만성 승모판 패쇄부전증 환자에서의 안지오텐신 전환효소 억제제의 장기 투여 효과

김대희,이명묵,이해영,조현재,박승정,서재빈,서정원,양한모,윤창환,조상호,이준희,김용진,김명아,손대원,오병희,박영배 대한순환기학회 2004 Korean Circulation Journal Vol.34 No.2

Rotavirus 전기영동형별을 위한 RNA 추출과정 및 조건의 비교연구

조은경,김은순,양재명,김경희,조양자 한양대학교 의과대학 1992 한양의대 학술지 Vol.12 No.2

Rotaviruses (Rv) are the major cause of severe dehydrating dearrhea among young children in Korea, where they are frequently responsible for 68% of the cases requiring hospitalization. Laboratory diagnosis of rotaviral infection in Korea depends mainly on enzyme-linked immunosorbent assay (ELISA) that employs expensive commercial diagnostic kits purchased from abroad and that shous the presence or absence of group A Rv only. PACE have however provided epidemiologic evidence to demonstrate the sxistence of different strains (both group A and atypical Rv) during diarrgeal outbreak, the appearance of new strains and desappearance of old ones from a community, shifts in the prenalence of strains, and the persistance of a single strain in newborns. For such reasons, we have felt that it is essential to raise the sensitivity and shorten the processing time for PAGE. We therfore have devised a rapid method for Rv RNA extration by directly extracting the Rv RNA from the specimens (that is, skipping freon extraction and running as ultracectrifuge) and using an urea-containing dissociation mixture to dissolve dried RNA for PAGE. We have also shorten the PAGE diagnostic results by sequential staining of the same gel with ethidium bromide and silver stain. With the use of the rapid method, it was feasible to increase the efficacy of the PAGE with reduced cost and working time. Phenol extration of stools, however, was essential in maintaining the sensitivity of the method.

Inhibition of Bluetongue Virus(BTV) Infectivity by cis-Diamminedichloroplatinum(Ⅱ) (CDDP)

Yang, Jai-Myung,Manning, JaRue S. 成均館大學校 科學技術硏究所 1991 論文集 Vol.42 No.1

블루텅 바이러스 감염성에 대한 CDDP의 영향을 조사하였다. 조직배양세포에서 기른 뒤 정제하지 않은 바이러스는 CDDP에 의해 급속히 감염성이 감소하나 완전이 없어지지는 않았다. CDDP를 처리할 때 non-ionic detergent를 같이 넣어 주면 BTV의 감염도가 감지할 수 없을 정도로 낮아진다. CDDP에 의해 감염성이 제거된 바이러스는 왁친으로 사용할 수 있는 가능성이 있다.

cis - Diamminedichloroplatinum(Ⅱ)(CDDP) induces denaturation and conformational changes in pBR322 DNA

Yang, Jai Myung,Koo, Ja Choon,Lim, Chang Soo,Hahn, Tae Ryong 한국농화학회 1990 Applied Biological Chemistry (Appl Biol Chem) Vol.33 No.4

pBR322 DNA exposed to CDDP was transformed into E. coli and plated on ampicillin containing media. The number of colonies formed on ampicillin agar plated was reduced to undetectable level after treat the DNA with l0μM of CDDP. The CDDP-treated pBR322 DNA was also showed changes in it's migration pattern on agarose gel electrophoresis and digested by single strand DNA specific S1 nuclease. These data suggest that CDDP adduction to pBR322 DNA resulted in denaturation and conformational changes which ultimately leads to the inactivation of the ampicillin resistant gene.

cis-Diamminedichloroplatinum(Ⅱ) Induces Con-formational Changes in Bluetongue Virus ds RNA

Yang, Jai-Myung,Manning, JaRue S. 成均館大學校 科學技術硏究所 1991 論文集 Vol.42 No.1

CDDP 처리한 블루텅 바이러스 ds RNA를 전자현미경으로 촬영한 사진은 RNA가 구부러져 짧아졌음을 보여주었다. 같은 RNA를 1% 아가로즈 겔에서 전기영동하면 이동 속도가 빨라진다. 이 실험결과는 CDDP가 ds RNA의 구조를 compact하게 변화시킴을 뜻한다. Form I과 Ⅲ의 pBR 322 DNA를 CDDP로 처리한 뒤 전기영동한 실험 결과는 CDDP가 긴거리의 염기 사이에 crosslink를 잘 형성하지 않음을 암시한다.

Construction of a Recombinant AcNPV Carrying E. coli β-galactosidase

Yang, Jai-Myung,Koo, Ja-Choon 成均館大學校 科學技術硏究所 1990 論文集 Vol.41 No.2

대장균의 β-galactosidase 유전자를 BamHI과 Aha Ⅲ로 절단한 DNA 단편을 pAcI 벡터의 BamHI 자리에 클론하여 pHBG-I을 만들고 야생형 AcNPV DNA와 pHBG-I DNA를 Spodoptera frugiperda 세포에 cotransfection한 뒤 extracellular virus (ECV)를 플라그 검정하여 polyhedral body를 형성하지 않는 재조합 AcNPV를 순수분리하였다. 재조합 AcNPV (β-gal-AcNPV)가 대장균의 β-galactosidase를 지니고 있음은 Southern blot 결과와 X-gal 존재하에 blue plaque이 형성되는 사실로 확인하였다. β-gal-AcNPV가 감염된 SF21 세포내에서 β-galactosidase는 감염된 후 약 18시간 뒤부터 합성되기 시작한다. β-galactosidase 유전자가 polyhedrin 촉진유전자의 제어를 받으므로 β-gal-AcNPV는 polyhedrin 유전자의 발현 조절 기작을 밝혀내는데 유용하게 사용될 수 있다. Plasmid pHBG-I was generated by cloning BamHl-Aha Ⅲ digested fragment of E. coli β-galactosidase into the BamHI site of plasmid pAcI. Wild type AcNPV DNA and supercoiled pHBG-I DNA were cotransfected into Spodoptera frugiperda, an insect cell line, and extracellular virus (ECV) was plaque assayed. A probable recombinant AcNPV from a plaque that did not generate occlusion body was purified three times. Southern blot analysis by using BglI digested 2.1 kb fragment of pHBG-I containing β-galactosidase coding region, as a probe, indicated that the plaque purified recombinant AcNPV carries E. coli β-galactosidase. The β-gal-AcNPV was able to form blue plaques in the presence of X-gal and β-galactosidase was highly produced in SF21 cell after 24 h p. i.

Secretory Production of Biologically Active Human Thrombopoietin by Baculovirus Expression System

Yang, Jai Myung,Lim, Seung Wook,Park, Myung Hwan,Park, Seung Kook,Koh, Yeo Wook,Na, Doe Sun The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.5

Human thrombopoietin (hTPO) was expressed to high levels in insect cells using the baculovirus expression system. Full-length hTPO cDNA containing a native signal peptide sequence was amplified by PCR from a human fetal liver cDNA library and cloned into the Autographa californica nuclear polyhedrosis virus (AcNPV) expression vector. Immunoblot analysis with antiserum against hTPO indicated that an approximately 55 kDa protein was produced in recombinant AcNPV infected insect cells. Recombinant hTPO was produced 4-fold higher in Trichoplusia ni(Tn5) cells than in Spodoptera frugiperda (Sf9) cells, with most of the hTPO produced in Tn5 cells secreted into culture medium. Addition of tunicamycin in the culture medium resulted in the reduction of the size of hTPO to 35-38 kDa, and most of the protein remained within the cell. These results suggest that N-glycosylation of hTPO is required for the secretion of the protein into the culture medium in insect cells. hTPO produced in insect cells induced proliferation and maturation of megakaryocyte progenitors, indicating that it is in a biologically active form.

Construction of a recombinant carring E . coli β - galactosidase

Yang, Jai Myung,Koo, Ja Choon 한국유전학회 1990 Genes & Genomics Vol.12 No.4

Plasmid pHBG-1 was generated by cloning BamHI-Aha III digested fragment of E. coli β-galactosidase into the BamHI site of plasmid pAcI. Wild type AcNPV DNA and supercoiled pHBG-1 were cotransfected into Spodoptera frugiperda, an insect cell line, and extracellular virus (ECV) was plague assayed. A plague purified recombinant AcNPV that did not generate polyhedra was able to form blue plaques in the presence of X-gal. This recombinant AcNPV carrying E. coli β-galactosidase gene would be useful to investigate the regulation mechanism of polyhedrin gene expression.

"Baculovirus"as a Eukaryotic Gene Expression Vector

Yang, Jai Myung 한국유전학회 1986 Genes & Genomics Vol.8 No.4

Autoqrapha californica nuclear polyhedrosis Virus (AcNPV) was used as an expression vector for a number of prokaryotic and eukaryotic genes. By using specially constructed transfer vector, the protein coding DNA sequences for β-galactosidase and other human hormonal genes were linked to the AcNPV promoter for the gene coding for polyhedrin, the major occlusion protein. Site-directed in vitro mutagenesis technique was applied to remove additional DNA sequences between polyhedrin promoter and initiation codon of inserted gene. Cotransfection of transfer vector and wild type AcNPV DNA resulted in the formation of recombinant AcNPV whose plaque morphology is different from that of wild type AcNPV. Plaques formed by recombinant AcNPV (occlusion minus) were picked and purified several times. The insertion of foreign DNA into AcNPV genomic DNA was confirmed by slot dot hybridization and southern blot analysis. The β-galactosidase inserted recombinant AcNPV produced blue plaque after the addition of X-gal into second agar overlay medium. Immunostaining of recombinant virus (AcNPV-TPR2) infected Spodoptera fruqiperda (fall armyworm), an established insect cell line, indicated that the foreign protein folded in immunologically proper conformation. ELISA test of intracellular and extracellular tissue culture fluids showed that majority of the produced foreign proteins were secreted into media. This result suggested that a human signal peptide was recognized by bisect cells and properly transported out of the cell. Further characterization of recombinant protein is undergoing at present time. Above results demonstrate that AcNPV should be suitable for use as a eukaryotic expression vector for the production of cloned gene that is not properly expressed in lower level organism.

cis - Diamminedichloroplatinum(Ⅱ)(CDDP) 에 의한 불루텅 바이러스 이중가닥 RNA 의 구조변화

양재명 한국농화학회 1991 Applied Biological Chemistry (Appl Biol Chem) Vol.34 No.2

cis-Diamminedichloroplatinum(II)(CDDP), an antitumor drug, did not generate crosslink between bluetongue virus (BTV) capsid protein at moderate concentration. Cesium chloride density gradient centrifugation study revealed that protein-RNA crosslink was not detectable in CDDP treated BTV. CDDP treated BTV ds RNA showed remarkable change in the migration pattern in polyacrylamide gel electrophoresis. These results suggest that the reduction of BTV core associated transcriptase activity is most likely by the CDDP adduction to the genomic ds RNA rather than by the protein-RNA crosslink and/or protein-protein crosslink.