합성펩타이드를 이용한 영양배엽세포-특이 가토 다클론 항혈청의 제작

이희섭,오재민,김정중,문형배,김원신,이황희 원광대학교 생명공학연구소 1995 생명공학연구소보 Vol.3 No.1

Within the last few years, a different approach to generating protein-reactive antibodies has been developed that has several advantages over conventional immunization. This involves synthesizing short peptide sequences, coupling them to immunogenic carrier molecules, and immunizing animals with the conjugates. 3βHSD(3β-hydroxy-5-ene steroid dehydrogenase; EC 1.1.1.145) is the enzyme of the plasma membrane of human trophoblast and it's cDNA sequence was identified by Nickon et al(Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3β-hydroxy-5-ene steroid dehydrogenase. J Reprod Fert 1991;149;156). For the production of trophoblast-specific antibody, we synthesized three oligopeptides that are epitope sites chosen from cDNA sequence of 3βHSD. Oligopetides were coupled with KLH(keyhole limpet hemocyanin) under 25% glutaraldehyde. The trophoblast-specific rabbit polyclonal antisera was produced by conventional methods. This antisera reacts with a 43kDa protein in human placental lysate by Western blotting analysis and The syncytiotrophoblasts and cytotrophoblasts from villi are stained positively with this antisera by immunohistochemistry. Villous trophoblasts were cultured in methionine-free media for 1 hour and [^(35)S]-Methionine for 24 hours. Media and cell lysate were immunoprecipitated with this antisera and 12% SDS-PAGE was performed. In fluorography, bend was not noted in media and 43kDa band was noted in medis and 43kDa band was noted in lysate. It was concluded that anti-3βHSD antibody produced by synthetic peptide was specific to trophoblasts and 3βHSD was membrane-bound protein of trophoblasts.

지치(Lithospermum erythrorhizon) 추출물의 멜라닌 생합성 억제효과

이황희(Hwanghee Blaise Lee),배석(Suk Bai),진종언(Jong-Eon Chin) 한국식품영양과학회 2005 한국식품영양과학회지 Vol.34 No.9

유전자 발현 조절 수준에서 멜라닌 색소 생합성 억제물질을 탐색하고자 지치 뿌리로부터 유용성 물질을 추출ㆍ분획하여 tyrosinase 프로모터를 지닌 B16 mouse melanoma cell에 처리한 결과 그 메탄올 추출물은 10 ㎍/mL의 농도에서 약 33% 이상의 tyrosinase 프로모터 활성 억제효과를 나타내었으며, 세포 활성능이 100 ㎍/mL의 농도에서 약 108%로 매우 안전하였다. 그리고 methylene chloride, ethyl acetate, butyl alcohol, 물 등의 용매 분획물들도 농도에 따라 차이는 있었지만 100 ㎍/mL 또는 500 ㎍/mL의 고농도에서 tyrosinase 프로모터의 활성을 억제하였다. 또한, 지치 메탄올 추출물의 농도를 달리하여 3일 동안 처리하였을 때 멜라닌 색소의 생성능은 10 ㎍/mL와 100 ㎍/mL의 농도에서 대조군 세포에 비해 각각 약 11%와 24%로 크게 감소하는 결과를 보여주었다. To estimate the inhibitory effect of Lithospermum erythrorhizon root extract on melanin biosynthesis, we tested its inhibitory effects on tyrosinase promoter in B16 mouse melanoma cells. Lithospermum erythrorhizon root extract had inhibitory effect above 33% on tyrosinase promoter at 10 ㎍/mL and exhibited no cytotoxicity under 100 ㎍/mL. Also, melanin biosynthesis decreased approximately 11% and 24% at 10 ㎍/mL and 100 ㎍/ mL, respectively. Therefore, Lithospermum erythrorhizon root extract would be considered very effective regulator of tyrosinase promoter and melanin biosynthesis.

뇌하수체 세포인 GH3세포에서 non-genomic action을 통한 Nonylphenol의 nitric oxide 증진효과

이경진(Kyung-Jin Lee),최철웅(Chul-Yung Choi),손현정(Hyun-Jung Sohn),정백진(Back-Jin Jeong),문소희(So-Hee Moon),이황희(Hwanghee Blaise Lee),이종빈(Jong-Bin Lee) 환경독성보건학회 2003 환경독성보건학회지 Vol.18 No.4

본 연구는 환경호르몬(endocrine disruptors)으로 분류되었으며,에스트로젠 화합물의 특성을 지닌 4-Nonylphenol(NP)이 설치류 pituitary 세포 중 성장호르몬을 분비하는 GH3 세포의 Nitric oxide (NO)을 증가시키는 작용기전을 규명코자 수행되었다. 먼저 GH3세포에 NP처리 농도에 따른 NO의 생성을 측정한결과 NP처리농도 의존적으로 증가시켰다. 이러한 NO의 증가가 genomic action인지를 확인하기 위해GH3 세포의 NO를 증가시키는 효소인 neuronal oxide synthase의 단백질량을 측정한 결과 CH3 세포에서NP에 의한 nNOS의 단백질의 변화는 없었다. 에스트로젠 화합물인 NP가 에스트로젠 리셉터 (ER)와의관계를 조사하기 위해 ER억제제(ICI 168,780)를 처리한 경우 NP에 의해 증가한 NO가 감소하였다. 또한유전자 전사억제제인 actlnomycin D 및 단백질 발현 억제제인 cycloheximide을 처리한 경우는 NP에 의한NO 증가억제효과가 없었다. 이러한 결과를 종합해 볼 때 GH3 세포에서 NP는 ER을 매개한 non-genomicaction에 의해 NO를 증가키는 것으로 사료된다.

EGF Inhibits Expression of WDNM1 and Sulfated Glycoprotein-2 Genes in Mammary Epithelial Cells

Lee, Mijoung,Hwang, Intaek,Choi, Yunjaie,Paik, Sanggi,Lee, Hwanghee Blaise,Baik, Myunggi 충남대학교 생물공학연구소 1998 생물공학연구지 Vol.6 No.-

We have previously shown that expressions of ferritin heavy chain (FHC), WDNM1, and sulfated glycoprotein-2(SGP-2)genes are induced at an livolution stage of mammary gland. Here we studied the effect of lactogenic hormones and EGF on the expression of involution-induced genes in HC11 mammary epithelial cells. Insulin, dexamethasone, prolactin, and its combinations did not affect expression of the genes. When cells were cultured in growth medium containing EGF, expression of WDNM1 and SGP-2 genes was strongly inhibited in a dose- and time- dependent manner, whereas expression of FHC gene was not influenced by EGF. Results demonstrate that EGF inhibits expression of WDNM1 and SGP-2 genes in mammary epithelial cells. ⓒ 1997 Academic Press

Antidiabetic effect of propolis: reduction of expression of glucose-6-phosphatase through inhibition of Y279 and Y216 autophosphorylation of GSK-3&agr;/&bgr; in HepG2 cells

Kang, Li-Jung,Lee, Hwanghee Blaise,Bae, Hyeun-Jong,Lee, Seong-Gene John Wiley Sons, Ltd. 2010 Phytotherapy research Vol.24 No.10

<P>Propolis is a sticky, resinous material that honey bees collect from various plants, and mix with wax and other secretions. The aim of this study was to evaluate the antidiabetic effect of propolis through an analysis of the expression and enzyme activity of glucose-6-phosphatase (G6Pase) and to elucidate the mechanism by which propolis inhibits G6Pase gene expression. When HepG2 cells were incubated in high glucose media (25 mm), G6Pase expression was induced. Propolis significantly reduced the expression and enzyme activity of G6Pase; however, the hypoglycemic effect was not abolished by the phosphoinositide 3-kinase inhibitor, LY294002, and by the mitogen-activated protein kinase (MAPK) inhibitor, U0126. Propolis inhibited the activity of GSK3&agr; and &bgr; via the inhibition of serine and tyrosine phosphorylation, specifically, Y279 for GSK3&agr; and Y216 for GSK3&bgr;. The phosphorylations of Y279 and Y216 occur through autophosphorylation by GSK3&agr;/&bgr; and are involved in their own activity. Although propolis showed antioxidant activity, antidiabetic effect of propolis was not influenced by hydrogen peroxide and N-acetylcysteine. These results suggest that propolis inhibits the expression of G6Pase by inhibiting the autophosphorylation of Y279 and Y216 of GSK3&agr; and &bgr;, respectively, which are involved in the activation of GSK3. These findings suggest that propolis may be a potential antidiabetic agent for the treatment of insulin-insensitive diabetes. Copyright © 2010 John Wiley & Sons, Ltd.</P>

Nitric Oxide Inhibits the Shedding of the glycosylphosphatidylinositol-anchored dipeptidase from porcine renal proximal tubules

PARK, Sung Wook,YOON, Hyun Joong,LEE, Hwanghee Blaise,Hooper, Nigel M.,PARK, Haeng Soon 전남대학교 약품개발연구소 2002 약품개발연구지 Vol.11 No.-

NO is related to the pathological condition acute renal failure, in which we previously observed that the level of soluble dipeptidase in urine was decreased. In this study the role of NO in the shedding of the glycosylphosphatidylinositol (GPI)-anchored form of renal dipeptidase (RDPase) was examined. The NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine rapidly inhibited the release of RDPase from porcine kidney proximal tubules. The substrate of NO synthase, ι-Arg, also inhibited the release of RDPase, and this effect was reversed by the NO synthase inhibitor N??-nitro-ι-arginine methyl ester. Western-blot analyses using antibodies raised against porcine RDPase and the inositol-1, 2-cyclic monophosphate moiety formed on phospholipase C cleavage of the GPI anchor demonstrated that SNP mediated its inhibitory effect on the release of RDPase via a GPI-specific phospholipase C (GPI-PLC). Peroxynitrite scavengers (deferoxamine and superoxide dismutase) or reducing agent (dithiothreitol) did not affect SNP's inhibition of the release of RDPase. Exposure to the G-protein activator AIF?? mimicked the ι-Arg effect in the presence of a low concentration of ι-Arg, and the effect was completely reversed by U73122, an intracellular phosphatidylinositol-specific PLC (PI-PLC) inhibitor. These results suggest a signal-transduction pathway involving NO, which is produced by NO synthase(s) following activation of a G-protein-coupled PI-PLC, resulting in inhibition of the GPI-PLC that cleaves and releases RDPase. Therefore, this indicates a role for NO as an inhibitory regulator of the shedding of the GPI-anchored RDPase in acute renal failure.

Grass Carp(Ctenopharyngodon idellus) Bile may Inhibit the Release of Renal Dipeptidase from the Proximal Tubules by Nitric Oxide Generation

Choi, Kyong,Park, Sung Wook,Lee, Kwang Jae,Lee, Hwanghee blaise,Han, Ho Jae,Park, Sung Kwang,Park, Haeng Soon 전남대학교 약품개발연구소 2000 약품개발연구지 Vol.9 No.1

There are many reports on acute renal failure (ARF) after ingestion of grass carp bile (CB; Ctenopharyngodon idellus). Renal dipeptidase (RDPase; EC 3.4.13.19) is a glycosylphosphatidylinositol-anchored ectoenzyme within the renal proximal tubules (PTs) and is proposed as a diagnostic enzyme of renal disease. We examined the release of RDPase following treatment with CB and various nitric oxide (NO) related compounds in porcine PTs. The RDPase release from PTs was inhibited by CB in a concentration-dependent manner and was also inhibited by sodium nitroprusside (direct NO donor) and L-arginine (NO synthase substrate) in the tested range (0-12 mM). CB-treated (0.1 ㎎/㎖) PTs showed a decreased RDPase activity in comparison with the control group. This inhibition was blocked by 2 mM L-NAME (NO synthase inhibitor) and U73122 (inhibitor of phosphatidylinositol-specific phospholipase C) in a concentration-dependent manner Eel bite (0-0.1 ㎎/㎖), used as the control, did not significantly affect the RDPase release from PTs. The NO concentration was observed as nitrite, the degradation product of the NO metabolism, increased in proportion to CB and L-arginine. The in crease of nitrite to 151.5% by CB treatment (0.1 ㎎/㎖) was blocked by 2 mM L-NAME (85.5%). When the phosphotipase C pathway was blocked by 70 and 20 PM U73122, the nitrite generation decreased to 122.7 and 89.4%, respectively. These results strongly suggest that NO generation and the phospholipase C pathway affect the RDPase release from the PTs and that they may be involved in the development of ARF in vivo following CB ingestion. The release of RDPase from PTs could be a useful tool not only for this CB-caused ARF, but also for the elucidation of other biochemical mechanisms.

피부암 및 피부암 전구증에서의 Acid Stable Trypsin Inhibitor ( ASTI ) 의 발현양상에 관한 연구

이종육 ( Jong Yuk Yi ),이동원 ( Dong Won Lee ),윤두희 ( Dou Hee Yoon ),조백기 ( Baik Kee Cho ),강창석 ( Chang Suk Kang ),이황희 ( Hwanghee Blaise Lee ),장원희 ( Won Hee Jang ),유욱준 ( Ook Joon Yoo ) 대한피부과학회 1996 대한피부과학회지 Vol.34 No.2

Background: Recently, much attention has been focused on the role of protease inhibitors such as acid stable trypsin inhibitor (ASTI) in the invasive growth of malignant tumors. However, there are no report on expression of ASTI from premalignant and/or malignant skin tumors. Objectives : In the present study the expression of ASTI was investigated in the different type of premalignant and/or, malignant skin tumors in attempt to clarify the relation between the expression of the ASTI and malignancy. Methods : For the detection of ASTI in the tumor tissue, the immunoperoxidase techniques that used mouse antibody raised against highly purified ASTI. The degree of ASTI immunoreactivity was semiquantitatively assessed for staining intensity as the percentage of ASTI-positive cells. Results : ASTI immunoreactivity was detected in most of the premalignant and malignant skin tissues. Especially, ASTI expression was present widespread in squamous cell carcinoma(SCC) with strong cytoplasmic membrane, where as in normal epidermis they were primarily present in the horny layer. The strong staining was the SCC, keratoacanthoma, Bowen's disease, actinic keratosis, basal cell epithelioma, Paget's disease in decreasing order. The significant difference in the staining intensity was observed between SCC and other groups. Conclusion : Results of immunohistochemical studies suggest that the tumor cells themselves could produce ASTI. Considering the suggestion that ASTI is a self protector, inhibitor to proteolytic protease as well as growth-stimulating factor. The present findings may indicate that ASTI expressed in malignant cells may play a role possibly closely associated with tumor development. (Kor J Dermatol 1996;34(2): 179-184)

SCI Identification of Urinary Dipeptidase as the Released Form of Renal Dipeptidase

We, Jeoung Soon,Kang, Bok Yun,Lee, Jae Cheon,Lee , Hwanghee blaise,Park, Haeng Soon 전남대학교 약품개발연구소 1997 약품개발연구지 Vol.6 No.1

Amphipathic and hydrophilic forms of human renal dipeptidase and urinary dipeptidase were purified by affinity chromatography using cilastatin, a dipeptidase inhibitor, as the ligand. The sequence analyses of the first ten amino acids of renal and urinary dipeptidases were shown to be identical, and they are Asp-Phe-Phe-Arg-Asp-Glu-Ala-Glu-Arg-Ile. Unambiguous results of amino acid sequencing, the molecular weight of native protein (190 kD), the molecular weight of subunit (47.7 kD) and a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzymes are composed of homotetramers. This is the most direct evidence that urinary dipeptidase is the released form of renal dipeptidase. In fact, they are the same enzymes.

Study of Chitosan on Fibroblast Growth Factor 2 (bFGF) from the Renal Proximal Tubular Cells

( Hyun Joong Yoon ),( Young Ho Kim ),( Hwanghee Blaise Lee ),( Jun Heung Lee ),( Haeng Soon Park ) 한국키틴키토산학회 2004 한국키틴키토산학회지 Vol.9 No.3