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Identification and Characterization of a Novel Bacterial ATP-Sensitive K+ Channel
최승범,Jong-Uk Kim,Hyun Joo,Churl K. Min 한국미생물학회 2010 The journal of microbiology Vol.48 No.3
Five bacterial species that are most likely to have putative prokaryotic inward rectifier K+ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K+ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K+ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K+ current with characteristics similar to those of the ATP-sensitive K+ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.
Kim, Han-Seop,Lee, Chang-Ho,Min, Churl K. The Korean Society for Integrative Biology 1998 Korean journal of biological sciences Vol.2 No.4
The G protein-activated inwardly rectifying $K^+$ channel (GIRK1) was coex-pressed in Xenopus oocytes along with the $5-HT_{1A}$ receptor, a 7-helix receptor known to be coupled to $K^+$ channels in many neural tissues. Thus, the activation of the $5-HT_{1A}$ receptor by its agonist leads to the opening of GIRK1. The GIRK1 current was measured using the two electrode voltage clamp technique with bath application of 5-HT in the presence of various external potassium concentrations $[K^+]_0$. GIRK1 showed a strong inward rectification since only hyperpolarizing voltages evoked inward currents. $K^{+}$ was the major ion carrier as evidenced by about 44㎷ voltage shift corresponding to a 10-fold external 〔$K^+$〕 change. 5-HT induced a concentration-dependent inward $K^+$ current ($EC_{50}{\equation omitted}10.7nM$) which was blocked by $Ba^{2+}$. Pertussis toxin (PTX) pre-treatment reduced the $K^+$ current by as much as about 70%, suggesting that PTX-sensitive G protein ($G_i or G_o$ type) are involved in the $5-HT_{1A}$ receptor-GIRK1 coupling in Xenopus oocytes.
Cho, Soo Churl,Yim, Seon-Hee,Yoo, Hanik K.,Kim, Mi-Young,Jung, Gyoo Yeol,Shin, Gi Won,Kim, Boong-Nyun,Hwang, Jun Won,Kang, Jason Jongho,Kim, Tae-Min,Chung, Yeun-Jun Lippincott Williams Wilkins, Inc. 2009 PSYCHIATRIC GENETICS Vol.19 No.4
OBJECTIVES: Autism spectrum disorder (ASD) has been thought to have strong genetic background, but major contributing genes or associated molecular–genetic pathways are yet to be identified. To explore the idiopathic ASD-associated copy number variations (CNVs), we conducted case–control study using whole-genome copy number analysis. METHODS: Whole-genome microarray-based comparative genomic hybridization was carried out on 28 children (24 boys and four girls) diagnosed as ASD and 62 Korean adults (45 males and 17 females) without any signs of abnormalities and family history of genetic disorders as normal controls. Fluorescence in-situ hybridization and capillary electrophoresis-single-strand conformational polymorphism were used for quantitative verification of the ASD-associated CNVs. RESULTS: Thirty-eight CNVs were identified. Among them, the distributions of copy number loss CNVs on 8p23.1 (odds ratio: 5.1, 95% confidence interval: 1.7–14.5, P=0.003) and on 17p11.2 (odds ratio: uncalculable because of zero cell, P=0.008) were found to be significantly different between ASD and control groups. DEFENSIN family occurs in a cluster at 8p23.1 region. Fluorescence in-situ hybridization and capillary electrophoresis-single-strand conformational polymorphism coherently showed reduced copy number of DEFENSIN in cases with 8p23.1 copy number loss CNV, which validated microarray-based comparative genomic hybridization results; but there are no known coding genes in the CNV on 17p11.2. CONCLUSION: Our approach as well as results can help to elucidate the genetic mechanism of idiopathic ASD.
김현진,최동순,장석준,한재호,장기홍,유희석,Churl K. Min 대한부인종양학회 2010 Journal of Gynecologic Oncology Vol.21 No.1
Objective: Aberrant expression of the cell surface proteoglycan, syndecan-1, is found in many malignancies. The current study describes the immunohistochemical study of syndecan-1 expression in normal, hyperplastic, and malignant endometrial tissues for evaluation of application as a parameter of cancer progression in patients with endometrial hyperplasia. Methods: Immunohistochemical staining of syndecan-1 was performed in 101 formalin fixed, paraffin embedded sections of normal, hyperplastic, and malignant endometrial tissues. We analyzed specimens from patients with normal endometrium (NE, N=10) as controls, and those of simple hyperplasia (SH, N=20), complex hyperplasia without atypia (CH, N=20), atypical hyperplasia (AH, N=20), and endometrial cancer (EC, N=31). Results: The mean rank of expression scores based on the frequency of syndecan-1 staining were 31.6, 20.5, 52.9,72.1, and 62.1 for NE, SH, CH, AH and EC, respectively (p<0.001). Syndecan-1 expression was significantly greater in CH (p<0.001) or AH (p<0.001) than in SH, and significantly greater in AH compared to CH (p=0.028). Syndecan-1 is more frequently expressed in CH (p=0.042), AH (p<0.001), or EC (p=0.002) than in NE. Syndecan-1expression did not differ significantly between NE and SH (p=0.248). Conclusion: Syndecan-1 expression appears to be useful as a predictive indicator in endometrial hyperplasia.
Oh, Jeong-Hyun,Lee, Hae-Sun,Park, Sang-Hyun,Ryu, Hee-Sug,Min, Churl K. BMJ 2010 International journal of gynecological cancer Vol.20 No.5
<B>Objectives:</B><P>Upregulation of syndecan-1, a member of the transmembranous proteoglycans that serves as a coreceptor for a wide pool of extracellular ligands, has been well documented in enabling the promotion of growth and invasion of endometrial cancer. As a step toward understanding a potential role for syndecan-1 in this process, we questioned whether syndecan-1 upregulates tumor-promoting characteristics, particularly, angiogenesis in an in vivo human xenograft tumor model.</P><B>Methods:</B><P>Human syndecan-1 was stably transfected into human endometrial adenocarcinoma 1A cells, and resulting transfectants were subcutaneously grafted into athymic mice; their outcomes were examined with respect to the enhancement of tumor growth and angiogenesis by immunohistochemistry, immunoblotting, and zymography.</P><B>Results:</B><P>Overexpression of syndecan-1 promoted tumor growth concomitant with increased angiogenesis in tumor xenografts as evidenced by an increase in immunoreactivity for vascular endothelial growth factor and vascular endothelial cell marker CD34. Furthermore, zymographic studies revealed that syndecan-1 overexpression markedly enhanced activities of matrix metalloproteinases 2 and 9.</P><B>Conclusions:</B><P>This is the first in vivo xenograft analysis providing evidence that supports that syndecan-1 has a critical role in carcinogenic progression, particularly, contributing to the development of angiogenesis and invasive phenotype in association with matrix metalloproteinases 2 and 9 activations in endometrial cancer.</P>
새로운 유전자 재조합 방법을 이용한 대장균에서의 인간 tissue inhibitor of mtrix metalloproteinase-2 (TIMP-2) 유전자의 가용성 발현
김종욱,최동순,주현,민철기,Kim, Jong-Uk,Choi, Dong-Soon,Joo, Hyun,Min, Churl-K. 한국생물공학회 2008 KSBB Journal Vol.23 No.3
암세포의 침윤은 숙주 조직의의 기저막과 세포외 기질을 침투함으로써 일어난다. 침윤과 전이과정에는 단백질가수분해 효소인 matrix metalloproteinases (MMPs)가 깊이 연관되어 있는 것으로 알려져 있으며, MMP의 가수분해 활성은 tissue inhibitors of metalloproteinases (TIMPs)라는 억제 단백질에 의해 억제된다. TIMP-2는 21kDa 크기의 포유류 단백질로 대장균에서 과발현 시 다른 많은 포유류 단백질과 마찬가지로 가용성이 낮은 봉입체 형태로 발현된다. TIMP-2 단백질의 접힘에 6개의 이황화결합이 필요하고, 이는 일반적으로 대장균 환경은 적합하지 않다. 본 연구에서는 대장균에서 불가용성으로 발현되는 TIMP-2 유전자를 유전자셔플링 기법의 한 가지인 StEP (staggered extension process)를 변형하고 동시에 $Mn^{2+}$ 농도 변화와 dGTP 불균형을 이용한 무작위 돌연변이 기법을 혼용하여 대장균에서 가용성 TMP-2 재조합 변이체를 생성하고자 하였다. 무작위로 재조합된 TIMP-2 유전자 중에서 가용성으로 발현되는 TIMP-2 유전자를 선별하기 위해서 chloramphenicol acetyltransferase (CAT)-융합 방법을 도입하였다. CAT 유전자가 가용성으로 발현되는 재조합 TIMP-2 유전자에 융합되면 이를 갖는 E. coli는 높은 chloramphenicol 환경에서 생존이 가능하게 된다. 이러한 in virro mutageuesis 기법과 CAT-융합 방법으로 대장균 가용성 TIMP-2 재조합 변이체를 14가지 얻을 수 있었다. 변이체 TIMP-2의 아미노산서열 분석과 구조 분석 결과 주로 소수성 아미노산이 친수성 아미노산으로 전환되었고, MMP와의 결합이 관여하지 않는 C-말단 부위에 돌연변이가 집중되어 있었다. 본 연구에서 개발된 간편하고 새로운 in vitro 재조합 방법과 CAT을 이용한 스크리닝 기법은 다른 많은 대장균 내 불가용성 단백질의 발현에도 사용될 수 있을 것으로 사료된다. The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.