Rho GTPase와 미오신 활성의 상관관계 조사

김응국, 신은영 충북대학교 의과대학 충북대학교 의학연구소 2012 忠北醫大學術誌 Vol.22 No.1

연구목적 : 본 연구는 PC12 세포에서 nerve growth factor (NGF) 처리에 의해 Rho GTPase Rac이 활성화 되었을 때 미오신 II와 guanine nucleotide exchange factor (GEF) PIX의 상호작용을 관찰하고자 하였다. 재료 및 방법 : PC12 세포는 10% 혈청이 포함된 DMEM에서 배양하였다. NGF는 처리 전 24 시간 동안 혈청을 제거하였다. NGF 20 ng/ml를 표기한 시간대로 처리한 후 단백질 용출액을 처리하여 세포균질액을 얻었다. 이 과정을 통해 얻은 세포균질액은 PBD-His beads로 pulldown assay를 실시하여 Rac의 활성화를 검증하였다. 또한 anti-PIX antibody로 면역침전한 후 미오신 II 항체를 이용하여 결합여부를 확인하였다. 결과 : Rac의 활성은 NGF 처리 후 5분부터 20분까지 꾸준히 증가하였으며 30분에는 감소 하는 경향을 나타냈다. 뿐만 아니라 PIX와 미오신 II와의 결합은 10분부터 감소하기 시작하 여 20-30분에는 결합한 두 단백질의 양이 상당히 감소함을 확인할 수 있었다. 결론 : 이상의 결과로부터 NGF 처리에 의해 Rac이 활성화 되는 것과 PIX와 미오신 II의 결합이 해리되는 시간대가 일치한다는 것을 알 수 있었다. 이는 NGF 처리가 PIX와 미오신 II의 결합 상태를 해리시켜 PIX의 GEF 활성을 유도한다는 것을 설명하고 있다.

사람 혈청트란스페린 수용체의 정제와 그 특성에 관한 연구

이상진,김응국,김승렬 충북대학교 의과대학 충북대학교 의학연구소 1997 忠北醫大學術誌 Vol.7 No.1

사람 트란스페린 수용체는 90-kDa의 분자량을 가진 당단백질이 둘이 결합한 단백질로 두 분자의 트란스페린과 결합할 수 있다. 본 실험에서는 사람 태반과 혈청에서 트란스페린 수용체의 고순도 정제법을 보고한다. 사람 암세포주(K-562와 SNU-1)에서 결합상수를 구해보았으며, 정상세포(적혈구와 단핵구)와 비교하여 차이가 없음을 보고한다. Human transferrin receptor is a disulfide-linked homodimer of 90-kDa glycoprotein, capable of binding two transferrin molecules. The study reports a high yield affinity purification protocol for transferrin receptor from human placenta and serum which produces the highly purified protein. We have examined the binding of human transferrin to cultured human cancer cell lines(K-562, SNU-1). Results indicate the presence of a high affinity saturable binding site(Kd=2.5 × 10-8). However, the difference between the cell line receptors in the dissociation constant have not been observed.

自然産物 精神病藥劑의 Dopamine-2 및 Serotonin-2 受容體 結合

鄭寅元,金應國,金承烈 大韓神經精神醫學會 1992 신경정신의학 Vol.31 No.5

In an effort to establish the theoretical basis of traditional medicine in Korea' the radioligand receptor binding study was conducted with natural products. Some natural products used as antipsychotics in traditional medicine displaced the radiolabeled probes from the receptors of dopamine-2 and serotonin-2 in central nervous system of male Sprague-Dawleyrats. The results were as follows : 1) Reviewing the books about the oriental medicine including Dong Eui Bo Gam, there were over 200 natural products in prescriptions and mono-drug regimen treating the mental disorders, mainly psychosis. 2) For D₂receptor analysis ₃〔H〕-spiperone as radio-ligand, the striatal tissues of male Sprague-Dawley rats were used. The Bmax of the receptor was 146.54fmole/mg protein and the Kd was 0.38 nM as the basic data of studies. Of the 36 natural products, Polygala tenuifolia only had shown the affinity to the D₂receptor and made the dilution-binding curve. 3) The 5HT₂receptor binding study was conducted with ₃〔H〕-ketanserin and the cortical tissue. The Bmax and Kd were 57.34 fmole/mg protein, 0.08 nM, respectively. There was Polygala tenuifolia, Machilus thunbergii, Erigeron canadensis, and Citrus unshiu which showed the affinities and the dilution binding curves. 4) There could be related follow-up studies. One is to establish the reference books about biochemical structures and pharmacological characteristics of components of natural products in traditional medicine. Another is to screen other natural products by radio-ligand receptor binding study. And the other is to identify the pharmacological and biochemical characteristics of the effective components of natural products which show affinities to the receptors in this study.

미오신 II에 의한 신경 성장추의 액틴 다이내믹스 조절

신은영, 김응국 충북대학교 의과대학 충북대학교 의학연구소 2012 忠北醫大學術誌 Vol.22 No.1

연구목적: 미오신 II는 액틴과 결합해서 세포의 다양한 활성, 수축, 이동, 부착 등을 조절한다. Rho G 단백질도 유사한 역할을 수행한다. 따라서 미오신 II와 Rho G 단백질의 활성 조절은 밀접하게 연 관되어 있다. 본 연구에서는 신경 성장추를 모델로 이 두 개의 단백질이 어떻게 액틴 다이내믹스를 조 절하는가에 관한 연구를 수행코자 한다. 대상 및 방법: 신경 성장추는 쥐의 해마세포를 초대배양하여 확보한다. 미오신 II에 대한 저해제인 blebbistatin (BBS)을 처리한 후 신경 성장추의 액틴 골격의 변화를 관찰한다. 또한 신경세포의 신경 돌기 및 성장추의 확장을 촉진하는 인자인 신경성장인자를 처리하고 미오신 II의 인산화를 면역블롯과 면역화학법을 통해 관찰한다. 결과: 초대배양한 해마세포의 신경 성장추에 BBS를 처리하면 30분 내에 질서정연한 액틴 골격이 무 질서한 액틴 골격으로 변한다. BBS를 배양액에서 제거하면 서서히 원래의 형태로 전환되는 것으로 미 루어, BBS에 의한 액틴 골격의 변화는 가역적인 반응으로 추정된다. 신경성장인자를 처리하면 BBS에 의해 유도되는 급격한 변화는 보이지 않았다. 그러나 미오신 II의 인산화를 면역블롯과 면역염색을 통 해 확인할 수 있었다. 결론: 미오신 II는 인산화를 통해 Rho G 단백질의 활성과 신경 성장추의 액틴을 재구성하는 것으로 보인다.

BMB Reports : Nuclear Rac1 regulates the bFGF-induced neurite outgrowth in PC12 cells

( Eung Gook Kim ),( Eun Young Shin ) 생화학분자생물학회 2013 BMB Reports Vol.46 No.12

Rac1 plays a key role in neurite outgrowth via reorganization of the actin cytoskeleton. The molecular mechanisms underlying Rac1-mediated actin dynamics in the cytosol and plasma membrane have been intensively studied, but the nuclear function of Rac1 in neurite outgrowth has not yet been addressed. Using subcellular fractionation and immunocytochemistry, we sought to explore the role of nuclear Rac1 in neurite outgrowth. bFGF, a strong agonist for neurite outgrowth in PC12 cells, stimulated the nuclear accumulation of an active form of Rac1. Rac1-PBR (Q) mutant, in which six basic residues in the polybasic region at the C-terminus were replaced by glutamine, didn`t accumulate in the nucleus. In comparison with control cells, cells expressing this mutant form of Rac1 displayed a marked defect in extending neurites that was concomitant with reduced expression of MAP2 and MEK-1. These results suggest that Rac1 translocation to the nucleus functionally correlates with bFGF-induced neurite outgrowth. [BMB Reports 2013; 46(12): 617-622]

Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method

Kim, So-Jung,Jung, Ji-Won,Ha, Hye-Yeong,Koo, Soo Kyung,Kim, Eung-Gook,Kim, Jung-Hyun Korean Society of Hematology; Korean Society of Bl 2017 Blood Research Vol.52 No.1

<P><B>Background</B></P><P>Embryonic stem cells (ESCs) can be expanded infinitely <I>in vitro</I> and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical <I>in vitro</I> methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials.</P><P><B>Methods</B></P><P>Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF.</P><P><B>Results</B></P><P>Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34<SUP>+</SUP>CD43<SUP>+</SUP> hematopoietic progenitor cells (HPCs) and CD34<SUP>+</SUP>CD45<SUP>+</SUP> HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells <I>in vitro</I>.</P><P><B>Conclusion</B></P><P>In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.</P>

Association Between Oral Health and Airflow Limitation: Analysis Using a Nationwide Survey in Korea

Kim Sun-Hyung,Gu Seonhye,Kim Jung-Ae,Im YoHan,Cho Jun Yeun,Kim Youlim,Shin Yoon Mi,Kim Eung-Gook,Lee Ki Man,Choe Kang Hyeon,Lee Hyun,Yang Bumhee 대한의학회 2023 Journal of Korean medical science Vol.38 No.31

Background: Although poor oral health is a common comorbidity in individuals with airflow limitation (AFL), few studies have comprehensively evaluated this association. Furthermore, the association between oral health and the severity of AFL has not been well elucidated. Methods: Using a population-based nationwide survey, we classified individuals according to the presence or absence of AFL defined as pre-bronchodilator forced expiratory volume in 1 second/forced vital capacity < 0.7. Using multivariable logistic regression analyses, we evaluated the association between AFL severity and the number of remaining teeth; the presence of periodontitis; the Decayed, Missing, and Filled Teeth (DMFT) index; and denture wearing. Results: Among the 31,839 participants, 14% had AFL. Compared with the control group, the AFL group had a higher proportion of periodontitis (88.8% vs. 79.4%), complete denture (6.2% vs. 1.6%), and high DMFT index (37.3% vs. 27.8%) (P < 0.001 for all). In multivariable analyses, denture status: removable partial denture (adjusted odds ratio [aOR], 1.12; 95% confidence interval [95% CI], 1.04–1.20) and complete denture (aOR, 1.52; 95% CI, 1.01– 2.05), high DMFT index (aOR, 1.13; 95% CI, 1.02–1.24), and fewer permanent teeth (0–19; aOR, 1.32; 95% CI, 1.12–1.52) were significantly associated with AFL. Furthermore, those with severe to very severe AFL had a significantly higher proportion of complete denture (aOR, 2.41; 95% CI, 1.11–3.71) and fewer remaining teeth (0–19; aOR, 2.29; 95% CI, 1.57–3.01). Conclusion: Denture wearing, high DMFT index, and fewer permanent teeth are significantly associated with AFL. Furthermore, a reduced number of permanent teeth (0–19) was significantly related to the severity of AFL. Therefore, physicians should pay attention to oral health in managing patients with AFL, such as chronic obstructive pulmonary disease.

Cytoskeleton Rearrangements and Human Diseases : Novel biological function of betaPIX guanine nucleotide exchange factor

( Eung Gook Kim ) 생화학분자생물학회 2008 생화학분자생물학회 춘계학술발표논문집 Vol.2008 No.-

Mechanism of Drug Resistance in Hepatoma tissues : Differential Expression of Gamma-gluamyl Transpeptidase, Glutathione Transferase and P-glycoportein Genes in vitro and in Vivo

Kim, Eung Gook,Kwak, Sahng June,Park, Sang Schul,Kim, Soo Tae Korean Society of Molecular Biology 1991 Molecules and cells Vol.1 No.2

Expression of a Soluble Extracellular Domain of Fibroblast Growth Factor Receptor-1 in E. coli

Eung Gook Kim 忠北大學校 遺傳工學硏究所 1994 遺傳工學硏究 Vol.8 No.1