Spermine and oxacillin stress response on the cell wall synthesis and the global gene expression analysis in Methicillin-resistance Staphylococcus aureus

Shrikant Pawar,Xiangyu Yao,Chung‑Dar Lu 한국유전학회 2019 Genes & Genomics Vol.41 No.1

Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly emerging bacteria causing infection, which has developed resistance to most of the beta-lactam antibiotics because of newly acquired low-affinity penicillin-binding protein (PBP2a), which can continue to build the cell wall when beta-lactams block other PBPs. Exogenous spermine exerts a dose-dependent inhibition effect on the growth of Escherichia coli, Salmonella enterica serovar, and S. aureus. Selection of an MRSA Mu50 derivative which harbors mutation on PBP2 gene (named as MuM) showing spermine resistance and which confers a complete abolishment of spermine-beta-lactam synergy was identified. To further investigate the gene expression changes, a transcriptome profiling of MuM against Mu50 (wild-type) without any treatment, MuM and Mu50 in response to high dose spermine and Mu50 in response to spermine-beta-lactam synergy at 15, 30 and 60 min time points was performed. Functional annotation was further performed to delineate the metabolic pathways associated with the significant genes. A significant down-regulation in the iron regulatory system, potassium channel uptake and polyamine transport system with an up-regulation in general stress response sigB dependent operon in MuM strain at 15, 30 and 60 min time points with spermine treatment compared to Mu50 strain was observed. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion pattern of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. A strong relation between PBP2 protein and general stress sigB response, iron, potassium and polyamine transport systems was observed. SigB regulon should be activated on stress, which was not seen in some of our previous studies where it was down-regulated in wild-type Mu50 strain with spermine stress. Here, an intriguing finding is made where there seems to be a correction of this abnormal response of no SigB induction to a significant induction by PBP2 mutation. In MuM strain, a significant downregulation of KdpABC operon genes at 15, 30 and 60 min time points on spermine stress is seen, which seems to be absent without spermine treatment. Since KCL has been found to protect the cell against spermine stress in wild-type strain by induction of KdpABC operon, it fails to do so in MuM strain underlying the importance of PBP2 protein in spermine stress. Analysis of spermine-dependent synergy with beta-lactams on cell wall synthesis revealed that it significantly reduces the degree of cross-linkage on cell wall with no change in trypsin digestion patterns of purified PBPs and without affecting PBPs expression or PBPs acylation by Bocillin. Furthermore, spermine does not help in enhancing the binding of beta-lactams to PBPs and binding of spermine to PBPs does not cause conformational changes to PBPs, as tested with trypsin digestion patterns. Future studies on the molecular mechanism of spermine interactions with these systems hold great potential for the development of new therapeutics for MRSA infections.

Role of Spermine in Regulating Gβγ-induced Effector Activation

Myung, Chang-Seon 관동대학교 의과학연구소 2001 關東醫大學術誌 Vol.5 No.1

G단백 βγ소단위들은 세포 외로부터의 생물학적 신호를 세포 내로 전달하여 세포내 특정 주효기 혹은 단백질들과 반응함으로써 생물학적 반응을 나타내는 매우 중요한 신호 전달물질로 알려져 있다. G단백의 α소단위가 여러 가지 기전에 의해 활성이 조절되는 것에 비해 βγ소단위들의 조절기전에 대해서는 아직 잘 알려져 있지 않은 실정이다. 이에 본 연구에서는 G단백 βγ소단위들에 의한 주효기의 활성이 내인성 polyamine인 spermine에 의해 조절되는지를 관찰하기 위하여, baculovirus/Sf9 insect cell system을 통하여 순수한 G단백 β1γ2 소단위를 분리, 정제하고, 이것이 대표적 주효기인 PLC-β와 type II adenylyl cyclase 활성화에 spermine이 미치는 정도를 측정해 보았다. G단백 β1γ2 소단위들은 이들 주효기의 활성도를 용량 의존적으로 각각 12배와 16배 증가시켰으며, 이들에 대한 EC50는 각각 3nM과4.1nM로 나타났다. Spermine이 G단백 β1γ2소단위에 의한 PLC-β의 활성에 미치는 영향을 관찰하기 위하여, 5nM의 β1γ2소단위 농도를 선택하여 여러 가지 농도의 spermine과 30분 먼저 반응을 시키고 효소의 활성도를 측정해보면 5nM의 β1γ2소단위 농도에 대한 효소의 활성도가 100% 감소하였으며 Spermine의 IC50는 약 0.11mM이었다. Spermine의 반응부위를 관찰하기 위하여 여러 가지 농도의 spermine을 PLC-β와 먼저 반응을 시킨 후 활성도를 측정해 보아도 비슷한 결과를 나타내었다. Spermine이 G단백 β1γ2소단위에 의한 type II adenylyl cyclase의 활성에 미치는 영향을 관찰하기 위하여 유사한 실험을 진행한 결과, 여러 가지 농도의 spermine을 β1γ2소단위와 혹은 G단백 αs소단위에 의해 활성화된 type II adenylyl cyclase와 30분 먼저 반응을 시킨 양쪽 모두의 경우에서 15nM β1γ2소단위 농도에 의한 cyclase효소의 활성도를 각각 100%와 67% 증가시켰으며 이들 각각에 대한 EC50는 약 1.01mM과 0.011mM이었다. 따라서,본 실험의 결과를 미루어 spermine은 G단백 βγ소단위에 의한 주효기의 활성도를 조절하는 내인성 polyamine으로서 PLC-β에 대해서는 억제성으로 또한 type II adenylyl cyclase에 대해서는 흥분성으로 작용하는 것을 알 수 있었으며, spermine이 PLC-β의 경우에서는 βγ소단위 혹은 효소에 모두 작용하지만, type II adenylyl cyclase의 경우 βγ소단위에 대해서 보다는 오히려 cyclase효소에 대하여 더 높은 친화성을 갖고 반응하리라 사료되는 바이다. The abbreviations used are: G proteins, guanine nucleotide-binding regulatory proteins; Sf9 cells, Spondoptera frugiperda cells (ATCC no. CRL 1711); PLC-β; phosphatidylinositol- specific phospholipase C-β isoform; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, D-myoinositol 1,4,5-trisphosphate; DAG, 1,2-diacylglycerol; LUVs, large unilamellar vesicles; DTT, dithiothreitol ; CHAPS, 3- [(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate ; EGTA, ethyleneglycol-bis-(β-aninoethyl ether) N,N'- tetraacetic acid ; EDTA, ethylenediaminetetraacetic acid; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethane-sulfonic acid; BSA, bovine serum albumin; Genapol C-100, polyoxyethylene (10) dodecyl ether; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate.

이상 저온 조건에서 Spermine 처리가 딸기의 생육과 수량 및 품질에 미치는 영향

이규빈 ( Gyu-bin Lee ),이정은 ( Jung-eun Lee ),최윤의 ( Yun-ui Choe ),박영훈 ( Young-hoon Park ),최영환 ( Young-whan Choi ),강남준 ( Nam-jun Kang ),강점순 ( Jum-soon Kang ) 한국환경과학회 2018 한국환경과학회지 Vol.27 No.3

The present study was carried out to investigate the effects of spermine treatment on the growth, yield and quality in strawberry under low-temperature condition, and thereby develop a chemical method to minimize damages by low temperature in greenhouse cultivation. Spermine treatment significantly improved the growth of strawberry in terms of leaf number, leaf area, leaf length, leaf diameter, plant height and plant weight. The highest effect was observed in the 250 uM spermine treatment and the effect tended to be maintained during the entire growth period of 90 days. Fresh weight and dry weight were significantly different depending on the concentrations of spermine. Strawberry plants treated with 250 uM spermine showed higher fresh weight and dry weight compared to untreated control plants during the growth period. Fruit weight, fruit length and fruit diameter were relatively higher when treated with 100 μM spermine compared to other treatments. The fruit yield was the highest with 14 fruits per plant at 250 μM spermine treatment and the coloration of the fruit was the best at this treatment with the Hunter a and b values of 46.56 and 28.75, respectively. The hardness of strawberry fruit tended to increase higher than 2N at 250 μM and 500 μM 250 uM spermine treatment. The sugar content of strawberries treated with 250 μM spermine was 9.5 ° Bx which was 1.6 ° Bx higher compared to that in untreated control. However, spermine treatment did not affect the acidity of fruit and it remained 0.68-0.76% regardless of treatment concentrations. These results suggest that spermine treatment has a positive effect on the growth and productivity of strawberry fruit under abnormal low-temperature condition. The positive effect was the highest at 250 μM spermine treatment and gradually decreased in the order of 100 μM, 500 μM, and untreated control.

RBL-2H3 세포에서 Spermine에 의한 히스타민 유리와 칼슘유입에 미치는 상백피의억제효과

채옥희,강경진,신인호,김영기,이무삼,전병득 대한해부학회 1994 Anatomy & Cell Biology Vol.27 No.2

Methylglyoxal-bis (Guanylhydrazone)와 에틸렌 생합성 저해제가 대두 자엽 부정근 형성에 미치는 영향

조형일,한태진,하건수,이순희,김응식 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.6

MGBG와 에틸렌 저해제가 대두 자엽 부정근 형성에 미치는 영향을 알아보고자 에틸렌 생성량과 내생 polyamine 함량을 조사하였다. 대두 자엽 절편을 부정근 형성 배지에 2주간 배양하면서 관찰한 결과 많은 양의 부정근이 형성되었으나 $10^{-3}$ M MGBG를 처리하면 부정근 형성이 거의 억제되었다. 그러나 MGBG + spermine, MGBG + Cocl$_2$ 및 MGBG + spermine + Coc1$_2$를 농도별로 처리한 경우 부정근 형성 억제가 회복되었다. $10^{-3}$ M MGBG에 spermine을 농도별로 처리한 경우 $10^{-5}$ M의 spermine 혼합 처리시 다소회복되었다. $10^{-3}$ M MGBG +$10^{-5}$ M spermine + $10^{-4}$ M Coc1$_2$ 처리구에서는 부정근 회복능이 $10^{-3}$ M MGBG +$10^{-5}$ M spermine 처리구에 비하여 현저히 증가하였다. 또한 에틸렌 생성량과 polyamine 함량을 $10^{-3}$M MGBG, $10^{-3}$ M MGBG +$10^{-5}$ M spermine, $10^{-3}$ M MGBG +$10^{-4}$ M CoCl$_2$및 $10^{-3}$ M MGBG +$10^{-5}$ M spermine + $10^{-4}$ M Coc1$_2$처리구에서 조사한 결과 $10^{-3}$ M MGBG 단독 처리구에서 에틸렌 생성량이 가장 높았으며, spermidine과 spermine의 함량은 가장 낮았다. 부정근 형성능이 다소 회복된 $10^{-3}$ M MGBG+ $10^{-5}$M spermine 혼합처리구에서의 에틸렌 생성량은 대조구보다는 다소 높게 나타났다. 부정근 회복능이 가장 높았던 $10^{-3}$ M MGBG+ $10^{-5}$ M spermine + $10^{-4}$ M Cocl$_2$ 혼합처리구에서 에틸렌 생성량은 가장 낮게 나타났으며, polyamine 함량은 가장 높게 나타났다. The effect of methylglyoxal-bis (guanylhydrazone) (MGBG) and ethylene synthesis inhibitors on adventitious root formation from soybean cotyledon in relation to ethylene production and endogenous polyamine content was investigated. Cotyledon explants cultured on rooting medium formed numerous adventitious rook on the cut surfaces after 2 weeks of culture. However when cultured on rooting medium supplemented with MGBG, the root formation was strongly inhibited, its inhibitory effect was reserved when cultured on medium with MGBG + spermine, MGBG + CoCl$_2$ and MGBG + spermine+CoC1$_2$. A slight reversion of the rooting inhibition was observed in 10$^{-3}$ M MGBG +10$^{-5}$ M spermine treatment, whereas it caused a significant effect in 10$^{-3}$ M MGBG +10$^{-4}$ M treatment .Ethylene production and endogenous polymine content was investgated in 10$^{-3}$ M MGBG , 10$^{-3}$ M MGBG +10$^{-5}$ Mspermine, 10$^{-3}$ M MGBG +10$^{-4}$ M CoCl$_2$and 10$^{-3}$ M MGBG +10$^{-5}$ M spermine +10$^{-4}$ M CoCl$_2$treatments. Ethylene production highest in 10$^{-3}$ M MGBG +10$^{-5}$ M spermine treatment was higher than control. In 10$^{-3}$ M MGBG +10$^{-5}$ M spermine + 10$^{-4}$ M CoCl$_2$ treatment, ethilene production was lowest, whereas polyamine level was highest.

Effects of Spermine on Changes in Chlorophyll-Protein Complexes and Plastid Membrane Proteins of Mung Bean Cotyledons during Greening

Hong, Jung-Hee,Park, Hong-Duck 부산대학교 유전공학연구소 1995 분자생물학 연구보 Vol.11 No.-

Spermine이 녹화중인 녹두자엽의 엽록소-단백질 복합체(CPs) 및 틸라코이드막 단백질의 변화에 미치는 효과를 조사하였다. 녹화가 진행됨에 따라 CPs형성이 촉진되었으며, 특히 광계 Ⅰ의 엽록소- 단백질 (CP Ⅰ)이 다량 축적되었다. 광수확 엽록소 단백질(LHCP)은 48시간의 초기녹화과정에서 중요한 단백질로 나타났다. Spermine처리는 초기녹화과정에서 틸라코이드막의 단백질은 전 엽록체에서 다량 관찰되었고 24 kD 단백질은 전 처리구에서 뚜렷한 증가를 보여주었다. Spermine처리에 의해 틸라코이드막 단백질 형성은 대조구에 비해 다소 증가되었다. 이러한 결과들로부터 spermine은 녹화과정에서 색소체의 발달과 색소체막의 안정화에 중요한 작용을 하는 것으로 생각된다. Developmental changes of chlorophyll-protein complexes (CPs) and plastid membrane proteins in greening mung bean cotyledons and the effect of spermine thereon were examined by SDS-polyacrylamide gel electrophoresis. The changes in the amounts of CPs became larger with the progress of greening and light-harvesting chlorophyll a/b protein (LHCP) was the main CP in the early greening stage up to 48 h. As the greening proceeded, chlorophyll-protein of the photosystem Ⅰ (CPⅠ) accumulated. Application of spermine were effective in accumulating CPs of the thylakoid membrane in the early phase of greening. In the profiles of the plastid membrane proteins, quantitative and qualitative changes were observed with the onset of greening up to 72 h. 56 kD protein of major intensity was observed in all greened chloroplasts and 24 kD protein increased remarkablly in both control and spermine-treated cotyledons. The thylakoids from spermine-treated cotyledons showed higher amounts of thylakoid proteins as compared to the controls. The results suggest that spermine may play a role in the regulation of plastid development and stabilizes the membrane function during greening.

Spermine에 의한 흰쥐 복강 비만세포의 히스타민 유리와 Camp, Cgmp 수준변동에 미치는 상백피의 효과

채옥희,양문식,이무삼 大韓免疫學會 1996 大韓免疫學會誌 Vol.18 No.3

Cortex Mori(Morus alba L.), the root bark of mulberry tree, has been used as an antiphlogistic, diuretic, and expectorant in Korean herbal medicine. Previous studies demonstrated that phenolic extract of Cortex Mori has a hypotensive effect in rabbits and hot water extract of Cortex Mori has an inhibitory effects on spermine and compound 48/80-induced mast cell degranulation and histamine release from rat peritoneal mast cells(RPMC). The purpose of this study is to determine whether Cortex Mori could affect the intracellular levels of cAMP and cGMP of RPMC by spermine which has been known as activators to increase cytoplasmic Ca" concentration by stimulating transmembrane Cat+ transport and mobilizing intracellular calcium, and release histamine from RPMC ultimately. Morphological changes of RPMC by spermine alone or Cortex Mori plus spermine were observed on the inverted light microscope. Various doses of spermine(10, 5, 1, 0.5, 0.1mM) were added into RPMC suspension (106cells/ml) pretreated with or without various doses of Cortex Mori(100, 50, 10, 5, lmg/ml) at 37°C for 10 minutes. After incubation, the amount of hitamine in supernatant, intracellular cAMP, and cGMP levels of RPMC were measured by radioisotope enzymatic histamine assay, cAMP and cGMP radioimmunoassays. Results were 1) the spermine(lOmM)-induced degranulation and histamine release from RPMC pretreated with Cortex Mori were significantly inhibited, compared to those of control without Cortex Mori pretreatment, 2) the cAMP and cGMP levels of unstimulated RPMC were 17.5 ± 1.9 and 120 x 10-3± 20 pmols, respectively, 3) after the addition of the spermine, the intracellular levels of cAMP were decreased and those of cGMP were increased, 4) dose dependent fashions of cAMP and cGMP levels of RPMC were induced by increased concentrations of spermine, 5) Cortex Mori increased the cAMP levels(200.0± 11.4%) and decreased the cGMP levels (22.5±4.2%) of RPMC remarkably, compared to those of normal control, and 6) the spermine-induced cAMP levels of RPMC pretreated with Cortex Mori weresignificantly increased, compared to those of positive control without Cortex Mori pretreatment and the spermine-induced cGMP levels of RPMC pretreated with Cortex Mori were remarkably decreased, compared to those of positive control without Cortex Mori pretreatment. From the above results, it is suggested that Cortex Mori contains some substances to inhibit the spermineinduced mast cell activation so as mast cell degranulation, histamine release, and that its inhibitory effect may occure via specific mechanisms of increasing of intracellular cAMP levels or blocking of increasing of intracellular cGMP levels.

Cyclohexylamine과 Polyamine이 대두 자엽의 부정근 형성에 미치는 영향

한태진,조형일 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.4

Cryclohexylarnine(CHA)에 의한 부정근 억제가 spermidine 과 spermine에 의한 부정근 형성 회복에 미치는 영향을 알아 보고자 대두 자엽 절편을 이용하여 조직내의 polyamine함량을 조사하였다. 부정근 형성 배지에 CHA를 처리한 결과 $10^{-2}$ M CHA 처리시에는 spermidine이나 spermine을 혼합처리함에도 불구하고 부정근이 형성되지 않았다. 또한 2.5 $\times$ $10^{-3}$ M CHA 단독처리시에는 부정근 형성이 억제되었으나, $10^{-4}$ M spermine 혼합처리시에는 부정근 형성능이 회복되었다. Putrescine 함량은 부정근이 형성돠지 않았던 $10^{-2}$ M CHA 단독 처리구 및 $10^{-2}$ M CHA와 $10^{-3}$ M Spermine 혼합처리구에서는 배양 3일 이후 대조구보다 감소하였지만 부정근이 다소 형성되었고, 부정근 회복능이 가장 높았던 2.5 $\times$ $10^{-3}$ M CHA와 $10^{-4}$ M spermine 혼합처리구에서는 대조구보다 높은 함량을 나타내었다. Spermidine 함량은 모든 처리구에서 대조구보다 낮아서 함량이 낮을수록 부정근 형성이 억제되었으며, spermine의 함량은 대조구에 비하여 모든 처리구에서 급격히 증가되어 대조구에 비하여 함량이 높을 수록 부정근 형성이 억제되었다. In order to study the effect of cyclohexylamine (CHA) and polyamines during adventitious root formation, the correlation of adventitious root formation with endogenous polyamine content was investigated in inoculated soybean cotyledons at the rooting medium supplemented with CHA, spermidine and spermine. Adventitious root formation was inhibited in medium containing 10$^{-3}$ -10$^{-2}$ M CHA. Adventitious root formation was not formed in treatment with 10$^{-2}$ M CHA plus polyamine. It was inhibited in 2.5 $\times$ 10$^{-3}$ M CHA alone and reverved in 2.5 $\times$ 10$^{-3}$ M CHA plus 10$^{-4}$ M spermine. Putrescine content was lower in 10$^{-2}$ M CHA and 10$^{-2}$ M CHAplus 10$^{-4}$ M spermine treatment than in control after 3 days of incubation. However, it was higher in 2.5 $\times$ 10$^{-3}$ M CHA and 2.5 $\times$ 10$^{-3}$ M CHA plus 10$^{-4}$ M spermine treatment than in control. Spermine content was higher in all CHA treatments than in control, while spermidine content was lower than in control.

Effects of dietary spermine supplementation on cell cycle, apoptosis, and amino acid transporters of the thymus and spleen in piglets

Cao, Wei,Wu, Xianjian,Jia, Gang,Zhao, Hua,Chen, Xiaoling,Wu, Caimei,Cai, Jingyi,Wang, Jing,Liu, Guangmang Asian Australasian Association of Animal Productio 2018 Animal Bioscience Vol.31 No.8

Objective: This study investigated whether spermine supplementation could regulate cell cycle, apoptosis, and amino acid transporter-related genes expression in the thymus and spleen of early weaned piglets. Methods: Eighty female piglets were randomly distributed to receive adequate nutrients supplemented with spermine (0.4 mmol/kg body weight/24 h) or to be provided with restricted nourishment supplemented with normal saline for 7 h or 3, 6, or 9 d in pairs. Results: Regardless of administration time, spermine supplementation significantly up-regulated cyclin A2 gene expression but down-regulated p21 and cyclin D3 mRNA levels in the thymus and spleen and reduced cyclin E2 gene expression in the thymus of piglets (p<0.05). Irrespective of the treatment period, the reduced Bax and caspase-3 gene expressions and improved Bcl-2 mRNA level were observed in the thymus and spleen of spermine-administrated piglets (p<0.05). Regardless of supplementation time, spermine intake significantly enhanced the expressions of amino acid transporter-related genes (SLC1A1, SLC1A5, SLC7A1, SLC7A7, and SLC15A1) in both thymus and spleen, as well as SLC7A9 in the spleen of piglets (p<0.05). In addition, extended spermine administration also markedly promoted cell proliferation, depressed apoptosis and modulated amino acid transport (p<0.05), and such effects were the greatest during prolonged spermine supplementation (6 d) compared to the other time periods (p<0.05). Conclusion: Spermine supplementation may regulate cell cycle during the G1/S phase, suppress apoptosis and modulate amino acid transport. A period of 6 d of spermine supplementation is required to produce the optimal effects on nutritional implications.

Cytoprotective Effects of Polyamines Against Oxidative Stress

안선이,이지영,정해영,유미애,김종민,김병기,Ahn Seoni,Lee Ji Young,Chung Hae Young,Yoo Mi-Ae,Kim Jong-Min,Kim Byeong Gee Korean Society of Life Science 2005 생명과학회지 Vol.15 No.4

폴리아민은 모든 진핵세포에서 발견되는 다가 양이온성의 저분자 물질이며 세포성장에 필수적인 것으로 알려져 있다. 본 논문에서는 폴리아민의 역할 중에서 산화적인 스트레스에 대한 세포보호 효과를 연구하였다. 쥐의 간세포주인 $Ac_2F$에 산화 스트레스를 유발하기 위하여 2,2'-azobis(2-amidinopropane)dehydrochloride (AAPH)를 처리하였을 때, 세포증식은 농도 의존적으로 감소하였다. 배지에 폴리아민을 첨가하였을 때 세포성장은 농도 의존적으로 증가하였으며 ROS 발생은 현저히 감소하였다. 폴리아민 가운데 특히 spermidine과 spermine이 뚜렷한 세포증식효과를 보였다. Spermine의 경우, $20{\mu}M$농도에서 AAPH에 의해 유도된 ROS발생을 $45\%$나 감소시켰다. 산화 스트레스에 관여하는 효소들 가운데 주된 효소인 superoxide dismutate (SOD)와 catalase (CAT)의 세포 내 단백질을 Western blotting으로 조사한 결과, AAPH는 이 두 가지 단백질의 생성을 억제한 것으로 나타났다. 그러나 spermine을 처리하였을 때 두 단백질의 생산은 모두 정상적으로 회복이 되었다. 또한 세포주기의 중요한 조절 단백질인 cyclin E 역시 AAPH에 의하여 생성이 억제되었다. 이는 AAPH에 의하여 생성된 ROS가 세포주기의 S phase의 진행을 억제한 것으로 생각된다. AAPH에 의한 cyclin E의 억제는 spermine에 의하여 정상적으로 회복되었다. 위와 같은 Spermine의 항산화 효과는 ethidium bromide와 acridine orange를 이용하여 형태학적으로도 증명되었다. The polyamines are essential components of all eukaryotic cells and absolutely necessary for cell growth. In the present study, the cytoprotective role of polyamine was characterized. When $Ac_2F$ rat liver cells were treated with 1M 2,2'-azobis (2-amidinopropane) dehydrochloride (AAPH), a water soluble free radical initiator, viability of the cells was noticeably decreased due to the increase of reactive oxygen species (ROS). The cytotoxic effect of AAPH as well as ROS generation were significantly inhibited by the treatment of polyamines. Among polyamines, especially spermine at $20{\mu}M$ concentration exerted over $45\%$ inhibition of AAPH-induced ROS generation. Western blotting was performed to determine whether superoxide dismutase(SOD) or catalase (CAT) expression was involved in oxidative stress. The AAPH treatment blocked both SOD and CAT protein expressions. Spermine could recover those protein expressions to the untreated control levels. According to the result of cycline E measurement, AAPH might block the entry of the cells into S phase of the cell cycle. The reduced expression of cyclin E protein could be fully recovered by the addition of spermine. The antioxidative effects of spermine was also further proved by the apopotitic morphological analysis using ethidium bromide and acridine orange.