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.

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.

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

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.

Chitosan Increases the Release of Renal Dipeptidase from Porcine Renal Proximal Tubule Cells

Hyun Joong Yoon,Young Ho Kim,Sung Wook Park,Hwanghee Blaise Lee,Haeng Soon Park 한국통합생물학회 2003 Animal cells and systems Vol.7 No.4

Renal dipeptidase (RDPase, membrane dipeptidase, dehydropeptidase 1, EC 3.4.13.19) has been widely studied since it was first purified from porcine kidney brush border membrane. It was reported that RDPase activity in urine samples of acute and chronic renal failure patients decreases. Nitric oxide (NO) is a highly reactive free radical involved in a number of physiological and pathological processes. NO is able to act in a dual mode, leading either to induction of apoptosis or to blunted execution of programmed cell death. NO inhibited the RDPase release from porcine renal proximal tubules, which could be blocked by L-NAME. Chitosan, the linear polymer of D-glucosamine in  (1  4) linkage, not only reversed the decreased RDPase release by NO but also increased NO production in the proximal tubule cells. The stimulatory effect of NO on RDPase release from proximal tubules in the presence of chitosan must be different from the previously proposed mechanism of RDPase release via NO signaling pathway. Chitosan stimulated the RDPase release in the proximal tubules and increased RDPase activity to 220% and 250% at 0.1% and 1%, respectively. RDPase release was decreased to about 40% in the injured proximal tubules and was recovered in proportion to the increase of chitosan. Chitosan may be useful in recovery of renal function from HgCl2 injury.

Release of Renal Dipeptidase from Rabbit Renal Proximal Tubules and Its Inhibition by Gentamicin

Kang, Bok-Yun,We, Jeoung-Soon,Choi, Kyong,Lee, Hwanghee-Blaise,Han, Ho-Jae,Park, Haeng-Soon The Pharmaceutical Society of Korea 1999 Archives of Pharmacal Research Vol.22 No.4

Effects of several durgs on rabbit renal proximal tubules were examined for the applicability of renal dipeptidase (RDPase, EC 3. 4. 13. 11) release as a model system to study nephrotoxicity. The proximal tubule prepared by the method of Taub (1990) released RDPase spontaneously in the control experiment which was confirmed by Western blotting. RDPase was also released form cisplatin, lipopolysaccardie (LPS), and indomethacin-treated tubules. Gentamicin inhibited RDPase release in a concentration-dependent manner. This RDPase release system may not be a general model to screen nephrotoxicity but could be a useful source of RDPase purification in a simple and inexpensive way.

Chitosan Increases the Release of Renal Dipeptidase from Porcine Renal Proximal Tubule Cells

Hyun Joong, Yoon,Kim, Young-Ho,Park, Sung-Wook,Lee, Hwanghee-Blaise,Park, Haeng-Soon The Korean Society for Integrative Biology 2003 Korean journal of biological sciences Vol.7 No.4

Renal dipeptidase (RDPase, membrane dipeptidase, dehydropeptidase 1, EC 3.4.13.19) has been widely studied since it was first purified from porcine kidney brush border membrane. It was reported that RDPase activity in urine samples of acute and chronic renal failure patients decreases. Nitric oxide (NO) is a highly reactive free radical involved in a number of physiological and pathological processes. NO is able to act in a dual mode, leading either to induction of apoptosis or to blunted execution of programmed cell death. NO inhibited the RDPase release from porcine renal proximal tubules, which could be blocked by L-NAME. Chitosan, the linear polymer of D-glucosamine in $\beta$(1\longrightarrow4) linkage, not only reversed the decreased RDPase release by NO but also increased NO production in the proximal tubule cells. The stimulatory effect of NO on RDPase release from proximal tubules in the presence of chitosan must be different from the previously proposed mechanism of RDPase release via NO signaling pathway. Chitosan stimulated the RDPase release in the proximal tubules and increased RDPase activity to 220% and 250% at 0.1% and 1%, respectively. RDPase release was decreased to about 40% in the injured proximal tubules and was recovered in proportion to the increase of chitosan. Chitosan may be useful in recovery of renal function from $HgCl_2$injury.

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