• 약학회지
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• 제51권5호
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• pp.291-295
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• 2007

The present study examines the effect of dominant-negative Akt on the insulin-mediated microsomal epoxide hydrolase (mEH) induction in rat hepatocytes. We also assessed the role of insulin in the expression of soluble epoxide hydrrolase (sEH). Insulin increased mEH levels and the enzyme activities, whereas sEH protein expression was unaffected by insulin. The specific PI3K inhibitors or p70 S6 kinase inhibitor ameliorated the insulin-mediated increase in mEH protein levels. Infection with adenovirus expressing dominant-negative and kinase-dead mutant of Akt1 effectively inhibited the insulin-mediated increase in mEH expression and mEH activity. These results suggest that mEH and sEH are differentially regulated by insulin and PI3K/Akt/p70S6K are active in the insulin-mediated regulation of mEH expression.

• 대한약리학회지
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• 제32권2호
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• pp.233-241
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• 1996

Previous studies have shown that glucocorticoid suppresses microsomal epoxide hydrolase(EH) gene expression and that EH expression is altered during pregnancy. The effects of progesterone on the expression of rat EH and certain glutathione S-transferase(GST) genes were examined in this study. Northern RNA blot analysis revealed that progesterone was effective in increasing hepatic EH mRNA levels at 12 h to 48 h after treatment with a maximal 9-fold increase being noted at 12 h time point. Nonetheless, multiple daily treatment with progesterone rather caused minimal relative increases in EH mRNA levels. GST Ya and Yb1/2 mRNA levels were also transiently elevated at 12 h after progesterone treatment, followed by gradual decreases from the maximal Increases at day 1, 2 and 5 post-treatment. These changes in EH and GST mRNA levels were noted only at a relatively high dose of progesterone. Furthermore, immunoblot analyses showed that rats treated with progesterone for 5 days failed to show EH or GST induction, indicating that progesterone-induced alterations in EH and GST mRNA levels do not reflect bona fide induction of the detoxifying enzymes. Concomitant progesterone treatment of rats with the known EH inducers including ketoconazole and clotrimazole failed to additively nor antagonistically alter EH mRNA levels. In contrast, dexamethasone substantially reduced ketoconazole- or clotrimazole-inducible EH expression. These results showed that progesterone stimulates the EH, GST Ya and Yb1/2 gene expression at early times followed by marked reduction in the RNA levels from the maximum after multiple treatment and that the changes in mRNA do not necessarily reflect induction of the proteins.

• 생명과학회지
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• 제18권1호
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• pp.136-142
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• 2008

Rhodotorula glutinis epoxide hydrolase (EH) 유전자를 pPICZ vector에 이중발현 cassette로 재조합하여 발현시킨 재조합균주 Pichia pastoris를 제작하였으며 라세믹 styrene oxide 혼합물로부터 고순도 광학활성 (S)-styrene oxide를 제조하는데 사용하였다. 본 연구에서 사용된 R. glutinis EH 유전자는 전보에서 사용한 pPICZ B/RgEH plasmid DNA를 주형으로 하여 얻었으며 PCR 방법으로 BglII 제한자리를 돌연변이 시키고 AOX1 promoter $(P_{AOX1})$-RgEH 유전자-전사종결서열($TT_{AOX1}$)을 이중으로 가진 이중발현 cassette를 만들어 P. pastoris의 염색체 DNA에 삽입시켰다. 반응온도를 $30^{\circ}C$로 하였을 때, RgEH를 이중발현 cassette로 발현시킨 재조합균주 P. pastoris의 (R)-styrene oxide에 대한 $V_{max}$ 값은 $2.2{\mu}mol\;min^{-1} (mg\;dcw)^{-1}$으로 단일발현 cassette로 발현시킨 P. pastoris의 $0.4{\mu}mol\;min^{-1}(mg\;dcw)^{-1}$에 비하여 6배 향상되었다. 광학순도가 높은 (S)-styrene oxide를 제조하는 최적조건을 찾기 위하여 입체선택적 가수분해 속도 및 수율에 미치는 detergent 및 온도의 효과를 실험하였으며, Tween 20을 0.5% 첨가하고 $10^{\circ}C$로 반응시킨 경우 10분 반응을 통해 99.9% ee 이상의 고순도 (S)-styrene oxide를 43.4% 얻을 수 있었다.

• 약학회지
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• 제56권1호
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• pp.42-47
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• 2012

Soluble epoxide hydrolase (sEH) is a metabolic regulator of epoxyeicosatrienoic acids (EETs). EETs have many beneficial effects, vasodilation, anti-diabetes, anti-inflammation, cardiovascular protection, renal protection. Therefore, selective sEH inhibitors have a potential for treating these diseases. In the present study, screening methods for sEH inhibitors using PHOME ((3-phenyl-oxiranyl)-acetic acid cyano-(6-methoxynaphthalen-2-yl)-methyl ester) and 14-15-EET as substrates were established. To determine selectivity, microsomal epoxide hydrolase (mEH) inhibition assay was also developed using styrene oxide as a substrate of microsomal epoxide hydrolase. Our results obtained from 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid (AUDA) used as a positive sEH inhibitor and valpromide used as a positive mEH inhibitor showed that these methods are useful for discovery of drug candidates.

• 한국미생물·생명공학회지
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• 제34권2호
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• pp.129-135
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• 2006

Zebrafish (Danio rerio)의 microsomal epoxide hydrolase(mEH)로 추정되는 유전자를 클로닝하고 그 특성을 연구하였다. D. rerio의 mEH 추정단백질은 포유동물의 mEH 및 세균의 EH들과 아미노산서열 상동성을 보였으므로 결정분자구조(1qo7 및 1ehy)를 template로 하여 homology modelling을 행하였다. 클로된 단백질은 $Asp^{233}$, $Glu^{413}$ 및 $His^{440}$으로 구성된 catalytic triad와 2개의 tyrosine 잔기 및 oxyanion hole이 보존되어 있었다. 생물정보학적인 분석 및 EH 활성시험은 추정단백질이 D. rerio의 mEH라는 것을 보여주었다. Racemic styrene oxide를 기질로 하여 활성시험을 행한 결과, 재조합 D. rerio mEH는 (R)-styrene oxide을 입체선택적으로 가수분해하였으며 45분 반응시간에 99%ee의 광학순도를 가진 (S)-styrene oxide를 33.5% 얻을 수 있었다.

• Natural Product Sciences
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• 제25권4호
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• pp.348-353
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• 2019

Soluble epoxide hydrolases (sEH) are enzymes present in all living organisms, metabolize epoxy fatty acids to 1,2-diols. sEH in the metabolism of polyunsaturated fatty acids plays a key role in inflammation. In addition, the endogenous lipid mediators in cardiovascular disease are also broken down to diols by the action of sEH that enhanced cardiovascular protection. In this study, sEH inhibitory guided fractionation led to the isolation of five phenolic compounds trans-resveratrol (1), trans-piceatannol (2), sulfuretin (3), (+)-balanophonin (4), and cassigarol E (5) from the ethanol extract of the seeds of Passiflora edulis Sims cultivated in Vietnam. The chemical structures of isolated compounds were determined by the interpretation of NMR spectral data, mass spectra, and comparison with data from the literature. The soluble epoxide hydrolase (sEH) inhibitory activity of isolated compounds was evaluated. Among them, trans-piceatannol (2) showed the most potent inhibitory activity on sEH with an IC₅₀ value of 3.4 μM. This study marks the first time that sulfuretin (3) was isolated from Passiflora edulis as well as (+)-balanophonin (4), and cassigarol E (5) were isolated from Passiflora genus.

• 대한환경공학회지
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• 제28권2호
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• pp.144-149
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• 2006

Hexane을 유일 탄소 에너지원으로 하여 농화배양한 배양액으로부터 hexane 분해능이 우수한 EH741 균주를 순수 분리 동정하였고, 분리균주에 의한 hexane 생분해 특성을 조사하였다. EH741 균주는 Rhodococcus sp.로 동정되었고, 액상 배양계에서 hexane 용해도를 향상시키기 위해 첨가한 계면활성제 Pluronic F68(PF68)은 hexane 생분해 속도에 영향을 미치지 않았다. Hexane을 유일 탄소원으로 첨가한 무기염 배지에서 EH741의 최대 비성장속도(${\mu}_{max}$)값은 $0.04h^{-1}$이었고, 최대 hexane 분해속도($V_{max}$)와 포화상수($K_s$)는 각각 $161{\mu}mol{\cdot}g-DCW^{-1}{\cdot}h^{-1}$ 및 10.5 mM이었다. Rhodococcus sp. EH741은 hexane 처리를 위한 생물학적 공정에 유용하게 활용될 것으로 사료된다.

• 공업화학
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• 제18권3호
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• pp.279-283
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• 2007

한 종류의 epoxide hydrolase (EH) 효소 자체가 광학수렴 가수분해(enantioconvergent hydrolysis) 활성을 가지는 Caulobacter crescentus의 epoxide hydrolase (CcEH) 유전자를 PCR로 클로닝하여 재조합시킨 Escherichia coli 생촉매를 개발하였다. 재조합 E. coli 세포 10 mg을 10 mM styrene oxide와 반응시킨 다음 기질과 반응생성물을 chiral GC와 HPLC로 각각 분석 한 결과, (S)-styrene oxide 기질에 대해서는 위치 선택적으로 에폭사이드 링의 ${\alpha}$-탄소를 공격하여 (R)-diol로 전환시켰다. 반면에 (R)-styrene oxide에 대하여는 ${\beta}$-탄소를 공격하여 (R)-diol로 전환시키는 위치선택성을 가지고 있었다. 재조합 CcEH를 단일 생촉매로 사용한 광학수렴 가수분해반응을 통해 20 mM racemic styrene oxide에 대하여 광학순도 85%의 (R)-phenyl-1,2-ethanediol을 수율 69%로 생합성 할 수 있었다

• 대한약리학회지
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• 제29권2호
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• pp.275-282
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• 1993

Microsomal epoxide hydrolase (mEH)은 epoxide형 중간대사물을 해독화하는 효소이다. 본 실험실에서는 thiazole 또는 pyrazine을 rat에 투여할 때 mEH mRNA수준이 증가되고 mEH가 유도증가한다는 것을 밝힌바 있다(Carcinogenesis, Kim et al, 1993). 본 연구에서는 Thiazole처리를 한 rat의 간 microsome 분획으로 부터 DEAE-cellulose column chromatography를 이용하여 mEH를 순수분리하였고, 이를 SDS-PAGE분석 및 N 말단 amino acid 서열분석으로 확인하였다. Pyrazine처리를 한 rat의 간 microsome분획에서는 mEH와 더불어 이와 관련된 43 kDa 단백질이 함께 정제되었다. 정제된 thiazole 유도성 mEH를 토끼에 주사하여 항체를 생산하였고, 이 항체를 이용한 immunoblot 분석을 하였을 때 간 microsome 분획의 mEH가 thiazole투여군에서는 대조군에 비하여 10배, pyrazine 투여군에서 7배 증가하였다. Pyrazine처치한 rat의 간 microsome 분획에서는 mEH 관련성 43 kDa 단백질이 동시 유도증가하는 것을 면역화학적 반응으로도 확인하였다. 이때 Pyrazine으로 유도된 rat의 간 microsome 분획 또는 정제분획에 존재하는 43 kDa 단백질과 mEH의 비율은 1 : 15로 나타났다. 정제된 mEH와 43 kDa 단백질의 N 말단 amino acid 서열을 분석하였을때 43 kDa 단백질의 N 말단이 mEH와 동일하게 나타나 관련 단백질임을 확인하였다. 이러한 mEN 유도현상에 종차가 있는지를 알아보기 위하여 thiazole과 pyrazine을 각각 rabbit에 투여하였을 때 rabbit에서 는 mEH의 유도증가가 일어나지 않았으며, pyrazine 투여군에서 43 kDa 단백질의 증가는 관찰 되었다. 본 연구는 thiazole 또는 pyrazine 투여후 mEH 발현이 유도증가되며, pyrazine 투여 후에는 mEH 및 이와 관련된 43 kDa 단백질이 동시유도되고, 이러한 mEH 유도발현에 rat와 rabbit간에는 종차가 있음을 보여준다.

• Toxicological Research
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• 제13권4호
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• pp.339-347
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• 1997

Disulfiram (DSF) and diethyldithiocarbamate (DDC), a reduced form of DSF, protect the liver against toxicant-induced injury through inhibition of cytochrome P450 2E1. The effect of DSF and DDC on the levels of major hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) expression was comparatively studied, given the view that these enzymes are involved in terminal detoxification events for high energy intermediates of xenobiotics. Treatment of rats with a single dose of DSF (20-200 mg/kg, po) resulted in 2- to 15-fold increases in the mEH mRNA level at 24 hr with the ED$_{50}$ value being noted as 60 mg/kg. The mEH mRNA level was elevated ~15-fold at 24 hr after treatment at the dose of 100 mg/kg, whereas the hepatic mRNA level was rather decreased from the maximum at the dose of 200 mg/kg, indicating that DSF might cause cytotoxicity at the dose. In contrast to the effect of DSF, DDC only minimally elevated the mEH mRNA level at the doses employed. DSF moderately increased the major GST mRNA levels in the liver as a function of dose, resulting in rGSTA2, rGSTA3/5 or rGSTM1 mRNA levels being elevated 3- to 4-fold at 24 hr post-treatment, whereas the rGSTM2 mRNA level was not altered. DDC, however, failed to stimulate the mRNA levels for major GST subunits, indicating that the reduced form of DSF was ineffective in stimulating the GST the expression. The effect of other organosulfides including aldrithiol, 2, 2'-dithiobis(benzothiazole) (DTB), tetramethylthiouram disulfide (TMTD) and allyl disulfide (ADS) on the hepatic mEH and GST mRNA expression was assessed in rats in order to further confirm the increase in the gene expression by other disulfides. Treatment of rats with aldrithiol (100 mg/kg, po) resulted in a 16-fold increase in the mEH mRNA level at 24 hr post-treatment. DTB, TMTD and ADS also caused 5-, 9- and 12-fold increases in the rnRNA level, respectively, as compared to control. Thus, all of the disulfides examined were active in stimulating the mEH gene in the liver. The organosulfides significantly increased the rGSTA2, rGSTA3, rGSTA5 and rGSTM1 mRNA levels at 24 hr after administration. In particular, aldrithiol was very efficient in stimulating the rGSTA and rGSTM genes among the disulfides examined. These results provide evidence that DSF and other sulfides effectively stimulate the mEH and major GST gene expression at early times in the liver and that DDC, a reduced form of DSF, was ineffective in stimulating the expression of the genes, supporting the conclusion that reduced form(s) of organosulfur compound(s) might be less effective in inducing the mEH and GST genes through the antioxidant responsive element(s).