미생물 페니실린 아미다아제에 관한 연구 ( 3 ) Escherichia coli 가 생산하는 페니실린 아미다아제의 정제 및 동력학적 특성

한문희,성백린,김봉희,민태익 ( Moon H . Han,Baik Lin Seong,Bong Hee Kim,Tae Ick Mheen ) 생화학분자생물학회 1981 BMB Reports Vol.14 No.3

Penicillin amidase of Escherichia coli was purified abot 3,200 folds by ammounim sulfate fractionation, DEAE-cellulose, CM-cellulose, and Sephadex G-100 colum chromatography. The overall recovery yield was 57%. As compared with the previous purification method, the purification factor and the recovery yield increased about 56 and 1.4 folds, respectively. The purified enzyme was found to be homogeneous in polyacrplamide gel electrophoresis. The molecular weight determined by SDS-gel electrophoresis was 55,000. The specific activity of penicillin amidase for benzylpenicillin was 2.22 μmoles/sec/㎎ protein and the catalytic rate constant was 122 sec^(-1). The optimum pH and temperature were 8. 5 and 50℃, respectively, This enzyme was rapidly deactivated at temperature above 50℃ and pH above 9.0. Almost all of the metal ions did not affect the enzyme activity except for Hg^(+2) ion. The Michaelis constant for benzylpenicillin was 2.9 mM, the substrate inhibition constants for 6-APA and phenylacetic acid were 10 mM and 4.1 mM, respectively A steady state equation based on the four-step ordered reaction with an acyl intermediate was derived. The inhibition modes of both products based on this equation were discussed with respect to the present experimental results.

방사능 확산 검증을 위한 악기상 조건에서의 추적자 야외확산 실증실험

한문희,김은한,정효준,정해선,박미선,황원태,Han, Moon Hee,Kim, Eun Han,Jeong, Hyo Joon,Jeong, Hae Sun,Park, Mi Sun,Hwang, Won Tae 대한방사선방어학회 2013 방사선방어학회지 Vol.38 No.4

The suitability of the site criteria is a basic requirement for securing safety of nuclear power plants. The suitability should be confirmed through the estimation of environmental radiation effects at the exclusion area boundary under the severe weather conditions. In this study, field tracer experiments over short range of 1 km radius under severe weather conditions were conducted at flat area in Daejeon. Severe weather conditions are represented with stable atmospheric condition and low wind speed. In general, the condition is appeared at clean night time with weak wind. The analysis of the measured distribution of the released tracer gas shows two big differences between the results of the past experiments conducted under the favorable weather conditions. One is the difficulty of finding the typical distribution of the released tracer gas with peak concentration in the downwind direction. The other one is the appearance of the contour of the concentration of tracer gas at several hundred meters even though the gas released at 10 m height over the ground. 원전의 안전성분석보고서에는 운영기간중 만일의 사고에 따른 방사선안전성 확보를 위해 부지의 적합성 평가가 요구된다. 부지 적합성은 극히 보수적인 가정을 적용하여 제한구역경계에서 방사선 피폭영향을 평가하여 판단하게 된다. 이들 평가는 방사선영향평가 모델을 보수적인 기상조건 등에 적용하여 수행된다. 본 연구에서는 보수적인 평가 결과의 타당성 검증을 목적으로 확산에 양호하지 못한 악기상 조건에서 추적자확산실험을 실시하였다. 원전부지 실험에 앞서 대전시 인근 유성구 학하리 평지에서 추적자 확산실험을 실시하였다. 확산실험의 분석결과 기존의 확산조건이 좋은 경우의 실험결과와 비교할 때 크게 두 가지 큰 차이를 발견할 수 있었다. 하나는 풍속이 매우 낮고 풍향의 변화가 심해 주 풍하 방향에서 나타나는 최대 농도를 찾기 어렵다는 것이고, 다른 하나는 대기가 매우 안정하여 지상 10 m 낮은 높이에서의 방출임에도 불구하고 방출점에서 수 백 m 떨어진 지점에서 처음으로 상대적으로 높은 농도 분포가 나타나는 경우가 있다는 것이다.

협측 공간 병변: CT와MR영상소견에 기초한 새로운 분류법

한문희,한문희,제환준,최혁재,김영준,김인원,장기현,Han, Mun-Hui,Han, Mun-Hui,Je, Hwan-Jun,Choe, Hyeok-Jae,Kim, Yeong-Jun,Kim, In-Won,Jang, Gi-Hyeon 대한영상의학회 2004 대한영상의학회지 Vol.50 No.2

목적: 협측 공간 병변의 CT와 MR 영상소견에 기초한 새로운 분류법을 소개하고, 협측 공간 병변의 감별진단에 지침을 제공하고자 한다. 대상과 방법: 조직병리학적 또는 임상적으로 진단된 62예의 협측 공간 병변을 대상으로 하였다. 모든 병변은 그 형태학적 모양과 주변 공간으로의 침범에 따라 다음과 같이 3가지 형으로 분류하였다. 협측 공간에 국한되어 있고 명확한 경계를 가진 경우를 1형, 협측 공간에 국한되어 있고 불명확한 경계를 가진 경우를 2형, 그리고 여러 공간을 동시에 침범한 경우를 3형으로 하였다. 결과: 다형성선종, carcinoma ex pleomorphic adenoma, B-cell 임파선암, 세엽세포암종, 횡문근육종, 사구종양, 사기질모세포종은 1형으로 분류되었고, 악성종양과 양성종양의 감별은 불가능하였다. 농양, 감염이 동반된 유피낭종, Kimura 병, 선낭종암은 2형으로 분류되었다. T-cell 임파선암, 신경섬유종, 혈관기형, 두경부 염증, 이물육아종은 3형으로 분류되었고, 각각은 다른 영상 소견을 보였다. 결론: CT와 MR 영상소견에 기초한 이 새로운 분류법은 협측 공간 병변의 감별진단에 지침이 될 수 있을 것으로 생각한다. Purpose: To present a new classification based on the CT and MR imaging findings of buccal space lesions, and to propose guidelines for their radiologic differentiation. Materials and Methods: Sixty-two histopathologically confirmed or clinically diagnosed buccal space lesions were classified on the basis of their morphologic appearance and extension to adjacent space as either (1) a mass with a distinct margin, confined to the buccal space; (2) a mass with surrounding infiltration, confined to the buccal space; or (3) a multi-space occupying mass. Results: Type 1 included pleomorphic adenoma, ex-pleomorphic adenoma, carcinoma, B-cell lymphoma, acinic cell carcinoma, rhabdomyosarcoma, glomus tumor and ameloblastoma, and differentiation between malignant and benign neoplasms was not possible. Type 2 included adenoid cystic carcinoma, abscess, complicated dermoid cyst, and Kimura disease. T-cell lymphoma, neurofibroma, vascular malformation, inflammation, and foreign body granuloma pertained to type 3, and each type-3 entity showed different imaging characteristics. Conclusion: This new classification based on CT and MR imaging characteristics may provide useful guidelines for predicting the differential diagnosis of buccal space lesions.

삼차원 회전혈관영상에서 발생하는 영상 왜곡 요인에 대한 모형연구

한문희,이승로,함창곡,김희찬,장기현,Han, Mun-Hui,Lee, Seung-Ro,Ham, Chang-Gok,Kim, Hui-Chan,Jang, Gi-Hyeon 대한영상의학회 2004 대한영상의학회지 Vol.50 No.3

목적: 모형 연구를 통하여 삼차원 회전혈관영상의 영상왜곡과 관련 있는 요인을 규명하고 삼차원 영상에 적합한 조영제 농도를 밝히고자 하였다. 대상과 방법: 네가지 모형(1. 교차금속막대, 2. 단금속막대, 3. 단조영제막대, 4. 수중조영제막대: 300, 250, 200, 150 Img/ml 농도)을 제작하여 C-arm의 회전면, 직교면(회전면과 90$^{\circ}$), 중간면(45$^{\circ}$), 근접회전(20$^{\circ}$), 근접직교면(70$^{\circ}$)에서 회전조영술을 시행하고, 삼차원 영상을 만들었다(삼차원 회전조영술). 각 평면에서 얻은 삼차원 영상은 두 명의 신경방사선의사에 의해 직교면에 있는 막대 영상과 비교하여 음영도, 균질도, 그리고 경계의 명확도를 등급화(저$.$중$.$고등급)하여 화질을 평가하였다. 영상영역의 부위별 왜곡을 평가하기 위하여 단금속막대 직교면, 근접직교면, 그리고 중간면 영상의 중심부, 중간부, 변연부의 절단면 영상을 평가하고, 직경(수직 및 수평)을 측정하여 차이를 분석하였다. 결과: 네 가지 모형의 삼차원 영상은 근접직교면과 중간면에서 직교면과 차이가 없이 모두 고등급의 화질을 보였으나 회전면과 근접회전면에서 모든 모형은 화질이 저하되었고, 특히 금속모형의 회전면 영상은 세가지 항목에서 모두 저등급이었다. 금속막대에 비하여 조영제막대가 그리고 300과 150 농도에 비하여 200과 250 농도가 등급이 더욱 높은 경향을 보였다. 단금속막대의 부위별 절단면 영상과 직경은 의미 있는 차이를 보이지 않았다. 결론: 삼차원 회전조영술 영상은, 직선형의 물체가 회전면에 평행할수록 그리고 물체 고유의 X선 감쇄 정도가 심할수록, 영상 왜곡이 증가하였고, 이는 X선경화인공물과 밀접한 관련이 있다. 이러한 왜곡은 조영제 농도가 200-250 Img/ml 인 경우에 가장 적었으며, 생체 외 삼차원 혈관 영상을 위한 가장 적합한 농도로 추정된다. Purpose: To determine, by means of a phantom study, the distortion-related factors and appropriate iodine concentration for three-dimensional reconstruction rotational angiography. Materials and Methods: Four phantoms were created: crossed metal rods, one metal rod, one contrast rod, and a contrast rod under water. Iodine concentrations were 300, 250, 200, and 150 Img/ml, respectively. For each phantom, rotational angiography was performed in the rotational, right-angled (90$^{\circ}$to rotational), intermedial (45$^{\circ}$), close to rotational (20$^{\circ}$), and close to right-angled (70$^{\circ}$) planes. Two-dimensional projection images were transferred to a workstation at which 3D images were produced using the volume rendering technique. Image quality in each plane was evaluated in terms of opacity, homogeneity, and margin sharpness, which were graded as low, intermediate or high by two neuroradiologists who used images obtained in the right-angled plane as the standard reference. The same assessors evaluated in terms of the same parameters, cross-sectional images obtained at the central, intermedial, and peripheral portions of one metal rod positioned in the right-angled, close to right-angled, and intermedial planes, and in order to compare the values at different sites, one neuroradiologist measured the horizontal and vertical diameters of each cut image. Results: Three-dimensional images of all four phantoms were high quality in the close to right-angled and intermedial plane, but in the rotational and close to rotational plane were degraded. In particular, metal rod images obtained in the rotational plane were poor for all three items. In these two planes, image quality was better for the contrast rod than the metal rod, and at 200 and 250 Img/ml concentrations than at 300 and 150 Img/ml concentrations. There was no significant difference in image quality, nor in measured values of the diameter between cut images. Conclusion: A three-dimensional image was more distorted when a linear object was placed at a lesser angle to the rotational plane and when inherent X-ray attenuation was greater, a finding which must be closely related to the beam-hardening artifact. Distortion was least at 200-250 Img/ml of iodine concentration, the concentration thought to be most appropriate for in-vitro 3D angiography.

Studies on Microbial Penicillin Amidase (III) : Purification and kinetic Properties of Penicillin Amidase of Escherichia coli

한문희,성백린,김봉희,민태익,Han, Moon-H.,Seong, Baik-Lin,Kim, Bong-Hee,Mheen, Tae-Ick 생화학분자생물학회 1981 한국생화학회지 Vol.14 No.3

E. coli에서 추출한 penicillin amidase를 황산암모니움에 의한 분별침전 및 DEAE-cellulose, CM-cellulose, Sephadex G-100 컬럼 크로마토그라피에 의해서 3,200배 정제하였고, 이때의 효소의 회수율은 57%이었다. 이것은 기존방법에 비해 비활성은 56배, 그리고 회수율은 1.4배 증가되었다. 이와 같이 얻은 penicillin amidase는polyacrylamide gel 전기영동에 의해 순수함이 증명되었고, SDS-gel 전기영동에 의해 분자량은 55,000임을 알았다. 정제된 효소외 비활성은 기질인 벤질페니실린에 대하여 2.22 ${\mu}$kat/mg protein 이었고 촉매속도항수는 122 $sec^{-1}$이었다. 이 효소의 최적온도 및 pH는 각각 $50^{\circ}C$와 8.5이었으며, $50^{\circ}C$ 이상의 온도와 pH 9.0 이상에서 효소불활성이 빨랐다. $Hg^{++}$를 제외한 대부분의 금속이온은 효소활성에 영향이 없었다. 벤질페니실린에 대한 $K_m$ 상수는 2.9 mM, 기질 저해상수는 35.4 mM, 리고 반응생성물인 6-aminopenicillanic acid (6-APA)와 페닐초산에 의한 저해상수는 각각 10 mM과 4.1 mM이었다. 아실중간체를 형성하는 4단계 반응을 가정하여 steady state 방정식을 유도하고 반응생성물의 효소에 대한 저해양상에 대해서 실험결과 및 보고된 결과들과 비교하였다. Penicillin amidase of Escherichia coli was purified abot 3,200 folds by ammounim sulfate fractionation, DEAE-cellulose, CM-cellulose, and Sephadex G-100 colum chromatography. The overall recovery yield was 57%. As compared with the previous purification method, the purification factor and the recovery yield increased about 56 and 1.4 folds, respectively. The purified enzyme was found to be homogeneous in polyacrplamide gel electrophoresis. The molecular weight determined by SDS-gel electrophoresis was 55,000. The specific activity of penicillin amidase for benzylpenicillin was 2.22 ${\mu}$moles/sec/mg protein and the catalytic rate constant was 122 $sec^{-1}$. The optimum pH and temperature were 8.5 and $50^{\circ}C$, respectively. This enzyme was rapidly deactivated at temperature above $50^{\circ}C$ and pH above 9.0. Almost all of the metal ions did not affect the enzyme activity except for $Hg^{+2}$ ion. The Michaelis constant for benzylpenicillin was 2.9 mM, the substrate inhibition constants for 6-APA and phenylacetic acid were 10 mM and 4.1 mM, respectively A steady state equation based on the four-step ordered reaction with an acyl intermediate was derived. The inhibition modes of both products based on this equation were discussed with respect to the present experimental results.

경막동맥-해면정맥동루의 임상양상과 치료결과

김철 ( Cheol Kim ) , 곽상인 ( Sang In Khwarg ) , 한문희 ( Moon Hee Han ) 대한안과학회 2003 대한안과학회지 Vol.44 No.7

Cloning and expression of human prethrombin 2 cDNA in Escherichia coli

최은화,김용주,김종명,홍효정,한문희,김지영,Choi, Eun-Hwa,Kim, Yong-Joo,Kim, Jong-Myoung,Hong, Hyo-Jeong,Han, Moon-Hi,Kim, Ji-Young 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

A 0.6 kbp of 3'-portion of human prothrombin cDNA was isolated from a liver cDNA library of ${\lambda}gt$ 10 by in situ plaque hybridization. A cDNA fragment corresponding to the N-terminal region of prethrombin 2 was obtained from another cDNA library synthesized by reverse transcription of human liver mRNA using a specific oligonucleotide as a primer. The 48 bp cDNA sequences starting from the first nucleotide to the AvaI site of human prethrombin 2 cDNA was chemically synthesized such that prethrombin 2 can be expressed directly in E. coli expression vector system. The open reading frame(ORF) for human prethrombin 2 was cloned in pUC18 by combining the synthetic fragment and the cDNA fragments. Expression plasmids were constructed in which the ORF for human prethrombin 2 was inserted into the site downstream from the thermoinducible leftward promoter $(P_L)$ and the cII ribosome binding site of bacteriophage ${\lambda}$. Upon temperature induction, the plasmids expressed 34,000 dalton polypeptide, which was accumulated to an amount corresponding to 5-10% of the total bacterial proteins. The 34,000 dalton polypeptide was identified as human prethrombin 2 by SDS-gel electrophoresis and Western blot analysis. 사람 간 cDNA library로부터 프로트롬빈 mRNA의 3'쪽에 해당하는 0.6kbp cDNA를 plaque hybridization에 의해 분리하였다. 프로트롬빈 2의 N-말단에 해당하는 cDNA 절편은 간 mRNA와 상보적인 올리고뉴클리오티드 primer를 이용하여 만든 다른 cDNA library로부터 분리하였다. 프로트롬빈 2를 E. coli에서 직접 발현시키기 위해 프리트롬빈 2cDNA의 첫 뉴클라오티드부터 Ava I 부위까지의 48bp DNA를 화학적으로 합성하였다. 합성 DNA와 cDNA 절편들을 이용하여 완전한 사람 프리트롬빈 2 번역프레임 (ORF)를 포함한 pUC18 을 클로닝하였다. 사람 트리트롬빈 2 ORF를 박테리오파지 ${\lambda}$의 $P_L$ promoter와 c II ribosome binding site 아래에 연결하여 프리트롬빈 2 발현벡터를 제조하였다. 온도에 의해 유도시킨 결과 이 플라스미드는 E. coli에서 약 34,000 dalton의 폴리펩티드를 생성하였다. 이 폴리펩티드는 전체 박테리아 단백질의 5-10% 정도로 만들어졌다. Western blot 분석에 의해 이 폴리펩티드가 사람 프리트롬빈 2 라는 것을 확인하였다.

경동맥 해면동루

김우중(Woo Jung Kim),김윤덕(Yoon Duck Kim),한문희(Moon Hee Han) 대한안과학회 1991 대한안과학회지 Vol.32 No.4

E . coli 에서 사람 프리트롬빈 2 cDNA 의 크로닝과 발현

최은화,김용주,김종명,홍효정,한문희,김지영 ( Eun Hwa Choi,Yong Joo Kim,Jong Myoung Kim,Hyo Jeong Hong,Moon Hi Han,Ji Young Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

A 0.6 kbp of 3`-portion of human prothrombin cDNA was isolated from a liver cDNA library of λgt 10 by in situ plaque hybridization. A cDNA fragment corresponding to the N-terminal region of prethrombin 2 was obtained from another cDNA library synthesized by reverse transcription of human liver mRNA using a specific oligonucleotide as a primer. The 48 bp cDNA sequences starting from the first nucleotide to the AvaI site of human prethrombin 2 cDNA was chemically synthesized such that prethrombin 2 can be expressed directly in E. coli expression vector system. The open reading frame(ORF) for human prethrombin 2 was cloned in pUC18 by combining the synthetic fragment and the cDNA fragments. Expression plasmids were constructed in which the ORF for human prethrombin 2 was inserted into the site downstream from the thermoinducible leftward promoter (P_L) and the cII ribosome binding site of bacteriophage λ. Upon temperature induction, the plasmids expressed 34,000 dalton polypeptide, which was accumulated to an amount corresponding to 5-10% of the total bacterial proteins. The 34,000 dalton polypeptide was identified as human prethrombin 2 by SDS-gel electrophoresis and Western blot analysis.

평지 야외 대기 확산실험

김은한(Eun Han Kim),서경석(Kyung Suk Suh),황원태(Won Tae Hwang),한문희(Moon Hee Han),김학성(Hag Seong Kim) 大韓環境工學會 1997 대한환경공학회지 Vol.19 No.5