호알칼리성 Bacillus pseudofirmus HS-54가 생산하는 알칼리성 Protease의 특성

방성호,정인실,Bang, Seong-Ho,Jeong, In-Sil 한국미생물학회 2011 미생물학회지 Vol.47 No.3

An alkalophilic bacterium producing alkaline protease was isolated from waste water and solar saltern sample and identified as Bacillus pseudofirmus HS-54 based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The HS-54 protease was purified to homogeneity using ammonium sulfate precipitation, DEAE cellulose column chromatography, and sephadex G-100 gel filtration with a 4.0 purification fold. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 27 kDa. The optimal pH and temperature for the purified protease activity were 10.0 and $50^{\circ}C$, respectively. The purified enzyme was relatively stable at the pH range of 6.0-11.0 and at the temperature below $50^{\circ}C$. This enzyme was activated by $Ca^{2+}$ and $Mg^{2+}$ and inhibited by $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Al^{3+}$, $Ag^{2+}$. And this enzyme was strongly inhibited by PMSF, suggesting that it belongs to the serine protease superfamily. 알칼리성 protease를 생산하는 호알칼리성 균주를 분리하여 Bacillus pseudofirmus HS-54로 동정하였고, HS-54가 생산하는 알칼리성 protease를 ammonium sulfate 침전, DEAE cellulose chromatography, sephadex G-100 gel filtration을 통과시켜 정제하였는데, 정제된 protease의 분자량은 27 kDa이었다. 정제된 효소의 반응최적 pH는 10.0이었고 pH 7.0-11.0에서 비교적 안정하였다. 또한 정제된 효소의 반응최적 온도는 $50^{\circ}C$이었고 $10-55^{\circ}C$에서 안정하였다. 금속이온에 대한 영향은 $Ca^{2+}$와 $Mg^{2+}$ 등에 의해 효소활성이 촉진되었으나, $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Al^{3+}$ 등에 의해서 효소활성이 저해되었다. 본 효소는 PMSF에 의해 강하게 저해를 받는 것으로 보아 serine protease에 속하는 것으로 판단된다.

ErbB 수용체를 이용한 난소암세포 표적 유전자치료 벡터의 개발

정인실,방성호,Joung, In-Sil,Bang, Seong-Ho 한국미생물학회 2011 미생물학회지 Vol.47 No.1

암의 유전자치료에서 암세포로의 선택적 유전자전달 매체의 부족은 치료효과의 감소를 야기하는 문제이다. 본 연구에서는 난소암 유전자치료의 효율을 높이기 위한 목적으로 난소암세포로 선택적인 유전자전달을 하도록 개량된 아데노바이러스 벡터를 제조하고, 그 효율성을 난소암세포주를 이용하여 조사하였다. 난소암세포에 과다발현하는 분자인 ErbB receptor를 표적하도록 아데노바이러스 외피단백질 fiber에 ErbB receptor에 대한 ligand인 heregulin으로부터 유래한 펩티드를 부착하였다. 53개의 아미노산으로 구성된 외부 펩티드를 fiber에 부착하였을 때 바이러스 감염에 중요한 기능을 하는 fiber 삼량체 구조 형성에 영향을 미치지 않았다. Fiber를 조작한 개량 아데노바이러스는 야생형 fiber를 가진 1세대 아데노바이러스 벡터에 비해 선택적으로 난소암으로 유전자를 전달하는 비율이 증가하였다. 특히 항암제에 저항성을 가진 난소암세포주 OVCAR3에서 유전자전달 효율이 약 5배 증가되었다. 따라서 난소암의 유전자치료에서 개량된 아데노바이러스로 치료 유전자를 전달하면 치료의 효율성을 향상시킬 수 있을 것이다. Inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches have been described to improve gene transfer efficiency but suffer from a number of limitations. Here we tested an adenovirus carrying the small peptide ligand derived from heregulin${\beta}$ EGF-like domain onto fiber, the adenoviral capsid protein, to deliver transgene to ovarian cancer cells which overexpress ErbB, the cognate receptors for heregulin. The attachement of 53 amino acids to fiber didn't affect on the fiber's trimer structure that is critical for the viral entry to cells. The fiber-modified adenovirus can mediate entry and expression of a ${\beta}$-galactosidase into cancer cells in an increased efficiency compared the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to ErbB receptor overexpressing cancer cells, and could be used for future cancer gene therapy.

[화학] 금속이온이 처리된 활성탄소섬유의 흡착과 항균성

오원춘(Won-Chun Oh),방성호(Seong-Ho Bang) 한국분석과학회 2000 분석과학 Vol.13 No.2

Microthrix parvicella에 의한 슬러지 벌킹과 거품문제 해결

강민기(Min Gi Kang),김영철(Young Chul Kim),방성호(Seong Ho Bang),이진우(Jin Woo Lee),하준수(Jun Su Ha) 大韓環境工學會 2006 대한환경공학회지 Vol.28 No.4

Isoflurane 의 MAC(Minimum Alveolar Concentration)증가가 심혈관계에 미치는 영향

방성호,곽일용,김용락 대한마취과학회 1987 Korean Journal of Anesthesiology Vol.20 No.3

To investigate the cardioyascular effects of 1 MAC-and 2 MAC-isoflurane anesthesia, eight Mongrel dogs were anesthetized with intravenous thiopental sodium and maintained with endotracheal nitrous oxide (2 l/min) -oxygen(2 l/min) -isoflurane-pancuronium. Dogs were kept normothermic by the use of Aqua-Therm and normocapneic with controlled ventilation employing Airshield Ventimeter with frequent measurements of PaCO_2. An 18G Medicut was inserted in the left femoral artery, a Swan-Ganz catheter was passed into the pulmonary artery through the right femoral vein, and cardiovascular parameters were measured during control, 1 MAC-and 2 MAC·isoflurane administration. The results are as follows : 1) Heart rate, mean arterial pressure, cardiac output, cardiac index, stroke volume, left ventricular stroke work index and left ventricular dp/dt max decreased during both MAC isoflurane anesthesia. These decreases were more prominent during 2 MAC-isoflurane administration. 2) Pulmonary capillary wedge pressure and pulnary vascular resistance did not change significantly during the 1 MAC phase, but did during the 2 MAC phase. 3) Mean pulmonary arterial pressue decreased slighter during both phases. 4) Central venous pressure and systemic vascular resistance did not change significantly throught the experiment. The above findings indicate that isoflurane does directly depress the myocardium and the hemodynamic function, and it is important to decrease isoflurane concentration acccordingly with the use of nitrous oxide.

Salmonella typhimurium의 산성-유도 유전자인 cad operon의 염기서열 분석

방성호 한서대학교 산학협력연구원 부설 환경연구소 1988 환경연구소 논문집 Vol.1 No.-

Salmonella typhimurium은 자연과의 상호작용뿐아니라 동물 숙주 세포내에서도 다양한 산성 pH stress를 경험하게 되며, 이러한 산성 환경에서 유전자 발현을 조절함으로서 산선 pH stress에 대해 적응·생존할 수 있다. 산성 pH에서 발현이 유도되는 S. typhimurum의 cadBA operon을 Escherichia coli의 cadBA를 probe로 이용하여 S. typhimurum 염색체 DNA library로부터 콜로니 잡종형성을 통해 cadBA를 클로닝하였다. cadBA operon은 pPF86의 BamHI-SalI 절편(4.3kb) 부위에 존재하였으며, 이 절편의 염기서열을 결정하였더니 BamHI-SalI 절편내에서 cadA, cadB 두 개의 구조유전자가 존재하였다. cadA는 2,142bp로 이루어져 있었다. CadA의 아미노산 서열은 E. coli의 CadA, Hafnia alvei의 lysine decarboxylase들의 아미노산 서열들과 높은 상동성을 가지고 있었으며, CadB의 아미노산 서열은 E. coli의 lysine/cadeverine antiporter로 제안된 CadB와 P. aeruginosa의 arginine/ornithine antiporter인 ArcD의 아미노산 서열들과 높은 상동성을 보였으며, 소수성 양상도 매우 유사하였다. Salmonella typhimurium resdonds to changes in external pH by altering its pattern of gene expression. To better understand how bacteria sense and respond to changes in external pH, we have studied the genetic elements involved in acid induction of the S. typhimurium cad operon encoding lusine decarboxylase. S. typhimurium cadBA was cloned by colony hybridization, using the probe of E. Coli cadBA gene. The 4.3kb BamHI-SalI fragment form pPF86 was subcloned into pBR322. We report the sequence of the cad operon encoding lysine decarboxylase which is a protein of 714 amino acids, and another protein, cadB which consists of 445 amino acids. The amino acid sequence of lysine decarboxylase showed high homology to that of lysine decarboxylase of E. coli, and Hafnia alvei. The CadB sequence showed homology to that of CadB of E. coli, adn ArcD of Peudomonas aeroginosa, encoding lysine/cadaverine antiporter and arginine/ornithine antiporter, respectivley.

체온순환 전후 체온변화에 관한 임상적 연구

방성호,김성덕,김광우,장호조,권무일,김봉덕 대한마취과학회 1979 Korean Journal of Anesthesiology Vol.12 No.4

Changes of body temperature were observed in the esophagus and rectum by telethermometer during extracorporeal circulation in 40 cases of open heart for detection of significant differences in survivals and non-survivals. The following results were obtained; 1) Esophageal and rectal temperature(just prior to extracorporeal circulation) were 36.4±0.69。C, 36.8±0.8。C respectiyely in non-survivals and 36.0±0.73。C, 36.4±0.8。C in survivals after open heart surgery 2) Normal differences between rectal and esophageal temperature came to disappear after 30 minutes of extracorporeal circulation in two groups. 3) Esophageal temperatures were higher than rectal temperatues after extracorporeal circulation for open heart surgery in both groups. 4) Changes of temperature diferences between rectum and esophagus have no correlation with mortality because of the variation in two areas in both groups.