Methicillin, Cefamandole, Oxytetracycline에 의한 사람 호중구 Elastase의 변화

김사열(Sa-Youl Ghim),정혜영(Hye Young Jeong),배성준(Sung Jun Bae),강구일(Koo Il Kang) 대한약리학회 1989 대한약리학잡지 Vol.25 No.1

사람 혈액속의 elastase와 관련된 질병에 대한 연구는 다양한 저해제의 개발을 동반해 왔으며, 최근 항생제도 그 관심대상이 되고 있다. 두 단계의 액체 크로마토그래피를 거쳐 얻은 고순도의 elastase에 12종의 항생제를 처리하였다. 세포벽합성 저해제로 알려져 있는 penicillin계와 cephalosporin계 항생제를 각각 3종씩 처리한 결과, methicillin과 cefamandole은 10mM 농도에서 elastase 활성을 50% 이상 저해하였지만, 나머지는 거의 10% 미만이었다. 단백질합성 저해제 중 oxytetracycline의 elastase에 대한 저해효과는 10mM 농도에서 95% 이상으로 매우 탁월하였으며 (IC₅₀=0.3mM), gentamicin도 50% 이상 저해하였으나, 다른 aminoglycoside나 chloramphenicol은 역시 10%미만이었다. 실험해 본 항생제 가운데, oxytetracycline, cefamandole, methicillin, gentamicin 등은 elastase에 대한 강력한 저해제였으며, 그 작용기전은 항생제의 알려진 약리학적 기전과는 다른 차원의 모델임이 분명하였다. Human neutrophil elastase (HNE, EC 3, 4 21, 11), a major causative factor in the induction of pulmonary emphysema, were purified by two steps of liquid chromatography. Purified elastases were cross-reacted with antibody to human neutrophil elastases. Methicillin and cefamandole, which are known as inhibitors of cell wall synthesis of microorganisms, could inhibit the activity of human neutrophil elastase up to 50% with 10mM of both agents and IC₅₀ of methicillin was 9.8 mM. Gentamicin, one of the aminoglycosides, also inhibits human neutrophil elastases up to 60% of original activity with 10 mM of this agent and IC₅₀ was 9.0 mM. We could demonstrate similar effects in oxytetracycline. 10 mM of oxytetracycline inhibited 95% of human neutrophil elastase and IC₅₀ was 0.3 mM. Overall, oxytetracycline, cefamandole and methicillin are strong inhibitors of human neutrophil elastase, and they could be a drug of cholice for the diseases which were known as pathogenesis related to elastase. We also suggest that the mechanism of action of these antibitics are different from the mechanism of antimicrobial effects like inhibition of both cell wall synthesis and protein synthesis.

Symposium 1 : Metabolic Pathway and Enzyme Regulation ; Molecular physiological regulation of pyrimidine nucleotide synthesis in bacterial cells

김사열 ( Sa Youl Ghim ) 생화학분자생물학회 2000 생화학분자생물학회 추계학술발표논문집 Vol.2000 No.-

Bacillus amyloliquefaciens 액화형 α- amylase 유전자의 클로닝 및 Bacillus subtilis 에서의 발현

김사열(Sa - Youl Ghim),송방호(Bang - Ho Song),이인구(In - Koo Rhee),서정훈(Jung - Hwn Seu),홍순덕(Soon - Duck Hong) 한국미생물생명공학회 1986 한국미생물·생명공학회지 Vol.14 No.6

RAPID PURIFICATION AND MOLECULAR CHARACTERIZATION OF HUMAN NEUTROPHIL ELASTASE

김사열(Ghim Sa-Youl),배성준,강구일 고신대학교(의대) 고신대학교 의과대학 학술지 1989 고신대학교 의과대학 학술지 Vol.5 No.1

Elastase에 의해 야기되는 질병의 예방 및 치료 응용을 위하여는, 먼저 이 효소가 고순도로 분리되고 그 근본적인 성질의 해명이 이루어져야 한다. 고순도의 elastase를 얻기위해, 사람의 전혈로부터 Hypaque-Ficoll step gradient에 의해 순수한 neutrophil을 얻은 다음 이것을 extract로 만들어 액체크로마토그래피를 실시하였다. Ultrogel AcA54 gel filtration을 거친 후 CM-sephadex 이온교환 크로마토그래피를 적용하였는데, 이때 유출 완충액 속에 0.1% Brij 35를 첨가하여 elastase의 순도를 더욱 높일 수 있었다. 이 정제된 elastase는 SDS-PAGE를 통해 MW 28, 000, 29, 000, 30, 000의 서로 다른 3개의 밴드로 확인되었으며, 그간 수많은 연구자들간에 elastase의 분자량에 대해 심한 견해 차이를 보여온 것은 그 분자적 성질이나 분자량이 유사한 cathepsin G가 혼재하거나 elastase분자내 혹은 분자간 S-S결합의 차이에 의함을 알 수 있었다.

독도 주변의 해수에서 분리한 세균의 다양성과 군집구조 분석

김사열 ( Sa Youl Ghim ),성혜리 ( Hye Ri Sung ) 한국미생물생명공학회 2010 한국미생물·생명공학회지 Vol.38 No.3

One hundred sixty three strains showing different colony morphological characteristics on different concentration of marine agar (MA) plates were isolated from ambient seawater near Dokdo island. Bacterial diversity and distributions were studied by phylogenetic analysis of the partial 16S rRNA gene sequences. One hundred sixty three strains were partially sequenced and analyzed phylogenetically. They were composed of 5 phyla, of which gamma-proteobacteria (58%), alpha-proteobacteria (20%), bacteriodetes (16%) were predominant. They were affiliated with 90 species. The 16S rRNA sequence similarity of the isolates was in 93.3 to 100 % range to reported sequence data. Thirty six isolates of among them were assumed to be novel species candidates based on similarity analysis of the 16S rRNA gene sequences. Overall, Proteobacteria and Bacteriodetes of the Dokdo coastal sea water showed a high diversity.

Antibiotics; Methicillin, Cefamandole and Oxytetracycline, Can Modulate the Activity of Human Neutrophil Elastases

김사열,정혜영,배성준,강구일,Ghim, Sa-Youl,Jeong, Hye-Young,Bae, Sung-Jun,Kang, Koo-Il The Korean Society of Pharmacology 1989 대한약리학잡지 Vol.25 No.1

Human neutrophil elastase (HNE, EC 3, 4 21, 11), a major causative factor in the induction of pulmonary emphysema, were purified by two steps of liquid chromatography. Purified elastases were cross-reacted with antibody to human neutrophil elastases. Methicillin and cefamandole, which are known as inhibitors of cell wall synthesis of microorganisms, could inhibit the activity of human neutrophil elastase up to 50% with 10mM of both agents and $IC_{50}$ of methicillin was 9.8 mM. Gentamicin, one of the aminoglycosides, also inhibits human neutrophil elastases up to 60% of original activity with 10 mM of this agent and $IC_{50}$ was 9.0 mM. We could demonstrate similar effects in oxytetracycline. 10 mM of oxytetracycline inhibited 95% of human neutrophil elastase and $IC_{50}$ was 0.3 mM. Overall, oxytetracycline, cefamandole and methicillin are strong inhibitors of human neutrophil elastase, and they could be a drug of cholice for the diseases which were known as pathogenesis related to elastase. We also suggest that the mechanism of action of these antibitics are different from the mechanism of antimicrobial effects like inhibition of both cell wall synthesis and protein synthesis. 사람 혈액속의 elastase와 관련된 질병에 대한 연구는 다양한 저해제의 개발을 동반해 왔으며, 최근 항생제도 그 관심대상이 되고 있다. 두 단계의 액체 크로마토그래피를 거쳐 얻은 고순도의 elastase에 12종의 항생제를 처리하였다. 세포벽합성 저해제로 알려져 있는 penicillin계와 cephalosporin계 항생제를 각각 3종씩 처리한 결과, methicillin과 cefamandole은 10mM 농도에서 elastase 활성을 50% 이상 저해하였지만, 나머지는 거의 10% 미만이었다. 단백질합성 저해제 중 oxytetracycline의 elastase에 대한 저해효과는 10mM 농도에서 95% 이상으로 매우 탁월하였으며 $(IC_{50}=0.3mM)$, gentamicin도 50% 이상 저해하였으나, 다른 aminoglycoside나 chloramphenicol은 역시 10%미만이었다. 실험해 본 항생제 가운데, oxytetracycline, cefamandole, methicillin, gentamicin 등은 elastase에 대한 강력한 저해제였으며, 그 작용기전은 항생제의 알려진 약리학적 기전과는 다른 차원의 모델임이 분명하였다.

Purification and Characterization of Human Neutrophil Elastase and Cathepsin G

강구일,김사열,정혜영,배성준,Kang, Koo-Il,Ghim, Sa-Youl,Joung, Hye-Young,Bae, Sung-Jun 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.4

사람의 중성구 elastase와 cathepsin G를 젤 여과법과 이온교환크로마토그라프에 의하여 분리하였다. 이들 두 효소는 각각 항체에 대한 특이한 면역반응을 보였다. Elastase의 분자량은 29K, 30K, 30.5K이며 cathepsin G의 분자량은 28.5K, 29K이었다. 이 두 효소들의 분자량보다 2 kilodalton 큰 분자들도 존재하나 이들 분자는 분자 내의 s-s 결합의 분해에 의한 SDS-PAGE상의 분자이동의 차이에서 생긴 현상으로 생각된다. 일가이온인 $Na^+$, $Li^+$, $K^+$ 및 $Cs^+$는 농도에 따른 elastase의 활성도에 영향을 주고 2가이온은 40 mM까지는 농도에 비례하여 활성도를 증가시키고 그 이후는 plateau에 도달하였다. lmM 미만의 DIFP, PMSF, ${\alpha}_1-PI$ 및 ${\alpha}_2-MG$은 사람 중성구 elastase의 활성도를 완전히 억제하였으나 같은 부류에 속하는 leupeptin은 별영향이 없었다. 이것으로 보아 elastase의 활성도 억제는 active site 이외의 다른 기전이 중요한 역할을 하는 것으로 사료된다. Human neutrophil elastase(HNE) and human neutrophil cathepsin G (HNCG) were purified by a two-step procedure involving gel filtration through Ultrogel AcA54 and ion-exchange chromatography through CM-sephadex C-25. The purified elastase and cathepsin G cross-reacted with anti-HNE antibody and anti-HNCG antibody respectively. Three elastases have molecular weights of 29,000, 30,000, and 30,500 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two cathepsin Gs have the molecular weights of 28,500 and 29,000. There was another group of elastases which showed molecular weights of 2000 dalton higher than the other group of elastase. The discrepancy or the diversity of molecular weights of HNE seems to be caused mainly by the disparity of intra-molecular disulfide bonds of HNE. Monovalent ions including $Na^+$, $Li^+$, $K^+$ and $Cs^+$ stimulated HNE by concentration dependency. Divalent ions also stimulated HNE very effectively at the concentration of less than 40 mM and then reached the plateau. HNE was completely inhibited by less than 1 mM of diisopropyl fluorophosphate (DIFP), phenylmethylsulfonyl fluoride (PMSF), ${\alpha}_1$-protease inhibitor $({\alpha}_1-PI)$, and ${\alpha}_2$-macroglobulin$({\alpha}_2-MG)$ which are catagorized as general endoprotease inhibitor. But leupeptin, which is known as serine and thiol-protease inhibitor, was ineffective on inhibition of HNE.

액체 크로마토그라피에 의한 Human Neutrophil Granular Proteases의 분리

정혜영,김사열(Ghim Sa-Youl),강구일 고신대학교(의대) 고신대학교 의과대학 학술지 1987 고신대학교 의과대학 학술지 Vol.3 No.1

Human leukocyte elastases were purified by gel filtration and ion exchange-chromatography. For gel filtration step, Ultrogel AcA 54 gave extremly good resolution and separation for highly positive charged proteins. Ultrogel AcA 54 also showed excellent separation characteristics of two different size of highly positive charged molecules like elastase and cathepsin G. Both CM-Cellulose and CM-Sephadex showed the possibility to separate the individual molecules of three isozymes. CM-Sephadex showed the better resolution for separation of individual isozyme without contamination of cathepsin G molecules.

미생물의 방해석 석출 작용을 이용한 자기보수 스마트 콘크리트 개발에 관한 연구

김화중(Wha-Jung Kim),김사열(Sa-Youl Ghim),박성진(Sung-Jin Park),최길준(Kil-Jun Choi),천우영(Woo-Young Chun) 한국콘크리트학회 2010 콘크리트학회논문집 Vol.22 No.4

독도서식 식물근권에서 분리한 포자형성세균과 질소고정세균의 군집구조 분석

전선애,성혜리,박유미,박재홍,김사열,Jeon, Seon-Ae,Sung, Hye-Ri,Park, Yu-Mi,Pak, Jae-Hong,Ghim, Sa-Youl 한국미생물·생명공학회 2009 한국미생물·생명공학회지 Vol.24 No.3

Bacteria were isolated from roots of plants belonging to family Solanaceae and Gramineae, inhabited in Dokdo island. Fifty six endospore-forming bacteria grown on tryptic soy broth (TSB) agar medium and 23 nitrogen-fixing bacteria (NFB) grown on nitrogen free agar medium were isolated, respectively. The isolates were partially identified by analyzing the 16S rDNA and categorized into phylogenetic groups. The 16S rDNA sequences of each identified isolates were compared with sequences of each type strains to analyze phylogenetic relationship by phylogenetic tree. As a result, endospore-forming bacteria and nitrogen-fixing bacteria were classified into 4 and 6 lineage groups, respectively. Among these isolated, 18 were presumed to be novel species candidates based on the similarity (lower than 98%) analysis of the l6S rDNA sequences.