Inhibitory Role of Polyamines in Dexamethasone-induced Apoptosis of Mouse Thymocytes

최상현,김용훈,홍기현,신경호,천연숙,전보권,Choi, Sang-Hyun,Kim, Yong-Hoon,Hong, Gi-Hyun,Shin, Kyung-Ho,Chun, Yeon-Sook,Chun, Boe-Gwun The Korean Society of Pharmacology 1996 대한약리학잡지 Vol.32 No.1

세포내 polyamine은 DNA 구조 뿐 아니라 전사과정, 세포의 성장, 분화, 및 증식 등에 간여하는 바, 배양 흉선세포의 apoptosis 을 억제한다고 한다. 따라서 dexamethasone에 의한 생쥐 흉선세포의 apoptosis 반응에 대한 polyamine의 억제작용을, polyamine 생성과 대사억제제들로 처치한 흉선세포의 일차배양실험에서 관찰하여, 그 결과를 A23187과 DHEA의 작용과 비교하였다. 1) 흉선세포 생존율이 dexamethasone, DHEA, A23187, DFMO, MGBG들에 의하여 직접 현저히 억제되며, aminoguanidine, putrescine, spermidine, 및 spermine들에 의해서는 영향을 받지 않았다. 2) 흉선세포 DNA의 분절화가 dexamethasone과 A2318T에 의하여 유의하게 증강되어 있으며 DHEA에 의하여도 다소 증가되었으나, DFMO, MGBG, aminoguanidine, putrescine, spermidine, 및 spermine들에 의하여는 크게 영향을 받지 않았다. 3) Dexamethasone에 의한 흉선세포의 apoptosis는 DHEA에 의하여 억제된 반면, DFMO, MGBG, 및 aminoguanidine에 의하여는 영향을 받지 않았다. Spermine은 dexamethasone과 A23187에 의한 세포생존율 감소를 유의하게 억제하였으며, A23187에 의한 세포생존율 감소는 putrescine과 spermidine에 의하여도 억제되는 경향을 보였다. 4) DFMO 및 MGBG에 의한 흉선세포 생존율 감소는 spermine에 의해 현저히 억제되었으나, putrescine과 spermidine에 의하여는 영향을 받지 않았다. 5) Dexamethasone을 DFMO 또는 MGBG와 병합처치하여 나타나는 흉선세포 생존율 감소는 각각 spermine과 putrescine에 의하여 유의하게 억제되었으나, aminoguanidine 또는 DHEA와 dexamethasone의 병합처치에 의한 생존율 감소는 polyamine 전처치에 의해 감소되지 않았다. 이상의 결과는 polyamine이 흉선세포의 apoptosis 반응을 억제할 수 있고, 이같은 억제효과의일부가 $[Ca^{2+}]_i$ 증가에 관련되는 신호전달과정과 연관될 뿐 아니라, 세포막의 polyamine transporter를 통한 polyamine 섭취가 이들의 생합성 또는 유리기능과 함께 세포내 polyamine 함량을 조정하므로, 흉선세포의 apoptosis에 억제적으로 작용할 수 있음을 시사하는 것으로 사료된다. It has been well known that polyamines ensure the stability of chromatin structure and the fidelity of DNA transcription. This study was carried out to evaluate the effect of polyamines on the apoptosis of mouse thymocytes induced by dexamethasone and polyamine synthesis inhibitors. 1) In the histological death findings of thymocytes double-stained with acridine orange and ethidium bromide, the apoptotic and the necrotic fractions (AF; NF) in the control group were $9.4{\pm}4.2%$ and $4.5{\pm}5.3%$, respectively. Dexamethasone $(3\;{\times}\;10^{-8}\;M:\;DX)$ in creased AF upto $52.0{\pm}8.1%$ and did not change NF, but A23187 $(5\;{\times}\;10^{-7}\;M:\;A2)$ increased AF and NF upto $45.0{\pm}8.9%$ and $20.5{\pm}10.6%$, respectively. 2) The thymocyte viability was significantly reduced by DX, DHEA $(1\;{\times}\;10^{-4}\;M)$, A2, DFMO $(1\;{\times}\;10^{-4}\;M)$, and $MGBG\;(1\;{\times}\;10^{-4}\;M)$, respectively. It was, however, little affected by $aminoguanidine\;(1\;{\times}\;10^{-4}\;M:\;AG)$, $putrescine\;(1\;{\times}\;10^{-5}\;M:\;PT)$, $spermidine\;(1\;{\times}\;10^{-5}\;M:\;SD)$, and $spermine\;(1\;{\times}\;10^{-5}\;M:\;SM)$. 3) The genomic DNA of mouse thymocyte was markedly fragmented by DX and A2, respectively, and to a lesser extent, by DHEA, but was little affected by MGBG, DFMO, AG, and each of polyamines. 4) The DX induced reduction of thymocyte viability was moderately attenuated by DHEA, but little affected by DFMO, MGBC, and AG. However, SM significantly attenuated the viability reduction induced by A2 as well as DX. 5) The thymocyte viability reduction by MGBG and DFMO was significantly attenuated by only SM among three polyamines applied in this study. 6) The thymocyte viability redution by combined treatments of DX with DFMO and MGBG, respectively, was significantly attenuated by SM, and moderately by PT. But the viability reduction by combined treatment of DX with AG or DHEA was not affected by polyamines. These results suggest that polyamines, particularly spermine, might play the inhibitory role in thymocyte apoptosis and the inhibitory effect can be ascribed in part to the increase of polyamine uptake by thymocytes pretreated with DFMO and MGBG.

뇌허혈 손상에 있어서 Polyamine 대사의 변동이 해마신경세포의 지연성괴사에 미치는 효과에 관한 연구

신경호(Kyung-Ho Shin),신화정(Hwa-Jung Shin),이영재(Young-Jae Lee),김형건(Hyung-Gun Kim),최상현(Sang-Hyun Choi),천연숙(Yeon-Sook Chun),전보권(Boe-Gwun Chun) 대한약리학회 1996 대한약리학잡지 Vol.32 No.3

웅성 모래쥐의 양경동맥을 7분간 폐쇄하여(OBC7) 뇌의 허혈을 유발하여, 해마의 microdialysate내 polyamine 함량과 조직내 polyamine 생합성효소(ornithine decarboxylase: ODC와 S-adenosylmethionine decarboxylase: SAM-DC)의 활성도를 분석하고, 해마의 cresyl violet(CV) 염색과 glial fibrillary acidic protein(GFAP) 면역염색소견들을 관찰하여, 허혈성 해마의 신경손상과 polyamine대사의 연관성을 검토하였다. 1) OBC부하 후, 해마의 dialysate내 polyamine 변동에서, putrescine(PT)은 현저히 증가되었으나, spermidine과 spermine은 다소 감소되는 경향을 보였고, 이에 해마조직내 ODC활성의 현저한 상승과 SAM-DC활성도의 유의한 저학 동반되었다. 2) Difluoromethylornithine(DFMO)는 OBC에 의한 PT증가와 ODC활성도 상승을 유의하게 억제하였으나, methylglyoxal bis(guanylhydrazone)(MGBG)는 각각 다소 억제하는 경향을 보였다. 3) OBC부하 7일후에 관찰한 조직소견에서, 해마의 CA1 부위의 유의한 신경손상이 유도되었으나 CA3와 dentate gyrus 부위에는 미약한 손상만 보였으며, GFAP 양성반응도 CA1 부위에서만 유의한 증가를 보였다. 이같은 소견들은 DFMO에 의하여 크게 영향을 받지 않았으나 MGBG에 의하여 유의하게 억제되었다. 이상의 성적들은 해마 polyamine의 과도한 허혈성 증가가 허혈성 신경손상에 관여할 수 있으나, 한편으로 polyamine대사의 과도한 억제도 허혈성 신경손상을 악화시킬 수 있으며, 허혈성뇌손상에 대한 MGBG의 보호작용은 polyamine 대사보다는 다른 작용에 매개되는 것으로 사료된다. Male Mongolian gerbils (60 ~ 80g) were given DL-difluoromethylornithine (DFMO; 250mg/kg, ip) and methylglyoxal bis(guanylhydrazone) (MGBG; 50 mg/k, ip), respectively, 1 h prior to transient (7 min) occlusion of bilateral common carotid arteries (OBC7) and a daily dose of one of them for 6 days after recirculation, and the polyamine contents, activities of ornithine and S-adenosylmethionine decarboxylases (ODC and SAM-DC), and light microscopic findings of the hippocampus were evaluated. The hippocampal putrescine (PT) levels of the control gerbils treated with saline (STGr), markedly increased after OBC7, showing a peak level at 24 h after recirculation. The peak PT level was reduced in DFMO treated gerbils (DTCr) and in MGBG treated gerbils (MTGr). And 7 days after recirculation, the PT level of DTGr was decreased to about 75% of the PT level in the sham operated group (nonTGr) and to about 55% of the STGr level, respectively. The hippocampal spermidine (SD) level of STGr tended to decline, showing the lowest value at 8 h after recirculation. But the spermidine (SD) level of DTGr was somewhat higher at 8 h after OBC7 than those of STGr and MTGr The hippocampal spermine (SM) levels of all the experimental groups were little changed for 7 days after OBC. OBC7 markedly increased the hippocampal ODC activity. reaching a maximum (about 3 times higher than preischemic level) at 8 h and rapidly recovered to the control value by 24 h in STGr gerbils, and the OBC7-induced increase of ODC activity was significantly attenuated by DFMO or MGBG treatment. Whereas OBC7 induced a rapid decrease of the hippocampal SAMDC activity follwed by gradual recovery to the preischemic level, and the decrease of the SAMDC activity was slightly attenuated by DFMO or MGBG treatment. 7 Days after OBC7 the histological finding of the hippocampal complex stained with cresyl violet showed an extensive delayed neuronal damage in the CA1 region and to a lesser extent, in the dentate gyrus, sparing the CA3 region. And the neuronal death was aggevated by DFMO but significantly attenuated by MGBG. The immunochemical reactivity of hippocampus to anti-GFAP antibody was significantly increased in the CA1 region and to a lesser extent, in the dentate gyrus 7 days after OBC7, but was little changed in the CA3. And the increase of the anti-GFAP immunoreactivity was moderately enhanced by DFMO and significantly suppressed by MGBG. These results suggest that the polyamine metabolism may play a modulatory role in the ischemic brain damage.

Changes of Polyamine Metabolism and Delayed Neuronal Degeneration of Hippocampus after Transient Cerebral Ischemia in Mongolian Gerbils

신경호,신화정,이영재,김형건,최상현,천연숙,전보권,Shin, Kyung-Ho,Shin, Hwa-Jung,Lee, Young-Jae,Kim, Hyung-Gun,Choi, Sang-Hyun,Chun, Yeon-Sook,Chun, Boe-Gwun The Korean Society of Pharmacology 1996 대한약리학잡지 Vol.32 No.3

웅성 모래쥐의 양경동맥을 7분간 폐쇄하여(OBC7) 뇌의 허혈을 유발하여, 해마의 microdialysate내 polyamine 함량과 조직내 polyamine 생합성효소(ornithine decarboxylase: ODC와 S-adenosylmethionine decarboxylase: SAM-DC)의 활성도를 분석하고, 해마의 cresyl violet(CV) 염색과 glial fibrillary acidic protein(GFAP) 면역염색소견들을 관찰하여, 허혈성 해마의 신경손상과 polyamine대사의 연관성을 검토하였다. 1) OBC부하 후, 해마의 dialysate내 polyamine 변동에서, putrescine(PT)은 현저히 증가되었으나, spermidine과 spermine은 다소 감소되는 경향을 보였고, 이에 해마조직내 ODC활성의 현저한 상승과 SAM-DC활성도의 유의한 저학 동반되었다. 2) Difluoromethylornithine(DFMO)는 OBC에 의한 PT증가와 ODC활성도 상승을 유의하게 억제하였으나, methylglyoxal bis(guanylhydrazone)(MGBG)는 각각 다소 억제하는 경향을 보였다. 3) OBC부하 7일후에 관찰한 조직소견에서, 해마의 CA1 부위의 유의한 신경손상이 유도되었으나 CA3와 dentate gyrus 부위에는 미약한 손상만 보였으며, GFAP 양성반응도 CA1 부위에서만 유의한 증가를 보였다. 이같은 소견들은 DFMO에 의하여 크게 영향을 받지 않았으나 MGBG에 의하여 유의하게 억제되었다. 이상의 성적들은 해마 polyamine의 과도한 허혈성 증가가 허혈성 신경손상에 관여할 수 있으나, 한편으로 polyamine대사의 과도한 억제도 허혈성 신경손상을 악화시킬 수 있으며, 허혈성뇌손상에 대한 MGBG의 보호작용은 polyamine 대사보다는 다른 작용에 매개되는 것으로 사료된다. 가장 많았고 습진과 소양증이 각각 19.5%였다. 30대 환자군에서는 습진이 30.3%였고 소양증이 19.1%였다. 40대에서는 소양증이 28.8%였고 지루성 피부염이 16.0%였다. 50대에서는 습진이 26.7%였고 60세 이상의 그룹에서는 습진이 50.0%, 바이러스 질환과 소양증이 각각 15.0%였다. 이들 결과는 통계적으로 유의한 차이를 보였다($X^2\;=\;74.995^{**}$, p = .001, df : 40). 결론 : 상대적으로 50세 이상 연령충의 대상 환자 수가 많지 않은 원인은 여리 가지 분석이 가능할 것으로 생각된다. 연령에 따라 피부질환이 다른 양상을 보임을 고려할 때 노인층의 신체적, 경제적, 사회적 특성을 이해하고 이에 따른 효과적이고 사회-경제학적 부담을 최소할 수 있는 한방 치료 영역의 확대 및 개발이 필요할 것으로 사려된다.주식 및 후식의 선호도에 남녀 간에 차이를 보이고 있으므로 단체급식을 위한 식단 작성 시 이러한 차이를 고려할 필요가 있으며 대학생 및 성인을 대상으로 한 영양 교육을 실시할 때 이러한 차이를 고려한 신생활 교정프로그램을 작성할 필요가 있다고 사료된다.$은 대조군에 비해 발아에 의하여 세포독성 효과를 증가되었지만, MCF-7와 Caco-2에 대한 항암효과는 없음을 알 수 있었다.것으로 사료된다.높게 인식할수록 재방문의도 및 추천의도가 커지는 것을 알 수 있었다. 대학교 급식소 운영주체에 대한 소비자 인지도 조사결과 향후 대학교 급식소를 운영하는 위탁급식 전문업체의 경우 그들의 브랜드를 알리기 위한 홍보전략이 절실히 필요함을 알 수 있었으며, 최근고객감소로 인하여 다양한 급식운영 마케팅전략을 수립하고 있는 단체급식 운영자들은 재방문 및 추천의도의 선행요건이 급식서비스 품질에 대한 소비자의 인식임을 명심하여 가장 기본이 되는 급식서비스 품질수준을 향상시키기 위하여 노력하여야 할 것이다. Male Mongolian gerbils $(60{\sim}80g)$ were given DL-difluoromethylornithine (DFMO; 250mg/kg, ip) and methylglyoxal bis(guanylhydrazone) (MGBG; 50 mg/k, ip), respectively, 1 h prior to transient (7 min) occlusion of bilateral common carotid arteries (OBC7) and a daily dose of one of them for 6 days after recirculation, and the polyamine contents, activities of ornithine and S-adenosylmethionine decarboxylases (ODC and SAM-DC), and light microscopic findings of the hippocampus were evaluated. The hippocampal putrescine (PT) levels of the control gerbils treated with saline (STGr), markedly increased after OBC7, showing a peak level at 24 h after recirculation. The peak PT level was reduced in DFMO treated gerbils (DTCr) and in MGBG treated gerbils (MTGr). And 7 days after recirculation, the PT level of DTGr was decreased to about 75% of the PT level in the sham operated group (nonTGr) and to about 55% of the STGr level, respectively. The hippocampal spermidine (SD) level of STGr tended to decline, showing the lowest value at 8 h after recirculation. But the spermidine (SD) level of DTGr was somewhat higher at 8 h after OBC7 than those of STGr and MTGr The hippocampal spermine (SM) levels of all the experimental groups were little changed for 7 days after OBC. OBC7 markedly increased the hippocampal ODC activity. reaching a maximum (about 3 times higher than preischemic level) at 8 h and rapidly recovered to the control value by 24 h in STGr gerbils, and the OBC7-induced increase of ODC activity was significantly attenuated by DFMO or MGBG treatment. Whereas OBC7 induced a rapid decrease of the hippocampal SAMDC activity follwed by gradual recovery to the preischemic level, and the decrease of the SAMDC activity was slightly attenuated by DFMO or MGBG treatment. 7 Days after OBC7 the histological finding of the hippocampal complex stained with cresyl violet showed an extensive delayed neuronal damage in the CA1 region and to a lesser extent, in the dentate gyrus, sparing the CA3 region. And the neuronal death was aggevated by DFMO but significantly attenuated by MGBG. The immunochemical reactivity of hippocampus to anti-GFAP antibody was significantly increased in the CA1 region and to a lesser extent, in the dentate gyrus 7 days after OBC7, but was little changed in the CA3. And the increase of the anti-GFAP immunoreactivity was moderately enhanced by DFMO and significantly suppressed by MGBG. These results suggest that the polyamine metabolism may play a modulatory role in the ischemic brain damage.

지베렐린과 Putrescine이 대두 절간의 세포 신장과 분열에 미치는 영향

윤상숙,박홍덕 효성여자대학교 부설 한국환경위생연구소 1991 환경위생연구 Vol.1 No.1

대두 제1절간에 지벨렌린, putrescine, MGBG 및 지베벨린과 'Put 혼합 물질을 농도 및시간에 따라 처리하여, 각 피층의 유조직 세포 크기를 측정하고 세포신장과 분열부위를 관찰하였다. 세포 신장은10^-6M지베렐린의 경우 Ⅱ부위, 10^-2MPut은 I부위,10^-2M MGBG는 I부위이며,처리시간은 모두 60시간에서 세포신장이 가장 활발하였다. 지베렐린과 Put을 섞어 처리한 경우는 단독으로 처리하였을 때 보다 세포 신장에서 큰 효과가나타났으나, 세포분열에는 나타나지 않았다. 대두 첫번째 절간에서 세포 신장 부위와 분열부위는 일치하지 않았다. The zone of cell elongation and cell division was observed in the parenchyma cell of cortex in Glycine max when it was treated with Gibberellin(GA),Putrescine (Put), methylglyoxal-bis(guanylhydrazone) (MGBG) and GA+ Put. The cell elongation was observed in all parts and specially enhanced in Ⅱ,I and I zone of internode treated with GA, Put and MGBG, respectively. Theoptimal concentration was 10^-6M GA, 10mM Put and 10mM MGBG. The cellelongation was enhanced in 60hrs. The cell division was prospered in Ⅲ zone of internode treated with GA,Put and MGBG and the optimal concentration was 10-4M,10mM and 10mM indi-vidually. The cell division was enhanced in 60 hrs. The cell elongation was enhan-ced more GA+ Put than GA and Put independently, but it was not effectivelyin the cell division. The cell elongation zone was not consistent with cell division zone in thefirst internode of soybean.

애기장대 잎 절편 배양시 NAA 농도에 따른 부정근, 모용 및 캘러스 형성에 미치는 Polyamine의 영향

한태진,홍종필,김준철,임창진,진창덕 한국식물생명공학회 2000 식물생명공학회지 Vol.27 No.2

애기장대 (Arabidopsis thaliana) 생태종 'Columbia'의 잎 절편 배양시 부정근, 모용 및 캘러스 형성에 미치는 polyamine의 역할을 알아보고자 putrescine, spermidine, spermine, cyclohexylamine (CHA) 및 methylglyoxal-bis(guanylhydrazone) (MGBG)의 영향을 조사하였다 애기장대 잎 절편을 부정근 (0.1 mg/L NAA), 모용 (2.0 mg/L NAA) 및 캘러스 (10.0mg/L NAA)를 형성하는 MS배지 각각에 putrescine, spermidine 및 spermine을 1∼100 mg/L 처리하였다. Polyamine 처리시 부정근 형성구에서는 부정근과 함께 모용이 형성되었으며, 모용 형성구에서도 모용과 함께 캘러스가 형성되었다 또한 CHA와 MGBG를 1∼100mg/L 범위에서 처리한 결과 CHA는 부정근 형성구에서 다소 부정근이 증가시켰으나 모용이나 캘러스 형성구에는 영향을 미치지 않았다. MGBG는 부정근 모용 및 캘러스 모두를 억제하였고 모용 형성구에서는 고농도에서 부정근을 형성시켰다. In order to study the role of polyamines on the formation of adventitious roots, trichomes and calli, the effects of putrescine, spermidine, spermine, cyclohexylamine (CHA) and methylglyoxal-bis(guanylhydrazone) (MGBG) were investigated in the leaf segment cultures from ecotype Columbia of Arabidopsis thaliana. When the leaf segments were cultured on the media for forming adventitious roots (0.1 mg/L NAA), trichomes (2.0 mg/L NAA) and calli (10.0 mg/L NAA), and then each cultures was treated with 1-100 mg/L of putrescine, spermidine and spermine, respectively. On the adventitious root-forming medium treated with polyamines the trichomes were induced with adventitious roots. And on the trichome-forming medium with polyamines calli were induced with trichomes. In orther hand each cultures was treated with 1-100 mg/L of CHA and MGBG, respectively. CHA promoted adventitious roots on the medium for adventitious roots, was not effected on media for trichomes and calli. MGBG inhibited adventitious roots, trichomes and calli in all cultures, and induced adventitious roots on medium for trichomes in high concentration.

Effect of Polyamines on Adventitious Root Formation from Tobacco (Nicotiana tabaccum) Leaf Segments

Jeon, Myoung Goo,Jo, Hyeong Il,Han, Tae Jin 한국식물학회 1998 Journal of Plant Biology Vol.41 No.1

To elucidate the effect of polyamines on adventitious root formation, we investigated the relationship between the frequency of adventitious root formation and the endogenous content of free polyamines in tobacco leaf segments which had been treated with polyamine biosynthesis inhibitors and polyamines. Adventitious root formation was inhibited in rooting medium (10 μM IAA) with methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine (CHA), and promoted with spermidine an dputrescine. Treatment with high IAA (100 μM) medium plus CHA or MGBG promoted rooting up reversion of the rooting inhibition than the one treated with high IAA concentration alone. Spermidine promoted adventitious root numbers on low IAA (1 μM) medium when applied during culture period. The rooting inductive phase (in the presence of IAA) was determined by periodical transfer of leaf segments from IAA-containing medium to IAA free medium, and by changing polyamine contents, to be inductive phase. Putrescine and spermidine were accumulated to a maximum during the inductive phase. Therefore, the results point out the involvement of polyamines in inductive phase of adventitious root formation in tobacco leaf segments.

Effects of Polyamines on Shoot and Root Development in Arabidopsis Seedlings and Carnation Cultures

Jang, Su Jin,Choi, Yu Jin,Park, Ky Young 한국식물학회 2002 Journal of Plant Biology Vol.45 No.4

Polyamines, a class of aliphatic amines, are active in the growth and development of bacteria, animals, and plants. To better understand their physiological role in plants, we used spermidine and the inhibitor of SAMDC to analyze the developmental patterns of roots and shoots from Arabidopsis seedlings and in-vitro carnation shoot cultures. We also monitored mRNA levels of the polyamine biosynthetic gene after adding various phytohormones to the growing media. Treating wild-type Arabidopsis seedlings with polyamine resulted in greater growth after four weeks; 1 mM spermidine increased root lengths by 39% and seedling weights by 44%. Spermidine was more effective in enhancing root growth in the det2 mutants than in the wild-type plants. In contrast, MGBG, an irreversible inhibitor of SAMDC activity, resulted in root lengths that were 29% of the controls, as well as seedling weights that were only 77% of normal. In our carnation cultures, shoot-growth rates were severely reduced by both spermidine and MGBG; root growth was also markedly inhibited by treatment with spermidine. Transcripts of ADC and SAMDC were significantly greater in cultures treated with kinetin and IAA, with levels being higher with the former. Previous research has shown that sensitivity and the appropriate cellular content for polyamines vary among plant species, and may also be organ- or tissue-specific. Polyamines may play an important developmental role because of their potentially stimulatory effects on phytohormonal signaling and/or synergism.