흑두로 제조한 청국에서 분리된 Bacillus subtillus BB-1으로 부터 혈전용해효소 유전자 크로닝 및 특성규명

이영훈,이성호,전주미,김홍출,조용운,박기훈,최영주,갈상완,Lee Young-Hoon,Lee Sung-Ho,Jeon Ju-Mi,Kim Hong-Chul,Cho Yong-Un,Park Ki-Hoon,Choi Young-Ju,Gal Sang-Wan 한국생명과학회 2005 생명과학회지 Vol.15 No.4

흑두로 제조한 청국으로부터 혈전용해력이 우수한 균을 선발하여 동정하였으며, 그를 Bacillus subtilis BB-1로 명명하였다. 이 균은 혈전용해효소 isozyme을 적어도 5개이상 생성하는 균주로 확인되었다. 이 균으로부터 크로모좀을 분리하여 shot gun법으로 혈전용해효소 유전자를 크로닝하였으며, 이 유전자를 BSF-1이라 명명하였다. 이 유전자는 714개의 아미노산을 암호화하고 있으며 기존에 밝혀진 혈전용해효소 유전자와 상동성은 검출되지 않은 새로운 혈전용해효소 유전자였다. 혈전용해효소활성 최적 pH 및 온도는 5.0과 $35^{\circ}C$였다. 기질특이성은 적혈구 배지 또는 skim milk, gelatin등에 전혀 분해활성이 없었다. 이는 혈전만을 특이적으로 분해하는 기질특이성을 보였으며, 혈전분해효소로서의 이용가능성이 충분한 것으로 판단된다. A bacterium producing five fibrinolytic isozymes was isolated from black bean chung kuk. The bacterium was identified as Bacillus subtilis BB-1 by 16s rDNA sequence homology search. A gene out of five fibrinolytic genes in the Bacillus subtilis BB-1 was cloned by shot-gun method. A Cla I DNA fragment of B. subtilis BB-1 chromosome was cloned in to pBluescript II SK(-) and showed the fibrinolytic activity to bacterial cells. The Cla I DNA fragment was sequenced and the sequences did not show homology with gene for protease or fibrinolytic enzyme genes in other organisms. The Cla I DNA fragment was reduced to 2,142 bp by activity-guided PCR cloning method. The optimum pH and temperature of the enzyme were 5.0 and $35^{\circ}C$, respectively. Substrate specificity of the fibrinolytic enzyme was detected in skim milk, casein, gelatin and blood agar plates. The activity of the enzyme was not detected with these substrates. Taken together, this enzyme is a new fibrinolytic enzyme and may be used to prevent thrombosis and arteriosclerosis.

Bacillus subtilis BB-1으로부터 나토키나아제 유전자 크로닝 및 대량발현

이영훈,이성호,박기훈,최영주,정영기,갈상완,Lee Young-Hoon,Lee Sung-Ho,Park Ki-Hoon,Choi Young-Ju,Jeong Yong-Kee,Gal Sang-Wan 한국생명과학회 2006 생명과학회지 Vol.16 No.2

흑두청국으로부터 분리된 혈전용해력이 우수한 Bacillus subtilis BB-1(KFCC 11344P)으로부터 혈전용해효소 유전자를 PCR법에 의해 크로닝하였고 이를 BCF-1으로 명명하였다. BCF-1의 DNA 염기서열결정 결과 1,145 bp 크기의 혈전용해 효소로, 일본의 natto로부터 분리된 nattokinase 유전자와 99%의 상동성을 보임을 확인하였다. 혈전용해효소 유전자의 발현을 위하여 Bacillus 발현계인 Bacillus-E. coli의 shuttle vector인 pEB vector에 크로닝 하고 host로서 B. subtilis 168에 형질전환시켜 대량 발현시켰다. 생산된 혈전용해효소의 최 적활성 pH와 온도는 7.0과 $35^{\circ}C$로 확인되었다, 기질에 대한 분해양상을 조사한 결과 fibrin에서만 특이적으로 강한 분해가 일어났으며, skim milk에서 아주 약한 분해능을 보였으나 blood agar, gelatin, casein에서는 전혀 분해능을 보이지 않았다. 특히 blood agar plate에서 분해능이 없는 것으로 보아 혈액 내에서의 적혈구 파괴현상과 같은 부작용에 대한 위험을 배제할 수 있을 것으로 사료된다. BCF-1에 의해 생산된 혈전용해효소는 fibrin 특이적으로 활성을 나타냄을 확인할 수 있으며, 이는 임상적이나 산업적으로 적용하였을 때 부작용에 대한 위험적인 문제는 배제될 수 있으리라 생각된다. A fibrinolytic enzyme gene was isolated from Bacillus subtilis BB-1 by PCR method. Primers for PCR cloning were designed according to pre-identified gene for fibrinolytic enzymes from B. subtilis. The primer sequences were 5'-CGG ATC CGT GAG AGG CAA AAA GGT G-3' and 5'-TGA ATT CTT AAT GTG CTG CTG CTT GTC C-3' as concensus sequences of the fibrinolytic genes of Bacillus species. The PCR product was 1,145 bp and the sequence homology was 99% with nattokinase gene isolated from Japanese natto. The cloned fibrinolytic gene was reconstructed in Bacillus-E. coli shuttle vector, pEB for bulk-production. The fibrinolytic enzyme was purified by FPLC from the cloned B. subtilis 168. The optimum pH and temperature of the enzyme were 7.0 and $35^{\circ}C$, respectively. The fibrinolytic enzyme did not show any activity toward to skim milk, gelatin, casein and blood agar plate. The enzyme specific polyclonal antibody was prepared in rabbit for further assays such as detection of the gene expression in plant cells. This means that the enzyme may be used for health-care such as thrombosis without any hamful effects in the blood vessel.

Antitumor Sterol Isolated from the Fruiting Body of Pleurotus eryngii

이영훈,박기훈,이병원,조용운,최영주,갈상완,Lee Young-Hoon,Park Ki-Hun,Lee Byong-Won,Cho Yong-Un,Choi Young-Ju,Gal Sang-Wan Korean Society of Life Science 2006 생명과학회지 Vol.16 No.2

새송이버섯으로부터 활성추적법으로 항암활성이 있는 물질인 에르고스테롤 프록사이드를 분리하였다. 이 스테롤의 구조는 분광법과 NMR법으로 확인하였으며 분자식은 $C_{28}H_{44}O_3$이었다. 폐암과 난소암에 $IC_{50}$값은 각각 $7{\mu}M$과 $14{\mu}M$이었다. DNA단편화 실험에서 이 화합물은 암세포의 chromosimal DNA 를 사닥다리모양으로 분해하였고, 세포 분열주기의 억제실험에서 G1단계를 억제함을 관찰하였다. Activity-guided fractionations led to the isolation of antitumor compound, ergosterol peroxide ($5{\alpha},\;8{\alpha}-epideoxy-24(R)-methylcholesta-6,\;22-dien-3{\beta}-ol$) from the fruiting body of Pleuratus eryngii that was cultivated artificially. This sterol structure was established by using spectroscopic methods ($^1H\;and\;^{13}C$ nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formular of $C_{28}H_{44}O_3$ displaying characteristic features of epidioxy sterols. The 50% inhibitory concentrations ($IC_{50}$) of ergosterol peroxide against human lung cancer cell line (A549) and human ovarian cell line (SK-OV3) were $7{\mu}M\;and\;14{\mu}M$, respectively. In the DNA fragmentation assay, the compound showed the programmed cell death causing the chromosomal DNA fragmentation. It reveals that ergosterol peroxide arrests G1 phase of the cell division cycle.

포도와 오이즙액을 이용한 동충하초(Paecilomyces japonica) 균사체 배양액의 미백효과

이영훈,박상상,이상원,이성호,박기훈,최영주,갈상완,Lee, Young-Hoon,Park, Sang-Sang,Lee, Sang-Won,Lee, Sung-Ho,Park, Ki-Hoon,Choi, Young-Ju,Gal, Sang-Wan 한국생명과학회 2006 생명과학회지 Vol.16 No.5

포도와 오이즙액에 동충하초 균사체 배양액의 미백화장품 원료로서의 이용가능성을 조사하였다. B16BL6 mouse melanoma cell에 동충하초 균사체 배양액을 배양기간 및 농도별로 처리했을 때, 15일 동안의 배양조건과 $50\;{\mu}l/ml$ (5%)의 농도로 처리하였을 때 멜라닌 생성억제 효과가 가장 높은 것을 확인하였다. In vitro tyrosinase 활성억제효과에서는 포도와 오이즙액에 동충하초 균사체 배양액을 10% 농도로 첨가시 50%의 멜라닌 생성 억제율을 보였으며, 30% 농도에서는 100% 억제를 확인하였다. 결과적으로 포도와 오이즙액에 동충하초 균사체 배양액은 멜라닌 생성의 주 효소인 tyrosinase의 작용을 억제함으로써 미백 효과를 나타내는 것을 확인하였다. 또한 세포독성실험에서 포도, 오이즙액과 그 즙액에 동충하초 균사체 배양액을 각각의 농도로 B16BL6 mouse melanoma cell에 처리 한 결과 포도, 오이즙액의 5% 처리 시에는 50% 사멸, 9% 이상에서는 100% 세포가 사멸하는 것을 확인 할 수 있었다. 그러나 포도, 오이즙액에 동충하초 균사체를 배양한 배양액은 20% 까지 처리 시에도 세포독성이 나타나지 않는 것을 확인 하였다. 따라서 포도와 오이즙액에 동충하초 균사체를 배양한 배양액은 세포 독성이 없으며, 미백 효과가 뛰어난 것으로 미백화장품 원료로서 사용가능함을 확인하였다. This study was carried out to investigate the whitening effect of mycelial culture broth of P. japonica in the mixture of cucumber and grape extracts. In the inhibition test of melanin biosynthesis of melanoma cell, B16BL6, the culture broth of P. japonica more then $50\;{\mu}l/ml$ (5%) concentration inhibited the melanin biosynthesis of the cell entirely without cytotoxicity. More then 10 days incubation of P. japonica in the mixture was required to have the inhibition activity. In vitro inhibition test of melanin biosynthesis of the culture broth of P. japonica was investigated in the concentration dependent manner of 10% to 50%. 30% concentration of the culture broth inhibited completely tyrosinase activity. In the cytotoxicity test, cucumber and grape extract itself has a strong cytotoxicity to the melanoma cell, B16Bl6. The value of $IC_{50}$ of the cucumber and grape extracts against the melanoma cells was 5% concentration. However, the culture broth of P. japonica incubated in the cucumber and grape extracts did not show the cytotoxcity up to 20% against melanoma cell, B16BL6. Therefore, we concluded that the culture broth of P. japonica in the mixture of cucumber and grape extracts can be used as a whitening cosmetic resource.

다양한 수술방법에 의한 진성악관절강직증의 치험례

이영훈,이상철,김여갑,류동목,이백수,윤옥병,Lee, Young-Hoon,Lee, Sang-Chull,Kim, Yeo-Gab,Ryu, Dong-Mok,Lee, Baek-Soo,Yoon, Ok-Byung 대한악안면성형재건외과학회 1999 Maxillofacial Plastic Reconstructive Surgery Vol.21 No.3

TMJ ankylosis is defined as a mobile disorder of jaw such as mouth opening limitation, limitation of anterior or/and lateral movement of TMJ. Kazanjian published first clinical report about classification of TMJ ankylosis dividing with intracapsular ankylosis and extracapsular ankylosis. TMJ ankylosis is resulted from trauma, infection, metastatic tumor, irradiation, burn and etc. When TMJ ankylosis is manifested in growing period, it affects to functional disorder and development and position of mandible, so it can result in maxillofacial deformity such as facial asymmetry, micrognathia, malocclusion. For treatment of TMJ ankylosis, various surgical interventions were devised ; condylectomy, gap arthroplasty, interpositional arthroplasty and TMJ reconstruction. So, we report our results with documental study and cases of true ankylosis in our department.

포도와 오이즙액을 이용한 동충하초(Paecilomyces japonica) 균사체 배양액의 피부 주름개선효과

이영훈,최우식,박기훈,최영주,갈상완,Lee Young-Hoon,Choi U-Sic,Park Ki-Hoon,Choi Young-Ju,Gal Sang-Wan 한국생명과학회 2006 생명과학회지 Vol.16 No.3

포도와 오이즙액에 동충하초 균사체 배양액을 화장품으로서의 이용가능성을 조사하였다. 콜라겐 생성촉진 효과에서는 사람의 섬유아세포에서 시험물질 $0.1{\sim}0.5%$ 농도에서 콜라겐 생합성을 증가시키는 것을 관찰하여 동충하초 균사체 배양액은 적절한 농도에서 세포내 콜라겐 생성을 증가시키는 것으로 판단하였다. 포도와 오이즙액에 동충하초 균사체 배양액의 항산화효과에서는 항산화제인 Vit. C와 함께 SOD 활성능을 측정한 결과 동충하초 균사체 배양액이 0.01% 부터 1.0% 범위에서 농도의존적으로 SOD 활성을 증가시켜 0.1% 에서는 약 50% 정도 1.0% 에서는 90% 정도의 슈퍼옥사이드 라디칼을 소거시키는 것으로 나타났다. 항 혈액응고제로 알려진 퀘르세틴의 함량증가에서는 최초 포도와 오이즙액보다 배양 후의 농도가 약 15배 이상 증가하여 피부의 항 혈액응고에도 효과가 있을 것으로 판단되며, 이를 인체에 대한 자극시험을 실시한 결과 30명의 피검자 모두에게서 피부자극이 전혀 일어나지 않음을 확인하여 인체에 무해한 것으로 판단되었다. 따라서 포도와 오이즙액에 동충하초 균사체 배양액은 피부에 대한 부작용이 전혀 없으며 주름개선효과 및 항산화 활성을 갖는 기능성 화장재료로서의 이용 가치가 매우 높을 것으로 사료된다. The possibility of usage as cosmetic resource of the mycelial culture broth of P. japonica in the mixture of cucumber and grape extracts was investigated. In the effect of collagen synthesis promotion in human fibroblast cells, the culture broth of P. japonica of 0.1, 0.5 and 1.0% concentration increased the amount of collagen synthesis than that of control cells. The culture broth increased the SOD activity in the concentration dependant manner of 0.01% to 1.0% in the antioxydation activity test. About 90% of superoxide radical was eliminated by 0.5% concentration of the culture supernatant in the antioxydation test. Anticoagulant quercetin in the course of mycelial growth in the mixture of cucumber and grape extract was accumulated to 15 folds than that of pre-culture. In the skin safety test of the culture broth, there is no any skin damage signal in the tested 30 people. Taken together, we concluded that the culture broth of P. japonica in the mixture of grape and cucumber extracts can be used as a cosmetic resource.

경동맥 내중막 두께 및 죽종과 심 혈관 질환 위험요인과의 관련성

이영훈,최련화,신민호,권순석,박경수,정슬기,정은경,최진수,Lee, Young-Hoon,Cui, Lian-Hua,Shin, Min-Ho,Kweon, Sun-Seog,Park, Kyeong-Soo,Jeong, Seul-Ki,Chung, Eun-Kyung,Choi, Jin-Su 대한예방의학회 2006 예방의학회지 Vol.39 No.6

Objectives : This study was conducted to examine the association between the carotid artery intima-media thickness (IMT), plaque and cardiovascular risk factors according to gender and age. Methods : The data used for this study were obtained from 1,507 subjects (691 men, 816 women), aged 20-74 years, who participated in 'Prevalence study of thyroid diseases' in two counties of Jeollanam-do Province during July and August of 2004. The body mass index (BMI) and waist hip ratio (WHR) were calculated by anthropometry The blood pressure, pulse rate, pulse pressure, total cholesterol, triglyceride, HDL cholesterol and fasting blood sugar level were also measured. Ultrasonography was used to measure the carotid artery IMT and plaque. IMT measurements were performed at 6 sites, including both common carotid arteries, and the bulb and internal carotid arteries. The definition of the 'mean IMT' was mean value obtained from these 6 sites. Results : The ${\pm}$ standard deviation IMT values were $0.65{\pm}0.14\;and\;0.60{\pm}0.13mm$ in men and women (p<0.001), respectively. The data were analyzed according to gender and the 50 year age groups. In a multiple linear regression analysis, age and hypertension were positively associated with the mean IMT in both men and women, aged<50 years. Age, total cholesterol and smoking (current) were positively associated with the mean IMT in men $(\geq50\;years)$. Age was positively associated with the mean IMT in women $(\geq50\;years)$, but the HDL cholesterol level was negatively associated. The prevalence of plaques was 44.2%(196/443) in men and 19.4%(89/459) in women, for those greater than 50 years of age. In a multiple logistic regression analysis, age (OR=1.090, 95%CI=1.053-1.129), HDL cholesterol (OR=0.964, 95%CI=0.944-0.984), total cholesterol (OR=1.009, 95%CI=1.002-1.017)and BMI (OH=0.896, 95%CI=0.818-0.983) were independently associated with plaques in men; whereas, age (OR=1.057, 95%CI=1.012-1.103), HDL cholesterol (OR=0.959, 95%CI=0.932-0.986), pulse pressure (OR=1.029, 95%CI=1.007-1.050) and triglycerides (OR=0.531, 95%CI=0.300-0.941) were independently associated with plaques in women. Conclusions: There were significant gender and aging differences in the association between the IMT, plaque and cardiovascular risk factors. Therefore, for the prevention of atherosclerosis, selective approaches should be considered with regard to gender and age factors.

임프란트 식립시 초기 안정성이 골유착에 미치는 영향

이영훈,김여갑,최병준,Lee, Young-Hoon,Kim, Yeo-Gab,Choi, Byung-Jun 대한악안면성형재건외과학회 2008 Maxillofacial Plastic Reconstructive Surgery Vol.30 No.6

Purpose: The long-term experience of using osseointegrated implants for prosthetic rehabilitation of the edentulous patients shows that high success rates can be predictably achieved. Primary implant stability has been identified to be a prerequisite to achieve osseointegration. In this study, we set up the amount of removed bone so that it differed on implant installation site for each group. The influence of each initial stability on secondary stability and osseointegration was compared with time lapse using resonance frequency analysis and histomorphometric analysis. Materials and methods: A total 27 US $II^{(R)}$ (Osstem, Korea) implants were placed in the mandibular edentulous area of 3 beagle dogs. The implant site was prepared by the conventional technique with drills, and three experimental groups were divided into under-drilling group, normal-drilling group and over-drilling group. The Implant Stability Quotient (ISQ) was measured at intervals of immediately, 4, 8, 12 weeks after placement using $Osstell^{(R)}$ mentor RFA. After the animals were sacrificed, histomorphometric evaluation was executed for measuring BIC and BD. Results: 1) The under-drilled group showed most high ISQ value for whole experiment period. 2) Bone-to-implant-contact(BIC) showed the tendency to be increased gradually as the experiment period passed except the 8 weeks of the normal group. 3) The under-drilled group showed most high bone density(BD) level for whole experiment period, and it was expressed the aspect to be increased gradually according to an experiment period passage in the average of all group. 4) Resonance frequency analysis and histomorphometric analysis are presumed by generally proportional. Conclusions: As this research result, it seems that there are some correlation between resonance frequency analysis and histomorphometric analysis. As are accomplished osseointegration stably so that more superior at the region which the overpressure comes to add, it will be applicable method in clinical field.

혈압 수준과 경동맥 내중막 두께 및 동맥경화반의 관련성

이영훈,권순석,최진수,이정애,최성우,류소연,신민호,Lee, Young-Hoon,Kweon, Sun-Seog,Choi, Jin-Su,Rhee, Jung-Ae,Choi, Sung-Woo,Ryu, So-Yeon,Shin, Min-Ho 대한예방의학회 2009 예방의학회지 Vol.42 No.5

Objectives : The aim of this study was to investigate the association of blood pressure levels with the common carotid artery intima-media thickness (CCA-IMT) and carotid plaques. Methods : Data were obtained from 2,635 subjects, aged 50 years and over, who participated in the Community Health Survey (a population-based, cross-sectional study) in Dong-gu, Gwangju city between 2007 and 2008. Participants were categorized into three groups according to blood pressure levels; normotensives (<120/80 mmHg), prehypertensives (120-139/80-89 mmHg), and hypertensives ($\geq$140/90 mmHg). Prehypertensives were further categorized as low prehypertensives (120-129/80-84 mmHg) and high prehypertensives (130-139/85-89 mmHg). Carotid intima-media thickness and plaques were evaluated with a high-resolution B-mode ultrasound. Statistical analyses were performed using chi-square test, ANOVA, and multiple logistic regression. Results : Prehypertensives had significantly greater maximal CCA-IMT values than normotensives, with a multivariate adjusted odds ratio of 1.78 (95% CI=1.36-2.32) for abnormal CCA-IMT (maximal CCA-IMT$\geq$1.0 mm), and 1.45 (95% CI=1.19-1.77) for carotid plaques. The multivariate adjusted odds ratio of low prehypertensives was 1.64 (95% CI=1.21-2.21) for abnormal CCA-IMT, and 1.30 (95% CI=1.04-1.63) for carotid plaques compared with normotensives. Subject with hypertension had higher frequency of abnormal CCA-IMT (odds ratio, 2.18; 95% CI=1.49-3.18), and carotid plaques (odds ratio, 1.98; 95% CI=1.46-2.67) compared with normotensives after adjustment for other cardiovascular risk factors. Conclusions : Our results indicate that there is a significant increase in the prevalence of carotid atherosclerosis in subjects with prehypertension (even in low prehypertensives) compared with normotensive subjects. Further studies are required to confirm the benefits and role of carotid ultrasonography in persons with prehypertension.

몬테카를로 시뮬레이션을 이용한 휴대용 의료기기 누적공차분석에 대한 사례연구

이영훈,문덕희,Lee, Young Hoon,Moon, Dug Hee 한국시뮬레이션학회 2016 한국시뮬레이션학회 논문지 Vol.25 No.2

Tolerances are defined as the allowable variations in the geometry and positioning of parts in a mechanical assembly for assuring its proper functionality. Tolerance analysis is the activity related to estimating the potential accumulated variation in assemblies. If the estimated variances go out of the specified ranges, it causes the quality problem. Thus, we should adjust the tolerances and this activity is called as tolerance design. In this paper, a case study on the accumulated tolerance analysis and design using Monte Carlo simulation is introduced, which is applied for developing a portable medical device. Using the simulation study, we can improve the assemblability and functionality of the product. 공차란 각 부품들이 기구적으로 조립되었을 때 원하는 수준의 기능성을 확보할 수 있도록 각 부품의 기하학적 형상이나 위치에 대해 허용하는 치수의 변동량을 의미한다. 또한 공차해석이란 부품들이 조립되었을 때, 각 부품의 공차가 조립품에서 어떻게 누적되어 미치는 영향을 분석하는 일련의 절차를 의미한다. 만일 누적된 공차의 분산이 규정된 일정 수준의 범위를 벗어나면 조립품의 품질에 문제가 발생하기 때문에 당초 설정한 부품의 공차를 수정해야 하며, 이러한 과정을 공차설계라고 한다. 이 논문에서는 휴대용 의료기기의 설계단계에서 공차해석과 공차설계를 위해 몬테카를로 시뮬레이션 기법을 활용한 사례를 소개한다. 이러한 시뮬레이션 연구를 통하여 제품의 조립성과 기능성을 개선시킬 수 있다.