agarose affinity chromatography by elution with free glutathione. The general properties of the purified GST-fusion protein were characterized by observing absorbance of released p-nitrophenoxide at 400 nm which was hydrolyzed from the substrate p-nitrophenyl palmitate. The optimum condition was observed at 25 $^{\circ}C$, pH 7.8 with 0.4 ${\mu}g$ of protein and 1.0 mM substrate in 0.6% (v/v) TritonX-100 solution. Also the lipase was activated by Ca+2, Mg+2, Ba+2 and Na+ but it was inhibited by Co+2 and Ni+2. pGEX-2T containing P. fragi lipase gene as expression vector was named pGL191 and used as a template for the site-directed mutagenesis by sequential PCR steps. A Ser-His-Asp catalytic triad similar to that present in serine proteases may be present in Pseudomonas lipase. Therefore, the PCR fragments replacing Asp217 to Arg and His260 to Arg were synthesized, and substituted for original fragment in pGL19. The ligated products were transformed into E. coli NM522, and pGEX-2T harboring mutant lipase genes were screened through digestion with XbaI and StuI sites created by mutagenic primers, respectively. No activity of mutant lipases was observed on the plate containing tributyrin. The purified mutant lipases were not activated on the substrate and affected at pH variation. These results demonstrate that Asp217 and His260 are involved in the catalytic site of Pseudomonas lipase.

• Asian Pacific Journal of Cancer Prevention
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• 제14권6호
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• pp.3925-3929
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• 2013

We aimed to investigate bladder cancer risk with reference to polymorphic variants of cytochrome p450 (CYP) 1A1, CYP1B1, glutathione S-transferase (GST) M1, and GSTT1 genes in a case control study. Polymorphisms were examined in 114 bladder cancer patients and 114 age and sex-matched cancer-free subjects. Genotypes were determined using allele specific PCR for CYP1A1 and CYP1B1 genes, and by multiplex PCR and melting curve analysis for GSTM1 and GSTT1 genes. Our results revealed a statistically significant increased bladder cancer risk for GSTT1 null genotype carriers with an odds ratio of 3.06 (95% confidence interval=1.39-6.74, p=0.006). Differences of CYP1A1, CYP1B1 and GSTM1 genotype frequencies were not statistically significant between patients and controls. However, the specific combination of GSTM1 null, GSTT1 null, and CYP1B1 codon 119 risk allele carriers and specific combination of GSTM1 present, GSTT1 null, and CYP1B1 432 risk allele carriers exhibited increased cancer risk in the combined analysis. We did not observe any association between different genotype groups and prognostic tumor characteristics of bladder cancer. Our results indicate that inherited absence of GSTT1 gene may be associated with bladder cancer susceptibility, and specific combinations of GSTM1, GSTT1 and CYP1B1 gene polymorphisms may modify bladder cancer risk in the Turkish population, without any association being observed for CYP1A1 gene polymorphism and bladder cancer risk.

• 한국초지조사료학회지
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• 제34권2호
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• pp.114-119
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• 2014

염 또는 건조 스트레스 처리에 의한 톨 페스큐 종자의 발아율 변화와 유식물체 수준에서의 유전자 발현을 조사하기 위하여 in vitro 조건에서 NaCl과 PEG를 처리하여 분석하였다. NaCl 처리시 톨 페스큐 품종별 발아율은 50 mM 농도에서 발아율이 서서히 감소하기 시작하였으며 350 mM의 농도에서는 모든 품종에서 발아가 되지 않는 경향을 보였다. NaCl 처리 농도에 따른 발아율 감소율은 Fawn 품종이 가장 큰 변화를 보였으며 Kentucky-31(E-) 품종이 가장 강한 내성을 보였다. 또한, PEG 처리시 톨 페스큐 품종별 발아율의 변화도 NaCl 처리시와 유사한 경향을 보였으며 고농도인 30% PEG 처리구에서는 모든 품종에서 발아가 되지 않는 경향을 보였으며 Kentucky-31(E-) 품종이 가장 강한 내성을 보였다. 톨 페스큐 유식물체 수준에서 염해와 건조 스트레스에 의한 유전자 발현양상을 조사하기 위하여 DEGs (differentially expressed genes) 탐색을 위한 ACP-based GeneFishing$^{TM}$ PCR 분석을 통해 NaCl 또는 PEG 처리에 따른 발현량의 차이를 보이는 총 4개의 DEG를 선발하여 클로닝하고 염기서열을 분석하였다. 무처리구에 비해 NaCl 처리시 4개의 DEG가 증가하였고 감소하는 DEG는 확인 되지 않았으나, PEG 처리에서는 3개의 DEG (DEG 1, 3, 및 4)가 증가하였고 1개의 DEG가 감소하는 경향을 나타내었다. 발굴된 DEG들을 blastx 검색에 의하여 rubisco large subunit (DEG1), microsomal glutathion S-transferase (GST) 3-like isoform 1 (DEG2) 유전자로 동정되었다.

• 생명과학회지
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• 제28권7호
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• pp.765-771
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• 2018

$PPAR{\gamma}$는 지방세포의 분화를 조절하는 핵심적인 전사 인자로서 이를 조절하는 후성유전학적 조절 기전이 비만억제 연구에서 중요하게 주목 받고 있다. 선행연구에서 CACUL1이 $PPAR{\gamma}$의 전사 활성 및 지방세포의 분화를 억제하는 corepressor로서 작용함을 밝힌 바 있으며 본 연구에서는 CACUL1의 새로운 결합 단백질로 발굴된 protein arginine methyltransferase 5 (PRMT5)의 $PPAR{\gamma}$ 조절 기능을 분석하였다. PRMT5가 CACUL1과 결합함을 immunoprecipitation assay in vivo와 GST-pull down assay in vitro를 통하여 확인하였다. Luciferase reporter assay 결과로 두 단백질이 상호 협력하여 $PPAR{\gamma}$의 전사 활성을 억제함을 확인하였다. PRMT5가 안정적으로 과발현 또는 knockdown되는 3T3-L1 세포주를 제작하여 지방세포 분화에 미치는 영향을 분석한 결과, PRMT5가 3T3-L1세포의 지방세포 분화를 억제함을 증명하였다. 같은 맥락으로 PRMT5는 $PPAR{\gamma}$의 타겟 유전자인 Lpl과 aP2의 발현을 억제하는 것을 RT-qPCR로 확인하였다. 이상의 연구 결과로 PRMT5이 CACUL1과 결합하여 $PPAR{\gamma}$의 전사 활성을 방해, 나아가 지방세포의 분화를 억제하는 기존에 알려지지 않은 분자적 기전을 처음으로 밝혔다. 따라서, PRMT5 효소 활성의 조절은 비만 억제를 위한 약물 개발에 단서를 제공할 것이다.

• 생명과학회지
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• 제16권5호
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.

• 생명과학회지
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• 제27권11호
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• pp.1233-1244
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• 2017

Calcium-dependent protein kinases (CDPKs)는 칼슘 이온을 매개로 한 신호전달 경로에서 중요한 역할을 한다. 벼(Oryza sativa)에는 29개의 CDPKs가 확인되었지만 그들의 기능은 완벽히 밝혀지지 않았다. 이 연구는 OsCPK11 유전자에 초점을 맞춰 그것의 기능적인 특징을 조사하였다. 벼의 어린 잎, 성장한 잎, 꽃에서 OsCPK11 유전자의 조직-특이적 발현이 확인되었고, 지베렐린이 처리된 벼의 호분층에서도 이 유전자의 발현을 확인할 수 있었다. Tos-17이 삽입된 oscpk11의 표현형에서 돌연변이체 각각의 키는 야생형과 구분되지 않았지만, 영과의 수나 무게는 통계적으로 유의미한 차이가 있었다. 덧붙여 많은 돌연변이체 낟알의 배젖에서 white belly materials이 확인되었다. OsCPK11의 cDNA가 cloning되었고, 약 60.5 kD인 OsCPK11 단백질이 GST affinity chromatography와 SDS-PAGE에 의해 얻어졌다. 아미노산 서열 분석을 통해 OsCPK11이 전형적인 CDPKs의 구조적 특징을 가짐을 알 수 있었다. 이 결과는 OsCPK11유전자의 기능과 식물에서 칼슘 이온을 매개로 한 신호전달 경로에 CDPK 역할의 유용한 정보를 제공해줄 것이다.

• Journal of Ginseng Research
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• 제47권1호
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• pp.106-116
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• 2023

Background: Pirarubicin (THP) is an anthracycline antibiotic used to treat various malignancies in humans. The clinical usefulness of THP is unfortunately limited by its dose-related cardiotoxicity. Ginsenoside F1 (GF1) is a metabolite formed when the ginsenosides Re and Rg1 are hydrolyzed. However, the protective effects and underlying mechanisms of GF1 on THP-induced cardiotoxicity remain unclear. Methods: We investigated the anti-apoptotic and anti-oxidative stress effects of GF1 on an in vitro model, using H9c2 cells stimulated by THP, plus trigonelline or AKT inhibitor imidazoquinoxaline (IMQ), as well as an in vivo model using THP-induced cardiotoxicity in rats. Using an enzyme-linked immunosorbent test, the levels of malondialdehyde (MDA), brain natriuretic peptide (BNP), creatine kinase (CK-MB), cardiac troponin (c-TnT), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione (GSH) were determined. Nuclear factor (erythroid-derived2)-like 2 (Nrf2) and the expression of Nrf2 target genes, including heme oxygenase-1 (HO-1), glutathione-S-transferase (Gst), glutamate-cysteine ligase modifier subunit (GCLM), and expression levels of AKT/Bcl-2 signaling pathway proteins were detected using Western blot analysis. Results: THP-induced myocardial histopathological damage, electrocardiogram (ECG) abnormalities, and cardiac dysfunction were reduced in vivo by GF1. GF1 also decreased MDA, BNP, CK-MB, c-TnT, and LDH levels in the serum, while raising SOD and GSH levels. GF1 boosted Nrf2 nuclear translocation and Nrf2 target gene expression, including HO-1, Gst, and GCLM. Furthermore, GF1 regulated apoptosis by activating AKT/Bcl-2 signaling pathways. Employing Nrf2 inhibitor trigonelline and AKT inhibitor IMQ revealed that GF1 lacked antioxidant and anti-apoptotic effects. Conclusion: In conclusion, GF1 was found to alleviate THP-induced cardiotoxicity via modulating Nrf2 and AKT/Bcl-2 signaling pathways, ultimately alleviating myocardial oxidative stress and apoptosis.

• Food Science and Biotechnology
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• 제17권2호
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• pp.367-372
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• 2008

Cellular protection against carcinogens could be achieved by the induction of phase 2 detoxifying and antioxidant enzymes such as glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO1). Nuclear transcription factor E2-related factor 2 (Nrf2) binds to antioxidant response element (ARE) in the promoter region of these genes and the resulting transactivation occurs. In the present study the effect of gujeolcho (Chrysanthemum zawadskii) roots on the Nrf2-ARE pathway were investigated. C. zawadskii root extract was fractionated with a series of organic solvents and their ability to induce Nrf2-ARE pathway was examined. We separated the most potent dichloromethane (DCM) fraction into 12 sub-fractions and found several sub-fractions with strong effects on the Nrf2-ARE pathway. Fraction 4 strongly induced the ARE-reporter gene activity as well as Nrf2 expression. Sitosterol was isolated as a major compound in fraction 4 although its activity was not as potent as its mother fraction. These results indicate that C. zawadskii roots might be used as a potential natural chemopreventive source.

• Asian Pacific Journal of Cancer Prevention
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• 제13권9호
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• pp.4413-4416
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• 2012

Aim: Individual differences in chemosensitivity and clinical outcome in non-small cell lung cancer (NSCLC) patients treatment with platinum-based chemotherapy may be due to genetic factors. Our study aimed to investigate the prognostic role of GSTP1, XRCC1 and XRCC3 in NSCLC patients treated with chemotherapy. Methods: A total of 460 cases were consecutively selected from The Affiliated Hospital of Nantong University between Jan. 2003 to Nov. 2006, and all were followed-up until Nov. 2011. Genotyping of GSTP1 Ile105Val, XRCC1 Arg194Trp, XRCC1 Arg399Gln and XRCC3 Thr241Met was conducted by duplex polymerase-chain-reaction with confronting-two-pair primer methods. Results: Patients with GSTP Val/Val exhibited a shorter survival time, and had a 1.89 fold greater risk of death than did those with the IIe/IIe genotype. For XRCC1 Arg194Trp, the variant genotype Trp/Trp was significantly associated with a decreased risk of death from NSCLC when compared with the Arg/Arg. Individuals carrying XRCC1 399Gln/Gln genotype had a longer survival time, with a lowered risk of death from NSCLC. Conclusion: This study indicated that GSTP1 Ile105Val, XRCC1 Arg194Trp and XRCC1Arg399Gln genes have a role in modifying the effect of platinum-based chemotherapy for NSCLC patients in a Chinese population. Our findings provide information for therapeutic decisions for individualized therapy in NSCLC cases.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2705-2709
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• 2012

Objective: To evaluate the predictive value of glutathione S-transferase (GST) gene polymorphisms for the prognosis of osteosarcoma patients receiving chemotherapy. Methods: A total of 159 patients were included in our study between January 2005 and December 2007., with follow-up until January 2012. Genotyping was based upon the duplex polymerase-chain-reaction with the PCR-CTPP method. Results: At the time of diagnosis, 15.4% of the patients presented with metastasis, while 22.3% developed metastasis during follow-up. At the time of final analysis on January 2012, the median follow-up was 45.5 months. Patients with null GSTM1 and GSTT1 had a higher event free survival rate than non-null genotype, but no significant association was found between the two genotypes and prognosis of osteosarcoma. Individuals with GSTP1 Val/Val genotype tended to live shorter than with the IIe/IIe genotype, and we found a significantly higher risk of death from osteosarcoma (adjusted HR=2.35, 95% CI=1.13-4.85). Conclusion: The GSTP1 gene polymorphism may have an important role in the prognosis of osteosarcoma patients with chemotherapy. Further analyses with larger samples and more genes encoding metabolizing and DNA repair enzymes are warranted.