Development of in-cell imaging assay systems for MMP-2 and MMP-9 based on trans-localizing molecular beacon proteins

이민전,조영아,황현진,김정희 대한약학회 2015 Archives of Pharmacal Research Vol.38 No.6

A sensitive in-cell imaging MMP-2 and MMP-9 detection systems that enables direct fluorescencedetection of a target protease and its inhibition inside livingcells has been developed. This in-cell imaging systemutilizes the concept of fluorescent molecular beaconreporter (MBR) protein comprising a masking protein, amitochondrial targeting sequence, a protease specificcleavage sequence and a fluorescent marker sequence,green fluorescent protein (GFP). The MBR protein isdesigned to change its intracellular location upon cleavageby either MMP-2 or MMP-9 from cytosol to mitochondria. Full and partial MMP-2 and MMP-9 were tested for optimalexpression and activity in the cell. The activity ofMMP-2 and MMP-9 was approximately 65–71 %. AmongMMP clones, MMP-2 catalytic domain and MMP-9 clonecontaining pro, catalytic and hemopexin domain were mostactive. Both MMP-2 and MMP-9 required divalent ions Caand Zn for its activity and MMP-9 was more active athigher Ca/Zn ratio. With the in-cell imaging assay theprotease activity can be measured in cellular environmentand cellular toxicity of candidate molecules can be monitoredat the same time. These are great advantage whencompared to other currently used in vitro biochemicalassays. The in-cell imaging assay developed in this studycan be modified for other MMPs and can be used in variouslife science and drug discovery researches including thehigh throughput screening and high contents screeningapplications.

Oct4 단백질의 DNA - 결합 영역에 융합된 HIV - 1 integrase

이민전,서진욱,김미라,신차균 中央大學校 遺傳工學硏究所 2002 遺傳工學硏究論集 Vol.15 No.1

Human immunodeficience virus type-1 (HIV-1) integrase (IN) mediates integration of viral DNA into the host cell genome which is an essential step during the life cycle of retroviruses. HIV-1 IN shows four enzymatic activities in vitro: non-specific DNA- binding, endonucleolytic (3'-processing), integration (strand transfer), and disintegration activities. Oct4 as a transcription factor recognizes and binds the specific sequences containing 5' -ATGCAAAT-3'. In order to study the specific binding and enzymatic activities of the integrase protein, we constructed the four different fusion proteins (OctIN1, OctIN2, INKOct1, INKOct1) using different combination of HIV-1 IN and Oct4 protein. The fusion proteins were overexpressed in Escherichia coli and purified by Ni-NTA and SP-sepharose columns. Analysis of the enzymatic activities showed that all the four fusion proteins has detectable endonucleolytic and integration activities, suggesting that the fusion proteins can be used to study derected integration and sequence specific DNA-binding of the integrase fusion protein.

면역결핍바이러스 인테그라제 억제제로서 Baicalein과 Baicalin

이민전(Min Jun Lee),김미라(Mi Ra Kim),이용섭(Yong Sup Lee),신차균(Cha Gyun Shin) 대한약학회 2003 약학회지 Vol.47 No.1

Baicalein and baicalin are flavonoid compounds isolated from medicinal herb Scutellaria baicalensis Georgi (Labiatae) and have been known to possess antiviral activities. In the present study; we investigated the in vitro effects of baicalein and baicalin on the three distinctive enzymatic activities of the human immunodeficlency virus type-1 (HIV-1) integrase - endonucleolytic, integration, and disintegration activities. Both compounds inhibited the three enzymatic activities in a dose-dependent manner The 50% inhibitory concentrations of baicalein and baicalin for endonucleolytic activities of HIV-1 integrase were 4.4 +/- 3.3 and 25.9 +/- 4.0μM, respectivel. In general, baicalein exhibited nearly 6- to 10-fold stronger inhibition than baicalin for the three enzymatic activities. These data demonstrate that baicalein or baicalin can be used as a leading compound to develop anti-AIDS chemotherapeutic agents targeting to the HIV-1 integrase.

다발성 선천성 기형을 동반한 내장 탈출아 1예

이민전(MJ Lee),안욱선(WS Ahn),전운천(WC John),문기성(KS Moon),지제근(JG Chi) 대한산부인과학회 1982 Obstetrics & Gynecology Science Vol.25 No.5

본 병원 산부인과에서 조기 분만된 1.8kg 다발성 선천성 기형이 동반된 미숙아 1예를 경험하였으며 출생 40분후에 사망하였다. 기형의 내용은 ruptured omphalocele, patent omphalomesentric duct, left cleft palate and lip, postaxial polydatylia with dystropicnail, aclrenal heteropia in perisplenic lymph node, Nodular excrescence of spleen, cystic lymphangioma involving thyroid, 등의 선천성 기형이 동반되었으며 이들 각각의 문헌적 고찰을 하였다. A Omphalocele associated with maltiple congenital anomalies was extremely rare complication of pregnancy. We had experienced a case of omphalocele associated with multiple congenital anomalies such as ruptured omphalocele, patent omphalomesenteric duct, left cleft palate and lip, postaxial polydactylia (Both hands and left foot) with dystrophic nails, adrenal heterotopia in left peritesticular region, pancreatic heterotopia in perisplenic lymphnode, nodular excrescence of spleen, cystic lymphangioma, involving thyroid and presented those with a brief case history and review of literatures.

자궁 경부에서 발생한 상피내 암종과 침윤성 암종의 간질 변화에 관한 병리조직학적 연구

고성민,노광을,이민전,김용임,이미자,전호종,서재홍 조선대학교 1993 The Medical Journal of Chosun University Vol.18 No.2

Connective tissue seems to be involved in malignancy by lytic processes as a crucial element in invasive growth. This looks relatively simple, but data have emerged that the stroma is not just a passive but rather an active participant. Recent advances in the borderland between cancer and connective tissue research have increasingly made it clear that the relationship between malignant tissue and its stroma is a very intricate one. The present study was performed in order to investigate the variety of stromal reactivity and alterations of basement membrane accompanying malignant growth and the distribution of Langerhans cells and T and B lymphocytes in cervical epithelium affected by intaepithelial neoplasia and invasive cervical carcinoma. The subjects in this were 14 cervicitis, 42 cervical intraepithelial neoplasia, 14 microinvasive cervical carcinoma and 14 invasive cervical carcinomas. A total of 84 formalin-fixed and paraffin-embedded specimens of normal, inflamed and neoplastic uterine cervix have been studied in order to correlate the epithelial changes with the expression of α-smooth muscle actin, S-100 protein, α_1-antichymotrypsin, lysozyme, LCA, CD20, UCHL1, OPD4, CD1, CD4, CD8, CD68 and typeⅣ collagen in stroma. The results of immunohistochemical and electron microscopical examinations yield virtually identical findings 1) The number of α-smooth muscle actin positive cells and the intensity of stain were related to the increasing grading of cervical intraepithelial neoplasia and invasive cervical carcinoma. 2) Normal cervix and cervical intraepithelial neoplasia grade Ⅰ and Ⅱ showed continuous basement membrane but invasive cervical carcinoma showed highly variable basement membrane deposition ranging from continuous to almost completely absent. 3) The cervical intraepithelial neoplasia Ⅲ and invasive cervical carcinoma showed an increase in spindle shaped Langerhans cells associated with increased numbers of stroma and intraepithelial lymphoid cells. The evaluation of collagen Ⅳ in basement membrane, S-100 protein and CD1 in Langerhans cells and α-smooth muscle actin in stromal cells of the uterine cervix may be an useful adjunct to diagnostic criteria of cervical intraepithrlial neoplasia and invasive cervical carcinoma, and may help understanding of the mechanisms of mesenchymal epithelial interactions during neoplasia.

자궁의 악성 혼합성 뮬러 종양 : 1예 보고 report of a case

기근홍,노광을,이민전 조선대학교 1993 The Medical Journal of Chosun University Vol.18 No.2

Malignant mixed mullerian tumor is rare tumor of the uterus. This tumor derived from mullerian mesoderm, but their histogenesis is still controversial. We report a case of homologous type of malignant mixed mullerian tumor of the uterus in 64 year-old female. The tumor composed of adencocarcinoma and leiomyosarcoma. After radical hysterectomy, this tumor is metastasize to lung 5 months later.

거대점막하 자궁근종의 1예

손순선,이영순,신성옥,이민전 대한산부인과학회 1981 Obstetrics & Gynecology Science Vol.24 No.8

상피종양의 keratin 생성능에 대한 전자현미경적 연구

이길선,이명주,장정수,이민전,노광을,서재홍 朝鮮大學校 附設 醫學硏究所 1993 The Medical Journal of Chosun University Vol.18 No.1

Keratins are characteristically present in all keratinizing and nonkeratinizing epithelial cells and their neoplasms. They consist of at least 19 different polypeptides ranging from 40 to 69 kilodaltons, which are developmentally related to specific epithelial cell types. Keratins of high and intermediate molecular weights are readily demonstrated in squamous cell carcinomas, which are characteristically rich in tonofilaments and tonofilament-desmosome complexes. Keratins of low molecular weights are expressed in adenocarcinoma including renal cell carinoma, hepatocellular carcinoma, small cell carcinoma, and carcinoid tumor. These tumors do not reveal any tonofilaments and sacrcely show discrete filaments on electron microscopy. An immunoperoxidase technique employing antibody to keratin was used to study distribution and staining pattern of keratin filaments in benign and malignant epithelial tumors (20 squamous cell carcinomas, 20 gastric adenocarcinomas, 20 transitional cell carcinomas and 15 non-neoplastic epithelia). These immunohistochemical results were compared with ultrastructural features of neoplastic diagnosis of squamous cell carcinoma, adenocarcinoma and transitional cell carcinoma, and estimation of degree of differentiation. The results are as follows : 1. The squamous epithelium, glandular epithelium and transitional epithelium disclosed the positive reaction for keratin. 2. Squamous cell carcinoma and transitional cell carcinoma exhibited positive reaction for keratin. 3. Gastric adenocarcinoma showed uniformly negative or weakly positive reaction for keratin. 4. On electron microscopy, heavy bundles of tonofilaments and desmosomes were more frequently encountered in well differentiated squamous cell carcinoma, In poorly differentiated squamous cell carcinoma, a few tonofibrils and desmosome were noted. 5. In the adenocarcinomas, the intermediate filaments were arranged randomly as nonaggregated, short filaments spread throughout the cytoplasm, although occasionally they formed a perinuclear whorl. These filaments did not become aggregated to form tonofilaments. 6. On the immunogold labeling for keratin of low molecular weight areas in gastric adenocinoma, in tense labeling of intermediate filaments for keratin was noted. From the above result, ultrastructural and immunohistochemical study on malignant epithelial cell were useful in differential diagnosis of squamous cell carcinoma, adenocarcinoma and transitional cell carcinoma, and estimation of degree of differentiation. It was also proved that immunogold labeling technique was especially valuable for diagnosis of vague case which showed negative or weakly positive reaction in immunohistochemical stain.

DA-125, a New Antitumor Agent, Inhibits Topoisomerase II as Topoisomerase Poison and DNA Intercalator Simultaneously

Jinwook Seo,Hak Sung Lee,이민전,Mira Kim,신차균 대한약학회 2004 Archives of Pharmacal Research Vol.27 No.1

DA-125, a novel derivative of adriamycin, is known for its anti-cancer activity. In this study, the inhibitory mechanism of DA-125 on topoisomerase was investigated in the simian virus 40 (SV40) replicating CV-1 cell by studying the SV40 DNA replication intermediates and DNAtopoisomerase complexes. DNA-protein complexes that were formed in the drug-treated cells were quantitated by using a glass filter assay. SV40 DNA replication intermediates that were accumulated in the drug-treated CV-1 cell were analyzed in a high resolution gel. DA-125 did not accumulate B-dimers of SV40 DNA replication intermediates which were found in the adriamycin- treated CV-1 cells. DA-125 induced a dose-dependent formation of the DNA-protein complexes, while adriamycin did not. When adriamycin and etoposide (VP16) were added to the SV40-infected cells at the same time, adriamycin blocked the formation of the DNA-protein complexes induced by VP16 in a dose-dependent manner. However, DA-125 blocked the formation of the DNA-protein complexes induced by VP16 up to the maximum level of the DNA-protein complexes that were induced by DA-125 alone. Adriamycin and DA-125 did not inhibit the formation of the DNA-protein complexes that were caused by camptothecin, a known topoisomerase I poison. DA-125 is bifunctional in inhibiting topoisomerase II because it simultaneously has the properties of the topoisomerase II poison and the DNA intercalator. As a topoisomerase II poison, DA-125 alone induced dose-dependent formation of the DNA-protein complexes. However, as a DNA intercalator, it quantitatively inhibited the formation of the DNA-protein complexes induced by a strong topoisomerase II poison VP16. Furthermore considering that the levels of the DNA-protein complex induced by VP16 were decreased by DA- 125 in terms of the topoisomerase II poison, we suggest that DA-125 has a higher affinity to the drug-binding sites of DNA than VP16 has.