흰쥐 뇌에서 발현되는 16kDa Vacuolar(H^+)-ATPase의 유전자 클로닝

신송우,유민 대한의생명과학회 2000 Journal of biomedical laboratory sciences Vol.6 No.3

Yacuolar (H+)- ATPase (V-ATDase)는 multi-subunit로 구성된 단백질로서, proton pumping을 통해 세포내 산성화반응에 관여를 한다. 최근에 이 단백질이 synaptic vesicle에서도 발견된 것으로 보아 뇌 신경전달에 중요한 역할을 수행할 것으로 추정하고 있다. 우리는 흰쥐 뇌에서 분리한 mRNA를 주형으로 한 PCR 반응에서 16 kDa subunit의 V-ATPase cDNA를 클로닝할 수 있었고, 이의 염기서열 또한 결정하였다. 분리된 뇌 16 kDa V-ATPase의 coding seqence는 전체 468 bp로서 간에서 보고되었던 것과 동일한 크기였다. 단지 3' 말단의 염기 하나가 A에서 C로 바뀌어 있었는데 모두 alanine (GCA, GCC)을 지정하기 때문에 단백질의 일차구조에는 변화가 없는 것으로 확인되었다. 한편 rat brain cDNA library 에서도 동일한 clone이 분리되었는데 역시 같은 부분에서 polymorphism이 발견되었고, RNA splicing 등 더 이상의 조직특이적 변화는 없었다. 본 연구는 16 kDa V-ATPase의 뇌에서의 기능과 신경말단에서의 neurotransmission 및 synaptic vesicle의 재순환 기전을 이해하는데 유용한 정보가 될 것이다. Vacuolar (H+)-ATpase (V-ATPase) is an intracellular protein which consists of multiple subunits. It carries out acidificatioon by pumping protons in the cell. This enzyme has also been found in the synaptic vesicles and may play an important role in the neurotransmission. We cloned cDNA figments encoding the 16 kDa subunit of V-ATPase from the rat brain by RT-PCR and PCR using total RNA or recombinant phage DNA as templates. They contained the full coding sequences (468 bp) and one nucleotide at 3' region turned out to be different (A to C) when compared to the liver counterpart. However, this polymorphic difference did not cause any significant change in the primary structure of the protein because both GCA and GCC code for alanine. Our study would contribute to the understanding of the function of 16 kDa V-ATPase in the brain and of the mechanisms of neuro transmission.

쥐 뇌에서 발현되는 S-100 Beta 유전자의 Polymorphism에 대한 분자생물학적 증거

유민,권오식,신송우 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1998 Journal of biomedical laboratory sciences Vol.4 No.2

쥐 뇌에서 발현되는 S-100 beta 유전자의 다형현상을 조사하였다. Polymerase chain reaction을 위한 주형으로는 뇌에서 분리한 mRNA를 직접 역전사한 cDNA, 또는 rat brain cDNA library에서 분리한 phage DNA를 사용하였다. 증폭된 DNA 절편들은 크기가 기존에 보고되었던 것과 일치하였으나 DNA sequencing을 통한 세부적인 분석 결과 coding region 내에 염기변화 (CAT가 CAC로 변함)가 있음이 확인되었다. 그러나 이들은 모두 histidine을 결정하는 유전암호이기에 단백질의 1차 구조에는 아무런 영향을 미치지 않는 다형현상으로 결론지어졌다. 본 연구는 S-100 beta 단백질에 그 동안 알려지지 않았던 다형현상이 존재함을 시사하는 것으로서 S-100 beta효소 유전자의 전체적인 구조를 이해하기 위한 학문적 자료가 될 것으로 기대된다. We examined mRNAs, isolated from the rat brain, to ascertain if there is any polymorphism for S-100 beta protein gene. As templates for polymerase chain reaction (PCR) the reverse-transcribed cDNA from the rat brain or phage DNAs isolated from the rat brain cDNA libraries were used. Although PCR products turned out to be exactly same as the expected size based on the previously reported mRNA sequence a single base substitution (CAT to CAC) was identified at nucleotide level. This change was considered as polymorphism since it did not cause any change of the primary structure for S-100 beta protein. This result should facilitate the understanding of the overall structure of the gene for S-100 beta protein.

사람의 게놈에 존재하는 Cytochrome P450 2E1의 Retropseudogene에 대한 분자유전학적 증거

유민,신송우 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1998 Journal of biomedical laboratory sciences Vol.4 No.2

Cytochrome P450 2E1 (CYP2E1)의 retropseudogene이 사람에 존재하는 지 확인하기 위해 혈액에서 분리한 DNA를 주형으로 한 polymerase chain reaction (PCR)을 수행하였다. Primer는 CYP2E1 유전자를 기초하여 여러개 제작하되 적절한 조합에 따라 정상유전자와 retropseudogene이 동시에 증폭될 수 있도록 고안하였다. 본 실험의 결과 그 동안 예상은 되었지만 DNA차원에서 미처 확인되지 못하였던 CYP2E1의 retropseudogene을 직접 증폭해낼 수 있었다. 세부 구조는 Southern blotting과 DNA 염기서열 결정에서 최종 분석되었다. PCR 반응으로 증폭된 부분에서는 염기서열이 mRNA와 완전히 일치하고 있는 점으로 미루어서 CYP2E1의 retropseudogene은 비교적 최근에 발생했을 것으로 추정되었다. We have carried out polymerase chain reaction (PCR) to investigate if retropseudogene for CYP2E1 is present in human genome. PCR primers were designed based on the structure of functional CYP2E1 gene and used to amplify both functional gene and retropseudogene in one reaction. From the repeated experiments we were able to amplify a previously unidentified CYP2E1 retropseudogene that was present in human genome. Its detailed structure was confirmed by Southern blotting and DNA sequencing. Nucleotide sequence of this retropseudogene was completely matched up to human liver CYP2E1 mRNA suggesting that the development of this retropseudogene might be a relatively recent event.

만성퇴행성 신경질환과 Apoptosis

유민,정영주,박영춘,신송우 啓明大學校 醫科大學 1996 계명의대학술지 Vol.15 No.4

쥐 뇌에서 발현되는 S-100 Beta 유전자의 Polymorphism에 대한 분자생물학적 증거

신송우(Song-Woo Shin),권오식(Oh-Sik Kwon),유민(Min Yoo) 대한의생명과학회 1998 Biomedical Science Letters Vol.4 No.2

쥐 뇌에서 발현되는 S-100 beta 유전자의 다형현상을 조사하였다. Polymerase chain reaction을 위한 주형으로는 뇌에서 분리한 mRNA를 직접 역전사한 cDNA, 또는 rat brain cDNA library에서 분리한 phage DNA를 사용하였다. 증폭된 DNA 절편들은 크기가 기존에 보고되었던 것과 일치하였으나 DNA sequencing을 통한 세부적인 분석 결과 coding region 내에 염기변화 (CAT가 CAC로 변함)가 있음이 확인되었다. 그러나 이들은 모두 histidine을 결정하는 유전암호이기에 단백질의 1차구조에는 아무런 영향을 미치지 않는 다형현상으로 결론지어졌다. 본 연구는 S-100 beta 단백질에 그동안 알려지지 않았던 다형현상이 존재함을 시사하는 것으로서 S-100 beta 효소 유전자의 전체적인 구조를 이해하기 위한 학문적 자료가 될 것으로 기대된다. We examined mRNAs, isolated from the rat brain, to ascertain if there is any polymorphism for S-100 beta protein gene. As templates for polymerase chain reaction (PCR) the reverse-transcribed cDNA from the rat brain or phage DNAs isolated from the rat brain cDNA libraries were used. Although PCR products turned out to be exactly same as the expected size based on the previously reported mRNA sequence a single base substitution (CAT to CAC) was identified at nucleotide level. This change was considered as polymorphism since it did not cause any change of the primary structure for S-100 beta protein. This result should facilitate the understanding of the overall structure of the gene for S-100 beta protein.

흰쥐 뇌에서 발현되는 16 kDa Vacuolar (H⁺)-ATPase의 유전자 클로닝

신송우(Song-Woo Shin),유민(Min Yoo) 대한의생명과학회 2000 Biomedical Science Letters Vol.6 No.3

Vacuolar (H?)-ATPase (V-ATPase)는 multi-subunit로 구성된 단백질로서, proton pumping을 통해 세포내 산성화반응에 관여를 한다. 최근에 이 단백질이 synaptic vesicle에서도 발견된 것으로 보아 뇌 신경전달에 중요한 역할을 수행할 것으로 추정하고 있다. 우리는 흰쥐 뇌에서 분리한 mRNA를 주형으로 한 PCR 반응에서 16 kDa subunit의 V-ATPase cDNA를 클로닝할 수 있었고, 이의 염기서열 또한 결정하였다. 분리된 뇌 16 kDa V-ATPase의 coding sequence는 전체 468 bp로서 간에서 보고되었던 것과 동일한 크기였다. 단지 3’ 말단의 염기 하나가 A에서 C로 바뀌어 있었는데 모두 alanine (GCA, GCC)을 지정하기 때문에 단백질의 일치구조에는 변화가 없는 것으로 확인되었다. 한편 rat brain cDNA library에서도 동일한 clone이 분리되었는데 역시 같은 부분에서 polymorphism이 발견되었고, RNA splicing 등 더 이상의 조직특이적 변화는 없었다. 본 연구는 16 kDa V-ATPase의 뇌에서의 기능과 신경말단에서의 neurotransmission 및 synaptic vesicle의 재순환 기전을 이해하는데 유용한 정보가 될 것이다. Vacuolar (H?)-ATPase (V-ATPase) is an intracellular protein which consists of multiple subunits. It carries out acidification by pumping protons in the cell. This enzyme has also been found in the synaptic vesicles and may play an important role in the neurotransmission. We cloned cDNA fragments encoding the 16 kDa subunit of V-ATPase from the rat brain by RT-PCR and PCR using total RNA or recombinant phage DNA as templates. They contained the full coding sequences (468 bp) and one nucleotide at 3' region turned out to be different (A to C) when compared to the liver counterpart. However, this polymorphic difference did not cause any significant change in the primary structure of the protein because both GCA and GCC code for alanine. Our study would contribute to the understanding of the function of 16 kDa V-ATPase in the brain and of the mechanisms of neurotransmission.

사람의 뇌에서 발현되는 Cytochrome P450 2E1 의 분자유전학적 증거

유민,신송우 한국유전학회 1998 Genes & Genomics Vol.20 No.3

We have carried out polymerase chain reaction (PCR) to investigate if cytochrome P450 2E1 (CYP2E1) is expressed in human brain. A 1. 4 kb DNA fragment was clearly amplified using recombinant phage DNAs, isolated from human brain cDNA library, as PCR templates. This DNA fragment was successively subjected to Southern blot analysis, nested PCR and DNA sequencing and finally its internal structure turned out to be exactly same as the previously reported liver CYP2E1. No deletions or insertions were identified. And there was no alternative RNA splicing event between these two tissues. Our results are indicative of the fact that CYP2E1 is actually expressed in human brain although its level is extremely low. Results strongly support the hypothesis that CYP2E1 has to be related to brain toxicity in any means and should facilitate the understanding of the function of CYP2E1 in the brain.

In Situ RT - PCR 로 결정한 쥐 뇌의 Cytochrome P450 2E1 의 발현 분포

유민,유관희,신송우 한국유전학회 1998 Genes & Genomics Vol.20 No.4

As a first step to understand cytochrome P450 2E1 (CYP2E1)-mediated metabolic processes in the brain we investigated the regional distribution of CYP2E1 at nucleotide level by direct in situ reverse transcription-polymerase chain reaction (RT-PCR). The mRNA of CYP2E1 was detected throughout the brain. Intense signals were localized in various brain regions such as hippocampus, thalamus, hypothalamus as well as piriform cortex. Highest expression was detected in hippocampal area. These results matched up to the previous finding by western blot analysis suggesting that CYP2E1 might be regulated at transcriptional level in the brain. A435 bp fragment for the brain CYP2E1 was also amplified from the reverse-transcribed cDNA templates. Nucleotide sequence of this fragment was exactly the same as corresponding region of liver CYP2E1 mRNA. This study would initiate the understanding of long-term memory loss due to chronic alcohol ingestion.

The Effect of Segmental Pedicle Screw Instrumentation on Actively Growing Spine-A Long-term Experimental Study-

김원중,이상호,신송우,Charles H.Rivard,Christine Coillard,Souad 대한신경외과학회 2004 Journal of Korean neurosurgical society Vol.35 No.5

Objective : Pedicle screw is gaining popularity in pediatric deformities. However, biological response of actively growing spine to rigid pedicle screw fixation remains unclear. The objective of this study is to determine the biological response of growing spine to rigid segmental fixation. Methods : Twelve mini pigs in actively growing period were subjected to posterior segmental screw -rod instrumentation spanning nine levels and creation of experimental scoliosis. There was no attempt of posterior arthrodesis. The pigs were subjected to periodic radiological examinations and were euthanized at 18 months for analysis. Results : There was no significant fixation failure despite conspicuous growth of the animals. Initial scoliosis of 31 5 was reduced to 27 8 at 18 months, but there was no statistical significance (p=0.37). Though there was no change in length of the implant construct, the vertebrae within the instrumented section showed mean longitudinal growth of 6 3 mm (p=0.000). The growth occurred at expense of the disc spaces that progressively narrowed with time. On necropsy, the instrumented region was completely fused posteriorly with crossing of the osseous traberculae across the former facet joints. Intervertebral discs were severely atrophic in all the discs with occasional spontaneous fusion. Conclusion : Even in the actively growing spine, the force of growth does not overcome the fixation offered by segmental pedicle screws. Longitudinal growth occurs at the expense of the joint spaces and leads to spontaneous intervertebral fusion. Our results may explain the favorable outcomes in pedicle fixations in pediatric population, showing little implant failure or nonunion.

요추 추간판 절제술 후 발생한 화농성 척추염에 대한 Chuinard & Peterson 골 이식술을 이용한 전방 유합술 및 경피적 후관절 나사못 고정술

장상범,이상호,이승철,신송우,김원중 대한척추신경외과학회 2004 Neurospine Vol.1 No.1

Objective: To describe the surgical techniques and the preliminary results of anterior fusion with Chuinard & Peterson bone graft and posterior percutaneous facet screw fixation for pyogenic spondylitis following lumbar discectomy. Methods: From April to October 2003, five consecutive patients underwent anterior lumbar interbody fusion with Chuinard & Peterson bone graft and posterior percutaneous facet screw fixation for the treatment of pyogenic spondylitis that developed after primary lumbar disc surgery. There were three males and two females. The mean age was 49.8 years(range, 28-75 years). Three patients had persistent inflammation with neurologic deterioration and two had severe back pain despite of less invasive interventions. The mean interval from the primary operation to fusion was 3.3 months(range, 1.5~7 months). All patients were allowed to ambulate immediate postoperatively with a lumbar orthosis, which was kept for three months. Intravenous and oral antibiotics were continued until hematologic profiles normalized. All patients were followed up clinically and radiologically. Results: The mean operating time, blood loss, and post-operative hospital stay were 210 mins, 890 ml, and 27 days, respectively. Four patients showed a solid fusion at 3 monthes and one patient showed fusion at 4 monthes postoperatively. M patients showed remission of infection and significant clinical improvement of back pain. Conclusions: Anterior fusion using Chuinard & Peterson bone graft and posterior facet screw fixation may be employed for postoperative pyogenic spondylitis without prolonged cast immobilization or risk of persistent infection caused by instrumentation.