• Entomological Research
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• 제48권5호
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• pp.390-399
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• 2018

In order to precisely assess gene expression levels, the suitable internal reference genes must be served to quantify real-time reverse transcription polymerase chain reaction (RT-qPCR) data. For armyworm, Mythimna separata, which reference genes are suitable for assessing the level of transcriptional expression of target genes have yet to be explored. In this study, eight common reference genes, including ${\beta}$-actin (${\beta}$-ACT), 18 s ribosomal (18S), 28S ribosomal (28S), glyceraldehyde-3-phosphate (GAPDH), elongation fator-alpha ($EF1{\alpha}$), TATA box binding protein (TBP), ribosomal protein L7 (RPL7), and alpha-tubulin (${\alpha}$-TUB) that in different developmental stages, tissues and insecticide treatments of M. separata were evaluated. To further explore whether these genes were suitable to serve as endogenous controls, three software-based approaches (geNorm, BestKeeper, and NormFinder), the delta Ct method, and one web-based comprehensive tool (RefFinder) were employed to analyze and rank the tested genes. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated according to normalized HSP70, and MsepCYP321A10 gene expression data. We found that the most suitable reference genes for the different experimental conditions. For developmental stages, 28S/RPL7 were the optimal reference genes, both $RPL7/EF1{\alpha}$ were suitable for experiments of different tissues, whereas for insecticide treatments, $28S/{\alpha}-TUB$ were suitable for normalizations of expression data. In addition, $28S/{\alpha}-TUB$ were the suitable reference genes because they have the most stable expression among different developmental stages, tissues and insecticide treatments. Our work is the first report on reference gene selection in M. separata, and might serve as a precedent for future gene expression studies.

• 한국식품영양과학회지
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• 제36권6호
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• pp.708-719
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• 2007

본 연구에서는 104명의 성인남녀를 대상으로 그들의 GSTT1, GSTM1, GSTP1의 유전적 다형성 분포도를 조사한 결과, 전체 대상자들 중 각각 60명(57.4%), 42명(40.6%)가 GSTT1 과 GSTM1의 유전자를 가지고 있었으며, GSTT1과 GSTM1 유전자가 둘 다를 가진 대상자는 27명(25.7%), 둘 중에 하나만 가지고 있는 사람은 47명(45.5%) 그리고 둘 다 없는 사람은 30명으로 28.7%에 해당되었다. GSTP1 유전자의 경우 homozygous(a/a) wild type은 79명(75.5%)으로 대부분의 대상자가 여기에 해당되었으며, heterozygous(a/b) heterozygous type은 22명(21.6%), 그리고 homozygous(b/b) wild type으로 나타난 대상자는 3명(2.9%)이었다. GSTT1 null type은 산화적 스트레스를 더 많이 받고 있는 것으로 알려진흡연자의 비율이 present 군에 비해 낮음에도 불구하고 적혈구 GSH의 농도는 유의적으로 낮고 임파구 DNA 손상정도는 유의적으로 높은 것으로 나타났다. GSTM1 유전자의 경우 적혈구 GSH-Px의 활성만이 GSTM1 null type이 presenttype에 비해 유의적으로 높은 것으로 나타났으며, 나머지 다른 지표에서는 GSTM1 null type과 present type 간의 유의적인 차이가 없었다. GSTP1 유전자 다형성에 따른 항산화 영양상태의 유의적인 차이는 없었다. 흡연과 GST 유전자 다형성의 상호작용결과, 혈장 ${\alpha}$-carotene 농도, 적혈구 GSH-Px와 GST 활성은 GSTT1 null type의 경우 흡연자에 비해 비흡연자가 유의적으로 높게 나타났으며, GSTT1 present type에서는 흡연자와 비흡연자간의 유의적인 차이가 없는 것으로 나타났다. GSTM1 유전자의 경우 GSTM1의 null type에서 흡연자의 경우 비흡연자에 비해 혈장 ${\gamma}$-tocopherol과 ${\beta}$-carotene 수준이 유의적으로 낮았다. GSTT1과 GSTM1 유전자가 둘 다 없는 경우(both null)에는 흡연자의 혈장 lycopene과 적혈구 GST 활성이 비흡연자에 비해 유의적으로 낮았다. GSTP1 유전자의 경우 혈장 ${\alpha}$-carotene과 적혈구 GSH-Px 활성은 a/a type의 흡연자가 비흡연자에 비해 유의적으로 낮았고, 적혈구 catalase의 활성은 a/b type에서 흡연자가 비흡연자에 비해 유의적으로 낮았으며, ${\beta}$-carotene 농도는 두 type 모두에서 유의적으로 흡연자가 낮았다. 이상의 연구 결과 GST 유전적 다형성에따라 산화, 항산화 관련 효소 활성 및 항산화 영양소 수준의상태가 차이가 있음을 알 수 있었으며, 특히 흡연자의 경우 GST 유전자 type에 따라 항산화 영양상태가 더 큰 영향을 받음을 알 수 있었다. 이러한 연구 결과는 유전적 다형성에따라 항산화 영양소의 권장수준을 책정하는데 기초연구자료로 활용될 수 있을 것이다. 즉, GSTT1 또는 GSTM1 null type의 경우 비흡연자에 비해 흡연자가 항산화 영양소 수준이 더 낮게 나타났으므로 그들의 체내 항산화 영양 상태를 증가시킬 수 있는 여러 방안들, 즉 항산화 비타민의 보충투여 및 야채와 과일의 섭취 권장 등이 고려될 수 있을 것이다. 또한 GST 유전자 다형성에 따라 항산화 영양상태의 차이가 있으므로 항산화 관련 in vitro 실험, 동물 및 인체 실험의 결과 해석 시 나타나는 개인 간의 변이차이는 GST 유전자 다형성으로 일부분 설명될 수 있으리라 기대된다. 향후대상자의 범위를 좀 더 넓혀서 GST 유전자 다형성과 항산화 영양상태의 관계를 규명하고자 하며 더불어 산화물질 해독에 관여하는 다른 유전자(CYP 450, SULT 등)의 역할에 대해서도 계속적인 연구가 필요하다고 사료된다.

• Journal of Microbiology and Biotechnology
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• 제27권5호
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• pp.983-989
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• 2017

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of $NADP^+$ in the affinity chromatography process. In the present study, the rat NPR clone containing a $6{\times}$ Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using $Ni^{2+}$-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

• Journal of Chest Surgery
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• 제41권3호
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• pp.354-359
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• 2008

배경: 와파린은 항응고제로 쓰이는 약물로서 주로 간 대사에 의해 배설되는 약물이다. 리팜핀은 결핵 혹은 심내막염 등에 쓰이는 항생제로 2C9과 3A4를 포함한 CYP계열 효소 유도를 일으키는 대표적인 약물이다. 따라서 두 약물을 병용할 경우 리팜핀의 효소 유도에 의한 와파린 대사율 증가로 와파린의 항응고 효과는 감소한다. 이에 따라 와파린의 적절한 용량 조절이 요구되나 정확한 증량과 감량 정도는 제시되지 못하고 있는 실정이다. 이에 본 연구에서는 와파린 복용 환자 중 리팜핀을 병용하게 된 환자를 대상으로 두 약물의 병용 전후, 상호작용의 정도를 시간 경과에 따라 평가하고, 상호작용에 영향을 미치는 요인을 분석하고 또한 이를 토대로 두 약물의 병용 전후, 임상에서 활용할 수 있는 와파린 용량 결정 방법을 설정하고자 하였다. 대상 및 방법: OO병원 항응고 치료 상담 팀의 상담기록지를 1998년 1월부터 2006년 9월까지 후향적으로 검토하여 리팜핀을 병용하게 된 환자를 대상으로 하였다(n=15). 결과: 리팜핀 병용 전 전체 환자의 평균 INR은 $2.25{\pm}0.52$이며 병용 초기 100일간의 평균 INR은 $1.98{\pm}0.28$이었다. 이 경우 병용 전과 병용 초기의 평균 INR은 유의한 차이가 없었다(paired t-test, p>0.05). 리팜핀 병용 중단 직전 2회 측정한 INR의 평균은 $2.19{\pm}0.34$이고 병용 중단 이후 INR의 평균은 $2.49{\pm}0.43$으로 병용 중단 전과 후의 INR 평균은 유의한 차이를 보였으나(paired t-test, p<0.05)모두 치료유효역 범위 내에 있었다. 결론: 항응고 치료 상담 팀의 용량 조절이 적절하다고 판단하여 항응고 치료 상담 팀의 조절을 근거로 병용 시작 시와 병용 중단시의 와파린 용량조절 수식을 도출해냈다

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.712-717
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• 2010

Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole-cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a hemeand diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. The surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of a heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins into practically useful whole-cell biocatalysts for extensive biotechnological applications, including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and biochip development.

• Asian-Australasian Journal of Animal Sciences
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• 제23권8호
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• pp.1118-1126
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• 2010

Male reproduction is influenced by a number of intrinsic and extrinsic factors, including environmental endocrine disruptors. Testosterone is a well recognized intrinsic regulator for development and function of the male reproductive tract, and thus male fertility. The testis and semen of many mammalians contain an unusually high concentration of estrogen. Testosterone is converted into estrogen by the enzymatic action of cytochrome P450 aromatase complex (Cyp19a1). Of the male reproductive tract, the efferent ductules (EDs) possess exceptionally elevated levels of estrogen receptors (ERs), ER${\alpha}$ and ER${\beta}$, indicating that estrogen, in addition to testosterone, would have a functional role in regulation of male reproduction. First, this review has focused on description and summary of what is currently known for functions of estrogen in the EDs. The biosynthetic pathway of estrogen occurring in the testis is briefly covered, following by detailed explanation of the morphology and physiology of EDs. In the next section, the sources and targets of estrogen in the male reproductive tract are highlighted, and possible functional roles of estrogen in the EDs are justified from the aspect of physiology, molecular biology, and morphology in adult animal models. Also, this section covers the importance of estrogen and ERs in maintaining normal function and morphology of the EDs during postnatal development. In the last part of this review, the effects of extrinsic factors, especially environmental endocrine-disruptors, on the EDs is summarized. The intent of this review is to emphasize the importance of estrogen for regulation of physiological function of the EDs, and thus male fertility.

• 한방재활의학과학회지
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• 제33권3호
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• pp.1-15
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• 2023

Objectives We aimed to find out the improvement effect of Baekhogainsam-tang (Baihu Jia Renshen-tang, BIT) on metabolic syndrome and alteration of microbiota and gene expression. Methods We used male C57BI/6 mice and randomly assigned them into three groups. Normal control group was fed 10% kcal% fat diet, high-fat diet (HFD) group was fed 45% kcal% fat diet and 10% fructose water. BIT group was fed same diet as HFD group and treated by BIT for once daily, 6 days per week, total 8 weeks. We measured their body weight and food intake every week and performed oral glucose tolerance test 1 week before the end of the study. Then we collected the blood sample to measure triglyceride, total cholesterol, high-density lipoprotein cholesterol, insulin, and hemoglobin A1c. We harvested tissue of liver, muscle, fat, and large intestine for quantitative polymerase chain reaction (qPCR) and histopathological examination. Fresh fecal samples were collected from each animal to verify alterations of gut microbiota and we used RNA from liver tissue for microarray analysis. Results The body weight and fat weight of BIT group were reduced compared to HFD group. The qPCR markers usually up-regulated in metabolic syndrome were decreased in BIT group. Bacteroides were higher in BIT group than other groups. There were also differences in gene expressions between two groups such as Cyp3a11 and Scd1. Conclusions We could find out BIT can ameliorate metabolic syndrome and suggest its effect is related to gut microbiota composition and gene expression pattern.

• Archives of Pharmacal Research
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• 제28권11호
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• pp.1287-1292
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• 2005

KR-33028 (N-[4-cyano-benzo[b]thiophene-2-carbonyl]guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. This study was performed to identify the metabolic pathway of KR-33028 in human liver microsomes and to compare its metabolism with that of cryopreserved human hepatocytes. Human liver microsomal incubation of KR-33028 in the presence of NADPH and UDPGA resulted in the formation of four metabolites, M1, M2, M3, and M4. M1 and M2 were identified as 5-hydroxy-KR-33028 and 7-hydroxy-KR-33028, respectively, on the basis of LC/MS/MS analysis with the synthesized authentic standard. M3 and M4 were suggested to be dihydroxy-KR-33028 and hydroxy-KR-33028-glucuronide, respectively. Metabolism of KR-33028 in cryopreserved human hepatocytes resulted in the formation of M1, M2, and M4. These data show a good correlation between major metabolites formed in human liver microsomes and cryopreserved human hepatocytes. In addition, KR­33028 was found to inhibit moderately the metabolism of CYP1A2 substrates. Based on the results obtained metabolic pathway of KR-33028 is proposed.

• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.44-54
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• 2019

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be $9.384{\AA}$. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH ${\rightarrow}$ FDR6 ${\rightarrow}$ FDX4 ${\rightarrow}$ Gel16.

• Clinical and Experimental Pediatrics
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• 제54권3호
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• pp.137-140
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• 2011

Testicular adrenal rest tumors (TARTs) are considered to be formed from aberrant adrenal tissue that has become hyperplastic because of elevated adrenocorticotropic hormone (ACTH) in male patients with congenital adrenal hyperplasia (CAH). A 6-year-old boy presented with testicular enlargement and pubic hair. He was diagnosed with CAH complicated by precocious puberty. However, he was not followed-up. At the age of 17, he visited the outpatient clinic because of testicular enlargement and short stature. His right and left testicles were $10{\times}6$ cm and $7.5{\times}4.5$ cm, respectively. His height was 155.1 cm (standard deviation score (SDS), -2.90). The diagnosis of CAH due to 21 hydroxylase deficiency was confirmed by mutation analysis of CYP21A2. Histological examination of the testes showed large, polygonal, eosinophilic cells with round nuclei and prominent nucleoli, which were suggestive of TARTs. He was treated with dexamethasone for 3 weeks and tumors regressed. Subsequently, dexamethasone was replaced by prednisolone and $9{\alpha}$-fludrocortisone; thereafter, the reduced testis size has been maintained.