갈조류 꽈배기모자반(Sargassum siliquastrum)과 쇠비름(Portulaca oleracea)으로부터 생리활성물질의 분리와 구조결정

The life of a human being has been extended even more than before due to improvement of medical technology. Nevertheless, the incidence rate of other new forms of diseases such as cancer, hyperlipidemia, diabetes, mental disorders, etc. has also increased greatly. Following extensive research, two common, widely available, natural plants have been found to have antioxidative and antiproliferative properties which may contribute to the prevention or treatment of some diseases or may inhibit the growth of some cancers. As a part of our search for new bioactive compounds from natural resources, the brown alga Sargassum siliquastrum and the terrestrial plant Portulaca oleracea were collected. Brown algae of the genus Sargassum (Sargassaceae) are widely distributed in the temperate and tropical oceans of the world, and often dominate benthic algal communities and occur in huge floating masses. Most species have cycles of vegetative growth and attrition. Portulaca oleracea L. (Portulacaceae) is an annual herb growing in warm areas of the world and has been used as traditional and folk remedies for thousands of years in many countries throughout the world. Each of the collected samples was briefly dried under shade and extracted with a mixture solvent of acetone-CH2Cl2 (1:1) at room temperature for 2 days and filtered. Then, the residue was re-extracted with MeOH in the same way. The combined crude extracts of each sample were partitioned between CH2Cl2 and water. The organic layer was further partitioned between n-hexane and 85% aq. MeOH, and the aqueous layer was partitioned with n-BuOH and H2O, successively. Previous study has shown 85% aq. MeOH solvent fraction of S. siliquastrum crude extracts has antioxidizing and antiproliferative effects. Therefore, six meroterpenoids, including three new ones were obtained from the 85% aq. MeOH fraction by bioactivity-guided separation. In our measurement for antioxidant activities, compounds 1-3 exhibited the strong scavenging effect on DPPH radical and peroxynitrite in a non-cellular system. In addition, ROS was generated in a cellular system. The antiproliferative effect of these compounds was also measured against AGS, HT-29, HT-1080, and MCF-7 human cancer cells. In comparative analysis, all compounds showed high growth-inhibitory effects on all cancer cell lines in a dose-dependent manner. 85% aq. MeOH solvent fraction of P. oleracea crude extracts showed a significant antiproliferative inhibitory effect against HT-29 colon cancer cells. On the basis of the above result, further purification of 85% aq. MeOH led to the isolation of homoisoflavonoids 7-13. The chemical structure of these compounds was established by extensive 2D NMR experiments such as 1H gDQCOSY, TOCSY, NOESY, gHMQC, and gHMBC and by synthesis of authentic compounds. The antiproliferative effect of compounds 7-10, 12 and 13 was also evaluated in HT1080, HT-29, AGS and MCF-7 human cancer cells using the MTT assay method. Among them, compound 10 exhibited the strongest antiproliferative inhibitory effect on the growth of human cancer cells in a dose-dependent manner. The antioxidant activity of these compounds was also evaluated by measuring intracellular ROS level and membrane lipid peroxidation. As a result, compounds 7-10 moderately decreased intracellular ROS level and compound 10 suppressed membrane lipid peroxidation. The latest report on extracts of P. oleracea suggested that it has a lypolitic effect through release of fatty acid and glycerol to the culture medium. Based on the above, the antiobesity effect of compounds 7-10 from P. oleracea was also examined by observing Oil-Red O stained lipid droplets in adipocyte and by measuring glycerol release and glucose contents in a culture medium. In addition, expression levels of several genes related to adipogenesis including transcription factors were examined using reverse-transcription polymerase chain reaction (RT-PCR) As a result, compounds 7 and 10 not only decreased production of lipid droplets and glucose contents but also increased glycerol release. Further, the effect of homoisoflavonoids (7-10) on the adipogenic differentiation was investigated at the gene expression levels, compounds 9 and 10 induced down-regulationion of adipogenic transcription factors (PPAR, C/EBP, PPAR, and SREBP1c) and adipogenic target adipocyte-specific genes (FABP4, FATP1, FAS, LPL, ACS1, perilipin and HSL). Moreover, compounds 9 and 10 down-regulated adipocytokine such as leptin and perilipin, and TNF-α. In short, compounds 9 and 10 have high inhibition effect on intracellular lipid accumulation and may be a valuable potential lead compound for the treatment of obesity. Therefore, these results suggest that S. siliquastrum and P. portulaca could be used as the valuable materials for developing biofunctional substances that may inhibit the growth of cancers, that may protect cells from the damaging effects of oxidation, or that may assist the body in maintaining a constant level of insulin, thereby preventing obesity.1. 서론 1.1. 해양생물 유래의 천연물질 탐색 1.2. 갈조 해조류 꽈배기 모자반(S. siliquastrum) 1.3. 쇠비름(Portulaca oleracea Linne) 1.4. homoisoflavonoids 1.4.1. Characters and bioactivity of Homoisoflavonoids 1.4.2. Synthesis of homoisoflavonoids 2. 재료 및 방법 2.1. 재료 2.2. 시약 2.2.1. 분리 및 합성 2.2.2 활성 2.2.3. 기기 2.3. 꽈배기모자반(S. siliquastrum)의 추출 및 분리 2.3.1. 추출 2.3.2 모자반의 활성 성분 분리 2.4. 쇠비름(P. oleracea)의 추출 및 분리 2.4.1. 추출 2.4.2. 화합물의 분리 2.5. Homoisoflavonoids 합성 2.5.1. 3,5-dimehoxyphenol합성 및 분리 2.5.2. 3-(3,5-dimethoxyphenoxy)propanoic acid합성 및 분리 2.5.3. 5,7-dimethoxychroman-4-one합성 및 분리 2.5.4. (E)-5,7-dimethoxy-3-[(2-methoxyphenyl)methylene]chroman-4-one 합성 및 분리 2.5.5. 5,7-dimethoxy-3-(2-methoxyphenyl)chroman-4-one 합성 및 분리 2.6. 항산화 활성 실험 2.6.1. DPPH radical 소거 활성 측정 2.6.2. Peroxynitrite 소거 활성 측정 2.6.3. 세포배양 2.6.4. 세포내 활성 산소종(ROS, reactive oxygen speices) 2.6.5. TBARS법을 이용한 lipid peroxidation 측정 2.6.6. GSH contents 측정 2.7. 암세포 증식억제 실험 2.8. 항비만 활성 실험 2.8.1. 3T3-L1 세포의 배양 및 지방세포로의 분화 유도 2.8.2. 3T3-L1전구 지방세포에서의 세포 분화능 측정 2.8.3. Glycerol 분비 측정 2.8.4. Glucose 소비 측정 2.8.5. Leptin 측정 2.8.6. 비만 관련 유전자 발현 측정 2.9. 통계분석 3. 결과 및 고찰 3.1. 이차대사산물 분리 및 구조 결정 3.1.1. 꽈배기 모자반으로부터 활성 성분의 구조 결정(1-3) 3.1.2. 쇠비름으로부터 활성 성분의 구조 결정(7-13) 3.1.3. Homoisoflavonoids 합성 3.2. S. siliquastrum 으로부터 분리된 이차대사산물의 생리활성 3.2.1. S. siliquastrum 으로부터 분리한 compounds 1-3의 인체유래의 암세포 증식억제효과 3.2.2. S. siliquastrum 으로부터 분리한 compounds 1-3의 항산화 효과 3.2.2.1. DPPH radical 소거 효과 3.2.2.2. Peroxynitrite 소거 효과 3.2.2.3. 세포 독성 효과 3.2.2.4. 활성 산호종(ROS) 소거 효과 3.2.2.5. 지질과산화 억제 효과 3.2.2.6 세포내 GSH(glutathion) 함량 3.3. P. oleracea의 추출물 및 분획물의 암세포 증식억제효과 3.4. P. oleracea로부터 분리된 이차대사산물의 생리활성 3.4.1. P. oleracea로부터분리된 compounds 7-10 및 12-13의 암세포 증식억제효과 3.4.2. P. oleracea로부터분리된 compounds 7-10 및 12-13의 활성 산소종(ROS) 소거 효과 3.4.3. P. oleracea로부터분리된 compounds 7-10 의 항비만 효과 3.4.3.1. 3T3-L1전구 지방세포에서의 세포 분화능 측정 3.4.3.2. Glucose 소비 측정 3.4.3.3. Glycerol 분비 측정 3.4.3.4. Leptin 측정 3.4.3.5. 비만 관련 유전자 발현 측정 3.4.3.5.1. 핵심 조절 전사인자 발현 3.4.3.5.2. 표적 유전자 발현 3.4.3.5.3. Adipocytokine 유전자 발현 3.4.3.5.4. 지방분해 유전자 발현 4. 결 론 참고문헌 부

갈조류 꽈배기 모자반(Sargassum siliquastrum)으로부터 생리활성 성분의 분리 및 구조결정

Marine macroalgae are widely recognized as very prolific sources of both biologically active and structurally unique secondary metabolites. Terpenoids, acetogenins, and mixed metabolites of uncommon carbon skeletons and functionalities belong to the major groups of metabolites from these plants. Meroterpenoids are mixed biosynthetic products which contain terpenoids and polyketides fragments. Particularly, meroterpenoids of chromene structural class were found abundantly whithin the brown algae and revealed a variety of bioactivities such as cytotoxicity, antioxidant activity, anthelmintic activity, and inducement of the larval settlement of a hydrozoan. As a part of our search for novel bioactive compounds from marine algae, the brown alga Sargassum siliquastrum was collected off the shore of Jeju Island. The collected samples of Sargassum siliquastrum were briefly dried under shade and repeatedly extracted for 2 days with a mixture (1:1) of acetone-CH2Cl2 (1.5 L X 2) and MeOH (1.5 L X 2), respectively. The combined crude extracts of Sargassum siliquastrum were fractionated into n-hexane, 85% aq. MeOH, n-BuOH, and water fractions. Antioxidant activities of n-hexane, 85% aq. MeOH, n-BuOH, and water fractions including crude extracts of Sargassum siliquastrum were evaluated using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical, authentic ONOO-, and ONOO- generated from SIN-1. Scavenging activity of each fraction on DPPH radical increased in the order of 85% aq. MeOH > n-hexane > n-BuOH > H2O fractions, while that of each fraction on authentic ONOO- and induced ONOO- from SIN-1 increased in the order of 85% aq. MeOH > n-BuOH > H2O > n-hexane fractions. On the basis of the above results, purification of the 85% aq. MeOH fraction using C18 reversed-phase column, followed by HPLC, resulted in the isolation of three new meroterpenoids (compounds 5-7) and four known meroterpenoids (compounds 1-4) of chromene class. The structure of the isolated compounds was established by extensive 2D NMR experiments such as 1H gDQCOSY, TOCSY, NOESY, gHMQC, and gHMBC, and by comparison with published spectral data. In our measurement for evaluating antioxidant activity of compounds 1-7 using DPPH radical, authentic ONOO-, and ONOO- generated from SIN-1, all of them exhibited significantly high activities. The protective effect of compounds from Sargassum siliquastrum on H2O2-induced oxidative stress was examined in HT1080 cells. The levels of intracellular reactive oxygen species (ROS) were measured using 2’,7’-dichlorofluorescin diacetate (DCFH-DA). All solvent fractions including crude extracts of Sargassum siliquastrum not only significantly decreased levels of intracellular ROS in a dose-dependent manner but also exhibited a protective effect on oxidative damage of purified genomic DNA. In addition, they increased intracellular GSH level in a dose dependent manner. Compound (1-7) significantly decreased those of intracellular ROS while they increased that of intracellular GSH at concentration of 5 ㎍/㎖. Therefore, these results suggested that these active compounds from Sargassum siliquastrum could be developed as a candidate for potential natural antioxidant related to oxidative stress. Antiobesity effect of each fraction was estimated by measuring levels of glycerol and triglyceride. When 3T3-L1 adipocytes are treated with a good antiobestic material, glycerol secretion is increased and triglyceride (TG) accumulation is reduced, compared to control adipocytes. Among tested samples, 85% aq. MeOH and n-BuOH fractions revealed very good antiobesitic effect, significantly increasing glycerol secretion and decreasing TG accumulation, respectively. Based upon these results, it is suggested that further investigation should be performed on the isolation and identification of the antiobesitic components in its active fraction.목 차 1.서론 2.실험 방법 2-1. 재료 2-2. 시약 2-3. 기기 2-4. 추출, 분획 및 분리 (1) 추출 및 분획 (2) 모자반의 활성 성분 분리 2-5. 항산화 활성 실험 (1) DPPH radical 소거 활성 (2) Peroxynitrire 소거 활성 2-6. 세포수준의 항산화 활성 실험 (1) 세포배양 (2) Cell viability의 측정 (3) ROS ( total free radical 측정) (4) DNA 산화 생성물의 측정 (5) GSH 함량 측정 2-7. in vitro 항비만 실험 (1) 3T3-L1 세포의 배양 및 지방세포로의 분화 유도 (2) Glycerol 함량의 측정 (3) TG 함량의 측정 3. 결과 및 고찰 3-1. 모자반에서 분리된 화합물들의 구조결정 3-2. 항산화 활성 (1) 꽈배기 모자반 조추출물과 용매분획의 활성 1) DPPH radical 소거 활성 2) Peroxynitrite 소거 활성 (2) 꽈배기 모자반에서 분리한 compounds 1-7 활성 1) DPPH radical 소거 활성 2) Peroxynitrite 소거 활성 3-3. 세포 수준에서 항산화 활성 효과 (1) 꽈배기 모자반 조추출물과 용매분획의 활성 1) MTT assay 2) ROS (Total free radical) 소거 활성 3) DNA 산화 4) GSH 함량 측정 (2) Compounds 1-7 의 활성 1) MTT assay 2) ROS (Total free radical) 소거 활성 3) GSH 함량 측정 3-4. in vitro 항비만 실험 (1) Glycerol 분비 및 triglyceride 축적 1) 꽈배기 모자반 조추출물과 용매분획의 효과 4. 요약 및 결론 5. 참고문

Methanol이 배양된 흰쥐 해마의 신경세포 및 신경교세포의 성장에 미치는 영향

Methanol은 산업체에서 흔히 사용되는 용매이고 사용량이 증가추세에 있으므로 인체가 이에 노출되어 중독될 가능성은 점점 높아지고 있다. Methanol에 중독되면 대사성 산증과 시각기능장애를 일으키고 치료되지 않거나 심한 경우 사망에 이르기도 한다. Methanol중독의 임상증산은 methanol 자체보다는 methanol의 대사 산물인 formic acid가 체내에 축적됨으로서 유발되는 것으로 알려져 있다. 그러나 설치류에서는 methanol 의 대사 및 흡수과정이 신속히 진행되므로 독성 대사 산물이 축적되지 않음에도 불구하고 임신중 methanol에 노출될 경우 태아에서 생식 및 발달 장애가 유발된다. 이는 ethanol 의 경우에 서와 같이 methanol도 그 자체가 직접적인 독성 작용을 갖기 때문이라고 생각된다. 이에 본 연구자는 흰쥐 태아의 해마 신경세포 및 신경교세포의 일차 배양세포에 농도별로 methanol을 투요하여 methanol 자체가 신경세포 및 신경교세포의 성장에 미치는 영향에 대하여 알아보고자 하였다. 연구 방법은 임신 17일된 Sprague-Dawley계 흰쥐의 태아에서 해마 조직을 분리하여 신경세포 및 신경교세포를 일차 배양한 후 methanol을 투여하지 않은 대조군과 maathanol 10, 100, 500, 1000 mM투여군으로 나누어 세포배양초기 (0, 18, 24 시간)의 신경돌기 성장 및 세포 생존율을 측정하였고 세포 배양 후기 (7일)의 신경교세포 수의 변화를 보기 위하여 단백 정량을 실시하였다. 연구 결과는 다음과 같다. 1. 본 실험 조건하에서 태령 17일의 흰쥐 해마의 신경세포 및 신경교세포는 성장 및 분화하였다. 2. 0-24시간동안의 신경세포 생존율은 대조군과 10 mM, 100 mM, 500 mM및 100 mM methanol 투여군간에 별다른 차이를 나타내지 않았다. 3. 신경세포돌기는 10 mM 및 100 mM methanol 투여군에서 18-24시간 동안 대조군에 비하여 유의한 길이 성장을 나타내었다. 4. 배양 7일째 단백량은 10 mM, 100 mM methanol 투여로 대조 군에 비하여 유의한 증가를 나타내었으나 1000 mM 투여군에서는 오히려 유의하게 감소하였다. 이상의 결과로 볼 때 methanol은 신경계 세포의 성장에 영향을 미칠 수 있다. 즉 methanol은 저농도(10mM 및 100 mM)에서는 세포배양 초기의 신경세포 성장 및 세포배양 후기의 신경교세포 성장을 촉진시키고 고농도 (1000 mM)에서는 세포배양후기의 신경교세포의 성장을 억제시킨다. 본 실험 결과 중 저농도의 methanol이 신경세포 및 신경교세포의 성장을 촉진시킨 결과는 효과와는 상반되는 의의를 갖는다. 그러므로 현재까지는 methanol 에 대한 연구는 주로 그 독성에 관한 연구가 이루어져 왔으나, 앞으로는 저 농도에서 methanol이 신경계 세포 성장을 촉진시키는 기전등에 대한 연구도 필요하리라 생각된다. ; Methanol has been widely used as an industrial solvent and environmental exposure to methanol would be expected to be increasing. In humans, methanol causes metabolic acidosis and damage to ocular system, and can lead to death in sever and untreated case. Clinical symptoms are attributed to accumulation of formic acid which is a metabolic product of methanol. In humans and primates, formic acid is accumulated after methanol intake but not in rodents due to the rapid matabolism of methanol. Nevertheless, the developmental and reproductive toxicity were reported in rodents. Previous reports showed that perinatal exposure to ethanol produces a variety of damage in human central nervous system by direct neurotoxicity. This suggest that the mechanism of toxic symptoms by methanol in rodents might mimic that of ethanol in human. In the present study I hypothesized that methanol can also induce toxicity in neuronal cells. For the study, primary culture of rat hippocampal neurons and glias were employed. Hippocampal cells were prepared from the embryonic day-17 fetuses and maintained up to 7 days. Effect of methanol (10, 100, 500 and 1000 mM) on neurite outgrowth and cell viability was investigated at 0, 18 and 24 hours following methanol treatment. To study the changes in proliferation of glial cells, protein content was measured at 7 days. The results were: 1. Under the present culture conditions, the fetal hippocampal neuronal and glial cells grew and differentiated well. 2. Compared to the control, neuronal cell viability in culture was not altered during 0-24 hours after methanol treatment significantly enhanced neurite outgrowth between 18-24 hours. 4. 7-day exposure to 10 or 100 mM methanol significantly increased protein contents but that to 1000 mM methanol decreased in culture. In conclusion, methanol may have a variety of effects on growing and differentiation of neurons and glial cells in hippocampus. Treatment with low concentration of methanol caused that neurite outgrowth was enhanced during 18-24 hours and the numbers of glial cell was increased for 7 days. High concentration of methanol brought about decreased protein contents. At present, the mechanism responsible for the methanol induced enhancement of neurite outgrowth is not clear. Further studies are required to delineate the mechanism possibly by employing molecular biological techniques.논문개요 ------------------------------------------------------------- ⅵ Ⅰ. 서론 ------------------------------------------------------------- 1 Ⅱ. 재료 및 방법 ----------------------------------------------------- 5 1. 실험동물 및 실험군 ----------------------------------------------- 5 2. 신경세포 배양 ---------------------------------------------------- 5 3. Methanol 투여 ---------------------------------------------------- 6 4. 신경돌기 성장 및 세포 생존율 측정 -------------------------------- 7 5. 단백질 정량 ------------------------------------------------------ 7 6. 자료분석 --------------------------------------------------------- 8 7. 시약 ------------------------------------------------------------- 8 Ⅲ. 결과 ------------------------------------------------------------- 9 1. 신경세포 배양 ---------------------------------------------------- 9 2. 신경세포 생존율 -------------------------------------------------- 9 3. 신경돌기 성장 ---------------------------------------------------- 13 4. 단백질 정량 ------------------------------------------------------ 13 Ⅳ. 고찰 ------------------------------------------------------------- 20 Ⅴ. 결론 ------------------------------------------------------------- 25 참고문헌 ------------------------------------------------------------- 27 영문초록 ------------------------------------------------------------- 3

ISOLATION AND PHYSIOLOGICAL CHARACTERISTICS OF EXTREMELY HALOPHILIC BACTERIA

In this thesis, halophilic bacteria were isolated and their physiological characteristics and salt-dependent activation of enzyme were studied. Fourteen halophilic bacteria were isolated from salterns by cultivating them in Sehgal & Gibbons medium supplemented with 15% and 25% NaCl. Of them, the morphological and physiological characteristics of nine strains which grew at 20-25% NaCl were investigated. The result was that six strains EH8, EH10, EH17, EH18, EH19 and EH23 were tentatively identified as Halobacterium sp. and three strains EH11, EH12, and EH13 were tentatively identified as Halococcus sp. Among them, typical two strains Halobacterium sp. EH8 and EH10 which grew fast at 25-30% NaCl were selected and their physiological characteristics and salt-dependent activation of enzymes were studied. The optimal NaCl concentration for growth of Halobacterium sp. EH8, EH10 were 25% and growth were inhibited when the NaCl concentration fell down to 10%. The optimal temperature for growth of Halobacterium sp. EH8, EH10 were 45˚C and 5O˚C, resprectively. But these strains did'nt grow when NaCl was replaced with KCl. Some enzymes involved in metabolic pathway such as Lactate dehydrogenase, Glucokinase, Glucose-6-phosphate dehydrogenase, Alanine dehydrogenase and Isocitrate dehydrogenase were also tested. Consequently, Glucose-6-phosphate dehydrogenase was not detected. But the rest enzyme activities were observed at 0-5M NaCl concentration. Especially, Lactate dehydrogenase activity was highest at 5M NaCl and 4M KCl. Other enzyme involved in electron transport system such as NADH oxidase and NADH dehydrogenase were tested, and their enzyme activities increased to a maximum level at 2M NaCl and observed the highest level when Na^(+) was replaced with cations like K^(+), NH_(4)^(+) and anions like sodium carbonate, sodium acetate. Their optimal pH was 9. Km value for NADH oxidase was 0.052mM. NADH oxidase activity increased when SDS and Tween 80 were added.;정상적인 환경이 아닌 높은 NaCl농도에서도 충분히 생육할 수 있는 호염성 세균을 분리, 동정하고 그들의 생리적 특성과 효소의 염 의존성을 조사하였다. 염전의 토양을 10％ 식염수에 현탁하여 15％와 25％ NaCl의 Sehagal & Gibbons 배지에서 배양하여 14균주의 호염성 세균을 분리하였다. 이렇게 분리된 호염성 세균중 20-25％ NaCl 농도에서 증식하는 9균주의 고도 호염성 세균을 대상으로 형태적, 생리적 특성을 조사하였다. 그 결과 EH8, EH10, EH17, EH19, EH23은 Halobacterium. sp. 로 EH11, EH12, EH13은 Halococcus sp.로 잠정적으로 동정되었다. 이 균주들 중 25% NaCl에서 비교적 빠른 증식을 나타내는EH8,EH10을 선별하여 생리적 특성과 효소의 염 의존성을 조사하였다. EH8, EH10의 최적 증식 NaCl농도는 25％이며 10％이하에서는 증식이 억제 됐다. 최적 증식 온도는 EH8이 45℃, EH10이 50％였으며 NaCl을 KCl로 대치했을때 두 균주 모두 증식하지 못했다. 세포내 대사경로에 관여하는 효소들인 Lactate dehydrogenase, Glucokinase, Glucose-6-phosphate dehydrogenase, Alanine dehydrogenase, Isocitrate dehydrogenase의 활성을 조사한 결과 Glucose-6-phosphate dehydrogenase는 활성이 없었고 나머지 효소들은 모두 활성이 있는 것으로 나타났으며, 특히 Lactate dehydrogenase의 경우는 5M NaCl일때 높은 활성을 가졌으며, KCl은 4M일 때 활성이 높게 나타났다. 전자 전달계에 관여하는 효소인 NADH oxidase와 NADH dehydrogenase의 염 의존성을 Halobacterium sp.EH10에서 조사한 결과 이들 효소는 2M NaCl에서 최적 활성을 나타냈으며, KCI, NH_(4)Cl등의 양이온과 sodium acetate, sodium carbonate등의 음이온에 의해서도 활성화 되었으며 최적 pH는 9였다. NADH oxidase의 Km치는 0.0526mM이었고 SDS와 Tween80의 첨가에 의해 효소의 활성이 증가되었다.목차 = ⅲ 논문개요 = ⅸ 서론 = 1 Ⅱ. 실험재료 및 방법 = 4 1. 호염성 세균의 분리법 = 4 2. 배지 = 4 3. 균주보존 = 4 4. 균주의 동정 = 6 1) 형태적 특성 = 6 2) 생리적 특성 = 7 5. 균배양 = 9 6. 세포내 대사 경로에 관한 효소 분석 방법 = 9 1) 효소 용액 조제 = 9 2) Lactate dehydrogenase(EC.1.1.1.27)의 활성 = 10 3) Glucotinase(EC.2.7.1.1)의 활성 = 10 4) Alanine dehydrogenase(EC.1.1.4.1)의 활성 = 10 5) Glucose-6-phosphate dehydrogenase(EC.1.1.1.49)의 활성 = 11 6) Isocitrate dehydrogenase(EC.1.1.1.42)의 활성 = 11 7. 전자 전달계에 관한 효소 분석 방법 = 11 1) 효소 용액 조제 = 11 2) NADH oxidase의 활성 = 13 3) NADH dehydrogenase 의 활성 = 13 4) 단백질의 정량 = 14 Ⅲ. 실험 결과 및 고찰 = 15 1. 호염성 세균의 분리 선별 = 15 2. 고도 호염성 세균의 동정 = 15 3. 고도 호염성 세균의 최적증식 조건 = 16 1) NaCl 농도의 영향 = 16 2) KCI 농도의 영향 = 23 3) MgSO_(4) 농도의 영향 = 23 4) 온도의 영향 = 26 5) 탄소원의 영향 = 26 4. Halobacterium sp. EH8. EH10의 세포내 효소활성 = 27 5. NADH oxidase의 염 의존성 = 38 1) NADH oxidase의 분포 = 38 2) 효소의 활성에 미치는 양이온의 영향 = 38 3) 효소의 활성에 미치는 음이온의 영향 = 39 4) pH의 영향 = 39 5) 계면활성제의 영향 = 44 6) NADH에 대한 Michaelies-Menten 상수의 결정 = 44 6. NADH dehydrogenase의 염 의존성 = 44 1) 양이온의 영향 = 44 2) 음이온의 영향 = 47 3) pH의 영향 = 47 Ⅳ. 결론 = 52 참고문헌 = 53 도판 = 60 ABSTRACT = 6

INTERRELATIONSHIP BETWEEN REFRACTIVE ERROR AND ANGLE KAPPA

kappa각은 시축과 안축이 이루는 각으로써 양성 kappa각이나 음성 kappa각이 클 때에는 비사시환자도 외관상 사시가 있는 것처럼 보일 수 있으므로 사시환자의 검사에 있어서 중요한 위치를 차지한다. 또 굴절상태에 따른 차이를 보여서 원시안에서는 양성 kappa각이 커지고 근시안에서는 작은 양성 kappa각이나 음성 kappa각을 나타낸다고 한다. 이에 저자는 한국인 소아에서의 kappa각의 정상치와 굴절상태에 따른 kappa각의 차이를 알아보기위하여 만 6세에서 만 11세까지의 아동 282명(564안)을 대상으로 조절마비하에서의 굴절검사와 대약시경(major amblyoscope)을 이용하여 kappa각을 측정한 결과 다음과 같은 결론을 얻었다. 1. kappa각의 빈도는 양성, 음성, 0의 순으로 감소하였으며 각 군간의 차이는 통계학적으로 유의하였다(p＜0.05) 2. 전체 피검자에서의 kappa각의 평균치는 +1.34°±1.33°로써 성별 및 좌우안별에 따른 차이는 없었다(p＞0.05). 3. 정시안에서의 kappa각의 평균치는 +1.71°±1.03°, 원시안에서는 +2.52°±0.81°, 근시안에서는 +0.90°±1.37°로써 원시안, 정시안, 근시안의 순으로 감소하였으며 각 군간의 차이는 통계학적으로 유의하였다(p<0.05).;Distributions of angle kappa and of cycloplegic refraction and their association were studied. The subjects were 282 nonstrabismic Korean children, aged from 6 years to 11 years, who visited Ewha Womans University Hospital from December 1990 to March 1991. The angle-kappa was measured by TOC major amblyoscope ED-011, and refraction was done under the cycloplegic state. The results were as follows : 1. The frequency of angle kappa was positive, negative and 0 in decreasing order. 2. The average values of refractive error and angle kappa were -1.03±2.20 diopter and +1.34 ˚±1.33 ˚. 3. The average values of angle kappa for hypermetropia, emmetropia and myopia were +2.52 ˚±0.81 ˚, +1.71 ˚±1.03 ˚, and +0.90 ˚±1.37 ˚. And these values were significantly different from each other.목차 논문개요 = ⅴ Ⅰ. 서론 = 1 Ⅱ. 대상 및 방법 = 2 A. 대상 = 2 B. 방법 = 2 Ⅲ. 결과 = 4 A. kappa각의 빈도 = 4 B. 성별, 좌우안별, 연령별 굴절이상도 및 kappa각치 = 4 C. 굴절이상도와 KAPPA각과의 관계 = 4 Ⅳ. 고찰 = 9 Ⅴ. 결론 = 11 참고문헌 = 12 ABSTRACT = 1

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