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      • 뱀독 Phosphodiesterase의 Endonuclease 활성에 대한 기질특이성

        염영나,김두식,Yum, Young-Na,Kim, Du-Sik 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

        뱀독 phosphodiesterase의 endonuclease 활성에 대한 pUC19 DNA의 절단부위를 확인하였다. Phosphodiesterase의 endonuclease 반응에 의하여 supercoil 형태의 pUC19은 linear형태 이외에 1579 bp와 1107 bp의 DNA 단편들로 분해된다. 제한효소 mapping 분석방법으로 결정한 pUC19의 절단부위는 1398과 2505였다. Cruciform을 이루고 있는 1398 위치의 절단부위는 single-strand specific nuclease S1의 작용부위와 일치하는 것으로 밝혀졌으며 2505 위치는 hairpin 구조를 이루는 것으로 예상된다. 그러므로 뱀독 phosphodiesterase의 endonuclease 활성은 DNA의 cruciform 또는 (A+T)-rich hairpin 구조를 인식하여 가수분해함을 증명하였다. Endonucleolytic cleavage sites of pUC19 by snake venom phosphodiesterase were identified. Limited digestion of supercoiled pUC19 DNA by the phosphodiesterase yields unit length linear form of the plasmid plus two discrete fragments which are 1579 by and 1107 bp. Studies of restriction enzyme mapping analysis indicate that the cleavage sites are 1398 and 2505 of pUC19. The cleavage site located at 1398 which is known as a cruciform structure, coincides with the position recognized by single-strand specific nuclease S1. The 2505 region appears to form a hairpin structure with (A+T)-rich sequence. It is concluded that the endonuclease activity of snake venom phophodiesterase recognizes certain specific secondary structures such as cruciform or (A+T)-rich hairpin region of DNA.

      • 한국산 뱀독의 Phosphodiesterase 정제와 특성

        염영나,최혜림,김두식,Yum, Young-Na,Choi, Hye-Lim,Kim, Doo-Sik 생화학분자생물학회 1991 한국생화학회지 Vol.24 No.1

        Phosphodiesterase (EC 3.1.4.1) has been purified to homogeneity from the venom of Korean snake Agkistrodon halys. A combination of ion-exchange, gel filtration and affinity chromatographic procedure leading to 3,600 fold purification of the phosphodiesterase with specific activity of 81.2 units/mg was established. Molecular weight of the purified enzyme, a monomeric glycoprotein, was identified to be 110,000. Apparent $K_m$, and $V_{max}$ values for the hydrolysis of bis-p-nitrophenyl phosphate (bis-PNP) were determined to be 2.44 mM and $4.5{\times}10^{-4}moles/l{\cdot}min$. respectively. Activation energy of the enzyme catalysis toward the bis-PNP hydrolysis was estimated to be 10.4 KJ/mol. The enzyme exhibited its maximum catalytic activity at pH 9.5. Both exonuclease and endonuclease activities of the purified enzyme were demonstrated using plasmid DNA as a substrate. It is clear that the phosphodiesterase is a bifunctional enzyme which retains intrinsic endonuclease function as well as exonuclease activity in a single protein molecule. 한국산 살모사(Agkistrodon halys)의 독으로부터 phosphodiesterase(EC 3.1.4.1)를 순수 하게 정제하였다. 5'-AMP 친화성 크로마토그라피를 포함하는 네 단계의 분리과정을 거쳐 3,600배 정제한 phosphodiesterase의 비활성도는 81.2 units/mg이었다. 정제된 효소는 monomer 구조의 당단백질로서 그 분자량이 110,000으로 확인되었다. 이 효소의 $K_m$과 $V_{max}$는 각각 $2.44{\times}10^{-3}M$, $4.5{\times}10^{-4}moles/l{\cdot}min$으로 측정되었고 bis-p-nitrophenyl phosphate의 가수분해를 촉매하는 활성화에너지는 10.4 KJ/mole이며 효소활성에 대한 최적 pH는 9.5로 나타났다. 순수하게 정제된 효소를 이용하여 plasmid DNA를 기질로 사용함으로써 phosphodiesterase의 exonuclease 및 endonuclease 활성을 모두 확인하였다. 따라서 한국산 뱀독의 phosphodiesterase는 exonuclease 활성은 물론 endonuclease 기능도 함께 보유하고 있는 이중성 효소임을 증명하였다.

      • 뱀독 Phosphodiesterase의 이중성 촉매기능

        염영나,김두식,Yum, Young-Na,Kim, Doo-Sik 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

        Exonuclease와 endonuclease 활성을 모두 가지고 있는 뱀독 phosphodiesterase의 촉매 부위에 대한 특성 연구를 수행하였다. 5’-AMP에 의하여 phosphodiesterase의 exonuclease 활성은 경쟁적으로 억제되며, 동시에 endonuclease 활성도 억제됨을 관찰하였다. N-bromosuccinimide 또는 diisopropylfluorophosphate를 이용한 효소의 화학변형 실험결과 역시 두 측매활성이 모두 비가역적2-.로 상실되지만 5’-AMP나 5’-ATP는 이와 같은 화학변형에 대해 현저한 효소활성 보호효과를 나타냈다. 실험적 증거를 토대로 하여 phosphodiesterase의 exo 및 endonuclease 활성은 110K dalton의 monomer 효소단백질 구조에 독립적으로 위치하는 것이 아니라 동일 촉매부위에 존재함을 입증하였다. Dual nuclease activities of snake venom phosphodiesterase from Agkistrodon halys were characterized. Exonucleolytic activity of the enzyme for the hydrolysis of synthetic substrate bis-p-nitrophenyl phosphate is competitively inhibited in the presence of adenosine 5’-mono-phosphate. At the same time, the endonuclease function is also inactivated by the mononuc-leotide. Chemical modification of the purified enzyme by either N-bromosuccinimide or diiso-propylfluorophosphate results in loss of the both catalytic activities. However, adenosine 5'-triphosphate or the corresponding mononucleotide could afford protection against the chemi-cal modification of the enzyme. Several lines of experimental evidence indicate that the catalytic site responsible for both of the exonuclease and endonuclease activities is located in a single functional domain of the monomeric enzyme molecule.

      • KCI등재

        말초혈액을 이용한 초고속 유전독성평가법 개발 연구

        안일영 ( Il Young Ahn ),김주환 ( Ju Hwan Kim ),임대현 ( Dae Hyun Lim ),양준영 ( Jun Young Yang ),김소영 ( So Young Kim ),이정선 ( Jung Sun Yi ),염영나 ( Young Na Yum ),임채형 ( Chae Hyung Lim ),이진하 ( Jin Ha Lee ),최기환 ( Ki Hw 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1

        To identify mutagenic potential of test substances, in vitro Ames tests are commonly used. Recently revised ICH S2(R1) guideline requires in vivo genotoxicity test if the result of the in vitro test is positive. In addition, a method testing multiple endpoints is required for animal welfare. Therefore we established a flow cytometry-based analysis such as Pig-a gene mutation assay and the micronuclei assay for detection of in vivo genotoxic potential using peripheral blood collected from repeated dose toxicity study. To evaluate these new methods, male Sprague Dawley rats were treated for 3, 14 or 28 days with N-nitro-N-ethylurea (ENU). ENU induced mutations in both reticulocytes (RET) and red blood cells of rats dose-dependently from the Pig-a gene mutation assay. ENU also increased micronucleated reticulocytes frequencies in flow cytometry based micronuclei assay, implying chromosomal damage to hematopoetic cells. These data show that both assays were well established. We additionally evaluated urethane and glycidol for applicability of Pig-a gene mutation assay and in vivo micronuclei assay by flow cytometry. Urethane, compared with vehicle control, did not increase Pig-a gene mutation and micronuclei frequency. Glycidol, compared with vehicle control, did not increase in micronuclei frequency, but Pig-a gene mutation significantly increased in the highest concentration for 28 days. Pig-a gene mutation assay for genotoxicity has many advantages: It can detect mutation in various species including humans, primates and rodents; and is integrated with repeated dose toxicity test without additional usage of animals; and has low spontaneous mutation frequency.

      • KCI등재

        연구논문 : 말초혈액을 이용한 초고속 유전독성평가법 개발 연구

        안일영 ( Il Young Ahn ),김주환 ( Ju Hwan Kim ),임대현 ( Dae Hyun Lim ),양준영 ( Jun Young Yang ),김소영 ( So Young Kim ),이정선 ( Jung Sun Yi ),염영나 ( Young Na Yum ),임채형 ( Chae Hyung Lim ),이진하 ( Jin Ha Lee ),최기환 ( Ki Hw 한국동물실험대체법학회 2014 동물실험대체법학회지 Vol.8 No.1

        To identify mutagenic potential of test substances, in vitro Ames tests are commonly used. Recently revised ICH S2(R1) guideline requires in vivo genotoxicity test if the result of the in vitro test is positive. In addition, a method testing multiple endpoints is required for animal welfare. Therefore we established a flow cytometry-based analysis such as Pig-a gene mutation assay and the micronuclei assay for detection of in vivo genotoxic potential using peripheral blood collected from repeated dose toxicity study. To evaluate these new methods, male Sprague Dawley rats were treated for 3, 14 or 28 days with N-nitro-N-ethylurea (ENU). ENU induced mutations in both reticulocytes (RET) and red blood cells of rats dose-dependently from the Pig-a gene mutation assay. ENU also increased micronucleated reticulocytes frequencies in flow cytometry based micronuclei assay, implying chromosomal damage to hematopoetic cells. These data show that both assays were well established. We additionally evaluated urethane and glycidol for applicability of Pig-a gene mutation assay and in vivo micronuclei assay by flow cytometry. Urethane, compared with vehicle control, did not increase Pig-a gene mutation and micronuclei frequency. Glycidol, compared with vehicle control, did not increase in micronuclei frequency, but Pig-a gene mutation significantly increased in the highest concentration for 28 days. Pig-a gene mutation assay for genotoxicity has many advantages: It can detect mutation in various species including humans, primates and rodents; and is integrated with repeated dose toxicity test without additional usage of animals; and has low spontaneous mutation frequency.

      • KCI등재

        OECD 급성경구투여독성시험 지침의 국내 확립 및 검증

        조영래 ( Young Rae Cho ),염영나 ( Young Na Yum ),한의식 ( Eui Sik Han ),곽승준 ( Seung Jun Kwack ),김형섭 ( Hyung Sub Kim ),강미선 ( Mi Sun Kang ),이진영 ( Jin Young Lee ),오재호 ( Jae Ho Oh ),임채형 ( Chae Hyung Lim ),김대성 ( Da 한국동물실험대체법학회 2008 동물실험대체법학회지 Vol.2 No.2

        As the study about the non-animal tests came to be internationally active and the interest in the animal welfare gradually became increased, OECD TG (Test Guideline) 401 that has been used since 1987 was abolished in 2002. Because TG401 is acute oral toxicity method depending on survival and death of many animals, it was heavily criticized. Therefore, the three alternative methods were developed. OECD TG420 and TG423 determine the class of chemicals according to GHS (Globally Harmonized Classification System for Chemical Substances and Mixtures) classification and OECD TG425 suggests the predicted LD50 of chemicals using AOT425 program. In this study, 10 chemicals were selected. The internationally admitted TG420, TG423 and TG425 were introduced and established through the method that these chemicals were orally administrated to SD female rats and then, the results were observed. Each chemical belonged to already known GHS class in the study using TG420 and TG423 and predicted LD50 was same or higher in the study using TG425 compared to already known LD50 value. In conclusion, the result of our study confirmed the decrease in the animal number and validated. The international harmonization of the non-animal tests will be pursued through this validation study.

      • KCI등재

        유세포분석기를 이용한 말초혈액에서의 소핵시험

        김주환 ( Joo Hwan Kim ),염영나 ( Young Na Yum ),김희윤 ( Hee Yun Kim ),김경아 ( Kyong A Kim ),김지은 ( Jee Eun Kim ),박순희 ( Sue Nie Park ) 한국동물실험대체법학회 2010 동물실험대체법학회지 Vol.4 No.1

        The micronucleus assay is the most widely utilized in vivo system for evaluating chemicals potential to induce chromosome breaks or to poison mitotic spindle apparatus. The bone marrow erythrocyte micronucleus assay is widely used for regulatory studies conducted to evaluate the potential for induction of chromosomal damage or chromosome loss due to exposure to drugs, food additives, pesticides, industrial chemicals, and other products. Peripheral blood reticulocytes(RET) have been accepted as an appropriate target for micronucleus (MN) assessment for both acute and cumulative damage. However, there is a problem when using rat peripheral blood reticulocytes, since the rat spleen selectively removes MN-RET from the circulation. The development of automated flow cytometric(FCM), anti-CD71-based methods for evaluating micronucleus frequencies in reticulocytes has great potential for improving the sensitivity, reproducibility, and throughput of the traditional in vivo rodent MN assay that uses microscopy-based methods. There were some validation studies of the FCM evaluation methods in many species. For instance it has been demonstrated that for mice, rats, dogs, monkey, and humans. Although spleen-dependent reduction in circulating MN-RET frequency resulting from genotoxic exposures attenuates the magnitude of MN-RET frequencies in peripheral blood relative to that in bone marrow, the analytical and statistical advantages of the FCM measurement in peripheral blood more than offset the higher frequencies observed for bone marrow smears analyzed. In addition to the analytical advantages of the FCM method, the ready accessibility of the small blood samples required for the FCM assay make possible the integration of the MN frequency assessment with routine toxicology studies. It is not necessary to conduct separate in vivo studies for the purpose of evaluating the potential for chromosomal damage in rodent bone marrow but that more reliable information can be obtained by FCM analysis of peripheral blood samples obtained from rodent studies during the course of routine toxicological evaluations.

      • SCOPUSKCI등재

        Diethylnitrosamine으로 유도한 랫드유래 간 배양세포(clone 9)의 발암화 과정에서의 효소변화

        김현배(Hyun-Bae Kim),염영나(Young-Na Yum),이미영(Mi-Young Lee) 한국독성학회 1999 Toxicological Research Vol.15 No.2

        The effects of diethylnitrosamine (DEN A) on the activities of various enzymes involved in aldehyde metabolism were examined in cultured rat liver clone 9 cell. The enzymes included alcohol dehydrogenase(ADH), aldehyde dehydrogenase (ALDH) , glutathione-S-transferase (GST) and catalase. The enzyme activity changes induced by various concentrations of DENA and repetitive treatment of fixed concentrations of DENA were analyzed in terms of specific activity and activity staining on the gel. About 2.5 times higher ADH activity was found when the cell was treated with DENA from 1 to 7 times. However, about 40% of ADH activity was increased at 40 mM DENA and then decreased thereafter as the doses of DENA were increased. On the other hand, the specific activity of ALDH was increased rapidly reaching to 2.5 times higher activity in concentration dependent and repetitive treatment of DENA. In case of GST level, only slight increases occurred with concentration dependent and repetitive treatment of DENA. Maximum ten folds increase of lipid peroxide level occurred in the DENA treated cell, whereas the increase of lipid peroxidation was not correlated with that of catalase activity. Specific activities of ADH, ALDH and GST in clone 9 cell were also compared with those of various human cancer cell lines.

      • KCI등재

        연구논문 : 유세포분석기를 이용한 말초혈액에서의 소핵시험

        김주환 ( Joo Hwan Kim ),염영나 ( Young Na Yum ),김희윤 ( Hee Yun Kim ),김경아 ( Kyong A Kim ),김지은 ( Jee Eun Kim ),박순희 ( Sue Nie Park ) 한국동물실험대체법학회 2010 동물실험대체법학회지 Vol.4 No.1

        The micronucleus assay is the most widely utilized in vivo system for evaluating chemicals potential to induce chromosome breaks or to poison mitotic spindle apparatus. The bone marrow erythrocyte micronucleus assay is widely used for regulatory studies conducted to evaluate the potential for induction of chromosomal damage or chromosome loss due to exposure to drugs, food additives, pesticides, industrial chemicals, and other products. Peripheral blood reticulocytes(RET) have been accepted as an appropriate target for micronucleus (MN) assessment for both acute and cumulative damage. However, there is a problem when using rat peripheral blood reticulocytes, since the rat spleen selectively removes MN-RET from the circulation. The development of automated flow cytometric(FCM), anti-CD71-based methods for evaluating micronucleus frequencies in reticulocytes has great potential for improving the sensitivity, reproducibility, and throughput of the traditional in vivo rodent MN assay that uses microscopy-based methods. There were some validation studies of the FCM evaluation methods in many species. For instance it has been demonstrated that for mice, rats, dogs, monkey, and humans. Although spleen-dependent reduction in circulating MN-RET frequency resulting from genotoxic exposures attenuates the magnitude of MN-RET frequencies in peripheral blood relative to that in bone marrow, the analytical and statistical advantages of the FCM measurement in peripheral blood more than offset the higher frequencies observed for bone marrow smears analyzed. In addition to the analytical advantages of the FCM method, the ready accessibility of the small blood samples required for the FCM assay make possible the integration of the MN frequency assessment with routine toxicology studies. It is not necessary to conduct separate in vivo studies for the purpose of evaluating the potential for chromosomal damage in rodent bone marrow but that more reliable information can be obtained by FCM analysis of peripheral blood samples obtained from rodent studies during the course of routine toxicological evaluations.

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