Vibrio vulnificus 균이 생산하는 Protease의 특성 및 Hemolysin활성에 미치는 영향

서수영(Su-Yeong Seo),정민호(Min-Ho Jeong) 大韓微生物學會 1992 大韓微生物學會誌 Vol.27 No.3

SCK 선암 세포주에서 방사선에 의한 apoptosis와 세포 주기

이형식(Hyung Sik Lee),박홍규(Hong Kyu Park),허원주(Won Joo Hur),서수영(Su Yeong Seo),이상화(Sang Hwa Lee),정민호(Min Ho Jung),박헌주(Heon Joo Park),송창원(Chang Won Song) 대한방사선종양학회 1998 Radiation Oncology Journal Vol.16 No.2

목 적 : SCK 선암 세포주에서 방사선 조사에 의해 일어나는 apoptosis와 세포 주기와의 연관성을 규명하고자하였다. 대상 및 방법 : SCK 선암 세포주를 apoptosis의 통상적인 정성 분석 방법인 agarose gel electrophoresis 방법을 이용하여 방사선 조사량과 배지 pH 환경과의 연구에서 2-12Gy의 방사선 조사량과 pH 7.5 및 6.6의 배양 배지 조건하에서 다양한 배양 시간의 경과에 따른 DNA fragmentation의 지표인 laddering을 관찰하였다. 실험 조작으로 apoptosis가 유발된 세포군을 정량적으로 분석하고 세포 주기 분석을 위해 FACScan을 이용하였다. 결 과 : apoptosis가 왕성히 발현되었던 pH 7.5 배지에서 배양하였던 세포에서는 방사선 조사직후부터 G2/M phase의 세포들의 분획이 증가하기 시작하여 12시간째 약 70%까지의 최고치를 보인 후 36시간째에 방사선을 조사하지 않았던 상태의 분획으로 정상화되었다. 하지만 pH 6.6 배지에서 배양하였던 세포에서의 G2/M phase의 세포들의 분획의 증가는 pH 7.5 배지에서 배양하였던 세포들에 비해 비교적 천천히 일어나고 그 최고치도 24시간째에 약 45%로 관찰되었다. 특이한 것은 G2/M phase의 세포들의 분획이 그 이후 감소되는 정도가 pH 7.5 배지에서 배양하였던 세포들에 비해 미약하여 48시간 배양 이후에도 약 30-35%의 세포는 G2/M phase의 세포들의 분획으로 관찰되었다. 결 론 : 연구자들은 이러한 현상이 세포들이 G2/M phase에서 많은 양의 세포들이 집적되어 세포주기를 순환하지 못하는 G2/M arrest 현상으로 이해하였다. 세포 내외의 산성환경 상태에서 방사선을 조사 받은 SCK 종양세포는 G2/M arrest 상태가 지속되며 이는 post- mitotic apoptosis를 억제한다고 추론하였다. Purpose : The relationship between environmental pH on the radiation induced-apoptosis in SCK mammary adenocarcinoma cells and cell cycle dependence was investigated. Material and Methods : Mammary adenocarcinoma cells of A/J mice(SCK cells) in exponential growth phase were irradiated with a 137Cs irradiator at room temperature. The cells were irradiated 1 hour after the media was replaced with fresh media at a different pHs. After incubation at 37℃ for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. Results : The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. When the cells were irradiated and maintained in pH 7.5 medium, the percentage of cells in G2/M phase rapidly increased to about 70% at 12 h after an exposure to 12Gy and returned to control level by 36 h. The percentage of cells in G1 phase decreased as the percentage of cells in G2/M increased. On the other hand, in pH 6.6 medium the percentage of cells in G2/M phases gradually increased to about 45% at 24 h after 12Gy irradiation and then slowly recessed and consequently, as much as 30-35% of the cells were still in the G2/M phase 48h after irradiation. The percent age of cells in G1 phase then increased as the G2/M arrest began to recede. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. Conclusion : Radiation-induced apoptosis in SCK tumor cells are reversely suppressed in an acidic environment. Radiation-induced G2/M arrest is prolonged in an acidic environment indicating that the suppression of radiation- induced apoptosis and prolongation of radiation-induced G2/M arrest in an acidic environment are related.

Murine Transfer factor의 정제와 활성도의 시험관내 측정에 관한 연구

오양효(Yang Hyo Oh),김영부(Young Bu Kim),정민호(Min Ho Jeong),최귀전(Gui Jeon Choi),서수영(Yeong Seo) 大韓微生物學會 1990 大韓微生物學會誌 Vol.25 No.6

Impact of Cyclooxygenase-2 Expression on the Survival of Glioblastoma

Youngmin Choi(최영민),Dae-Cheol Kim(김대철),Ki-Uk Kim(김기욱),Young-Jin Song(송영진),Hyung-Sik Lee(이형식),Won-Joo Hur(허원주),Sun-Seob Choi(최순섭),Su-Yeong Seo(서수영) 대한방사선종양학회 2007 Radiation Oncology Journal Vol.25 No.3

목 적: 다형성아교모세포종 환자들에서 cyclooxygenase-2 (COX-2) 단백의 발현 정도와 생존율에 미치는 영향을 조사 하고자 한다. 대상 및 방법: 1997년부터 2006년까지 다형성아교모세포종으로 수술 및 방사선치료를 받은 환자들 중에서, 의식 상태의 악화로 40 Gy 전에 방사선치료가 중단된 3명을 제외한 30명을 대상으로 하였다. 조직에서의 COX-2의 발현은 면역조직화학염색으로 검사하였다. 생존 분석과 성별, 나이, 활동도, 수술 정도, 방사선량, COX-2 발현 정도 등이 생존율에 미치는 영향을 Kaplan Meier 법과 log rank test로 분석 및 검증하였다. 결 과: 중앙추적관찰기간은 13.3개월이었다(6∼83개월). 전체 환자들에서 COX-2의 발현이 관찰되었고, 종양 세포의 5% 이상에서 COX-2가 양성이었던 환자가 24명이었다: 종양 세포의 25% 미만, 3명(10.0%); 25∼50%, 1명(3.3%);50∼75%, 2명(6.7%); 75∼100%, 24명(80.0%). 중앙생존기간이 13.5개월이었고, 2년 생존율은 17.5%였다. 수술 정도(50% 이상 종양 제거)와 방사선량(59 Gy 이상 조사)이 생존율에 유의하게 영향을 주었다(p<0.05). 종양 세포의 75% 미만에서 COX-2가 발현되었던 환자군과 75% 이상에서 발현되었던 환자군에서 중앙생존기간은 각각 15.5개월과 13.0개월이었고(p>0.05), 2년 생존율은 각각 33.3%와 13.3%였다(p>0.05). 결 론: 다형성아교모세포종에서의 COX-2 양성도는 높았지만, 다형성아교모세포종 환자들에서 COX-2 발현의 정도와생존율 간에는 통계적인 유의성이 없었으므로, 향후 보다 많은 환자들을 대상으로 COX-2 발현 정도가 생존율에 미치 는 영향에 대한 연구가 필요하다. Purpose: To investigate the degree and effect of cyclooxygenase (COX)-2 expression on the survival of patients with glioblastoma multiforme (GM). Materials and Methods: Between 1997 and 2006, thirty consecutive GM patients treated with surgery and postoperative radiotherapy (dose range: 44∼65.1 Gy, median dose: 61.2 Gy) were included in the study. Three patients were excluded that discontinued radiotherapy before receiving a dose of 40 Gy due to mental deterioration. The expression of the COX-2 protein in surgical specimens was examined by immunohistochemical analysis. Survival analysis and verification were performed with respect to sex, age, performance status, resection extent, radiotherapy dose, and degree of COX-2 expression using the Kaplan-Meier method and the log rank test. Results: The median length of follow-up was 13.3 months (range: 6∼83 months). Staining for COX-2 was positive in all patient samples. Staining for COX-2 that was positive for over 75% of the tumor cells was found in 24 patients. Staining for COX-2 that was positive in less than 25% of tumor cells was found in 3 patients (10.0%), staining for COX-2 that was positive in 25 to 50% of tumor cells was found in 1 patient (3.3%), staining for COX-2 that was positive in 50 to 75% of tumor cells was found in 2 patients (6.7%) and staining for COX-2 that was positive in 75 to 100% of tumor cells was found in 24 patients (80.0%). The median survival and two-year survival rate were 13.5 months and 17.5%, respectively. The survival rate was influenced significantly by the degree of resection (tumor removal by 50% or more) and radiotherapy dose (59 Gy or greater) (p<0.05). The median survival of patients with staining for COX-2 that was positive in less than 75% of tumor cells and in at least 75% of tumor cells was 15.5 and 13.0 months, respectively (p> 0.05), and the two-year survival for these groups was 33.3 and 13.3%, respectively (p>0.05). Conclusion: The absence of a statistical correlation between the degree of COX-2 expression and survival in GM patients, despite the high rate of COX-2 positive tumor cells in the GM patient samples, requires further studies with a larger series to ascertain the prognostic value of the degree of COX-2 expression in GM patients.

생쥐 대식세포에 대한 SNP의 Apoptosis 유발 효과

정민호,이상화,서수영,송진미,김화숙,박선미,이성태,윤식 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.3

Endogenously generated Nitric oxide(NO) redox species by the activation of inducible NO synthase(iNOS) induce apoptotic cell death in macrophages. In this experiments to exclude possible interference with NOS induction, we examined the ability of NO generating compound, sodium nitroprusside(SNP) to induce apoptotic cell death of murine macrophages(J774A.1, RAW 264.7, and thioglycollate-induced peritoneal macrophage). The induction of apoptosis was sup-ported by the absence of significant trypan blue staining but the occurrence of biochemical and morphological apoptotic markers of the increased DNA fragmentation. For the quantitation of apoptosis we used relatively simple and effective method, diphenylarnine(DPA) colorimetric as-say which was confirmed by DNA ladder and TUNEL stain. Incubation of J774A.1 macrophages with increasing concentrations of SNP(0.5-3mM) for 8 hr led to concentration de-pendent apoptotic response, but with higher concentrations cell viability and DNA fragmentation started to decline. Apoptosis was rapid at 1mM and 2mM SNP in J774A.l macrophages, yielding specific DNA fragmentation after 4 and 3 hr with no further increase or slight decrease in fragmentation after 8 and 10 hr, respectively. As the concentration of SNP was increased, the time course of apoptosis was shortened. Increased apoptotic cells of late stage cause decreased cell viability and DNA fragmentation by the loss of cell membrane integrity and DNA de-gradation. As a results incubation with 1mM SNP for 5 hr is sufficient to induce apoptosis in J 774A.1 macrophages. Detailed kinetic studies revealed that there was no direct correleation between nitrite(NO2-) accumulation and death-eliciting potency. Nitrite , a final NO oxidation product, cannot correlated with various NO-related redox forms possibly involved in a apoptotic signaling. In different murine macrophage cell line, RAW264.7, apoptosis was also induced by the incubation of 1mM SNP for 5 hr and the response was not inhibited by the addition of 1mM NG-monomethyl-L-arginine(NMMA), establishing a definite link between exogenously applied NO and apoptotic cell death. In contrast to cell lines there was no induction of apoptosis in peritoneal macrophages and J774A.1 cultured with thioglycollate. Angry state of thioglycollate induced in-Rammed macrophages may suppress NO-induced apoptosis compared to fully activated ma-crophages which express a high level of iNOS, produce large quantities of NO, and perform self destruction. The balance between apoptosis-promoting and opposing signals may regulate the susceptibility of macrophages to NO-mediated apoptosis. Therefore under inflammatory conditions, there may be a tendency to maintain cytotoxic activities of macrophages against pathogens before elimination of activated macrophages by an apoptotic process after massive stimulation.

Klebsiella pneumoniae에 의해 유도된 생쥐 복강 대식세포의 Apoptosis

이상화,김소선,정민호,서수영,송진미,김화숙,이성태,박선미,김정만 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.4

Klebsiella pneumoniae, a member of the family Enterobacteriaceae, is considered an important etiological agent in nosocomial infection. In this study, we show that K. pneumoniae strains were cytotoxic for the murine peritoneal macrophages, and cell death induced by these bacteria occurred through apoptosis, as shown by the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether the progress of apoptosis induced by these bacteria could be influenced by the inoculum size of K pneumoniae. Apoptotic cell death induced by K. pneumoniae was relatively affected by the inoculum size of bacteria. During the cultivation of murine peritoneal macrophages infected with K. pneumoniae, the time course experiment of nitrite concentration in the culture medium was not consistent with other experiments. Therefore we investigated the source of nitrite in the culture medium. Nitrite, a member of reactive nitrogen intermediates as antimicrobial and antitumoral effector molecules, could be produced profoundly by not only mammalian cells but also microorganisms. The amount of nitrite produced by six K. pneumoniae ranged from 50 to 326 M/ml and each strain produced nitrite maximally during exponential growth period. In these experimental system, it should be considered that the ability of bacteria to produce and consume nitrite might confuse the interpretation of experimental results. The ability of K. pneumoniae to promote the apoptosis of murine peritoneal macrophages may be important for the initiation of infection and the development of nosocomial infection. The understanding of the mechanism of apoptosis induced by K. pneumoniae could be useful in prevention and therapy of infectious diseases caused by this microorganism.

Dye-Ligand Affinity Chromatography를 이용한 Staphylococcus aureus의 장독소 B 분리

박선미,정민호,이상화,서수영,송진미,김화숙,이성태,임영진 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.4

Staphylococcal enterotoxins, which cause staphylococcal food poisoning, have been purified by several methods. These procedures have usually included various combinations of ion-exchange chromatogram and gel filtration; such procedures can be time-consuming and may also result in low recoveries of enterotoxins. Although high recoveries of staphylococcal enterotoxins have been reported with chromatofocusing, this technique is expensive and cannot easily be adapted for large-scale purification. In this study we applied a simple, single-step procedure for the purification of Staphylococcal enterotoxin B (SEB) from culture supernatant fluids, namely, dye ligand affinity chromatography in which dye is coupled to an agarose support matrix. Dye ligand affinity chromatography has been used to purify a wide range of enzymes and some blood proteins. We used ten kinds of dye ligand affinity columns. The results showed that SEB had the binding ability to Red A, Reactive Green 19, Reactive Green 5, Reactive Brown 10 and Cibacron Blue 3GA-Agarose. We had the highest resolution by using the Reactive Green 19 dye affinity column. The purified SEB produced a single band when subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blot using anti-SEB. We could also confirm that the purified SEB produced superantigenic effect. Therefore, it could be concluded that SEB was produced purely in single step process by dye-ligand affinity chromatography. Such a method will form the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.