대장균에서 재조합 Rat Guanine deaminase 유전자의 발현, 정제 및 분석

성연선,곽상준,박대성,김향원,이희영 단국대학교 1998 論文集 Vol.33 No.-

Guanine deaminase(EC, 3.5.4.3, ;Guanine aminohydrolase, GAH, GDA) catalyzes the deamination reaction of guanine to xanthine irreversibly. The cDNA encoding rat guanine deaminase had been isolated from a λZAPII rat brain expression library using antibody generated against purified rat guanine deaminase. Toe obtain recombinant GDA and analyze the property of catalytically conserved retion, here we expressed the recombinant GDA in E.coli and showed the retention of its catalytic activity similar to native rat GDA. To make the construct carrying coding region to GDA in pGEX4T prokaryotic expression vector, the region encompassing open reading frame of pBlue-GDA was PCR amplified and subcloned into pGEX4T prokaryotic expression vector with correct reading frame of fusion carrier glutathione S-transferase. After transformation to E.coli DH5a. The bacteria carrying pGEX-GDA was grwon in the condition of IPTG induction. The fusion protein GST-GDA was purified with GSH-sepharose affinity chromatography. The purified GST-GDA was subsequently digested with biotin-conjugated thrombin, and thrombin was removed by streptavidin agarose. Then sample were treated with GSH-sepharose affinity chromatography to remove the contaminant GST and GST-GDA. The purified recombinant GDA showed 70% of specific activity relative to that of purified rat GDA. The recombinant GDA showed identical molecular weight with rat GDA in SDS-PAGE.

순위 정렬 선다형 평가 문항을 적용한 초등학교 4∼6학년 학생들의 물의 순환에 대한 학습 발달 과정

성연선 ( Yeon Seon Seong ),맹승호 ( Seung Ho Maeng ),장신호 ( Shin Ho Jang ) 한국초등과학교육학회 2013 초등과학교육 Vol.32 No.2

This study investigated elementary students` (grade 4∼6) learning progressions for water cycling drawn from iterative assessments using ordered multiple-choice (OMC) items. An assessment system, which consisted of construct map, item design, outcome space, and measurement model, was employed in this study to examine children`s learning progressions. At the first stage of the assessment system, a construct map was designed on which children`s conceptual understandings from naive to most sophisticated were represented. At the item design stage, 8 OMC items were drawn from the construct map. Each item option of the OMC items was scored from 0 to 3 according to its level of understanding at the stage of outcome space. As a measurement model, Rasch model, a branch of item response theory, was applied to interpreting the outcomes of the OMC items. This cycle of assessment system was furtherly implemented iteratively in order to elaborate on the first version of water cycling learning progression. In conclusion, children`s understanding of water cycling could be described in two aspects: water distribution and water movement. We identified children`s conjectural developmental pathways about water cycling existed from superficial and naive accounts to more complex and abstract accounts.

β-TrCP1 degradation is a novel action mechanism of PI3K/mTOR inhibitors in triple-negative breast cancer cells

이용원,성연선,Edward Jeong Bae,오성훈,배인수,강효진 생화학분자생물학회 2015 Experimental and molecular medicine Vol.47 No.-

An F-box protein, β-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulationby β-TrCP has been widely studied, the regulation of β-TrCP itself is not well understood yet. In this study, we found that the level of β-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of β-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of β-TrCP1 prior to its degradation. In addition, knockdown of β-TrCP1 inhibited the proliferation of TNBC cells. We furtheridentified that pharmacological inhibition of mTORC2 was sufficient to reduce the β-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of β-TrCP1 in TNBC cells and targeting β-TrCP1 is a potential approach to treathuman TNBC.

부분 요도폐색이 방광의 콜라겐 유형 I, III, IV 및 Matrix- Metalloproteinase(MMP)-2 유전자 발현에 미치는 초기 영향

이안기,성연선,김광명,최황,김시황 大韓泌尿器科學會 1998 Korean Journal of Urology Vol.39 No.3

자궁경부암에서 FHIT 유전자의 단백질 발현 및 이형성 소실

김수연,성연선 대한산부인과학회 2000 Obstetrics & Gynecology Science Vol.43 No.4

FHIT는 1996년 Ohta group에 의하여 처음으로 밝혀진 가족성 신세포암에서의 염색체 전좌 부위이며, 여러 암에서 microsatellite의 결손이 보고된 염색체 3p14영역에서 positional cloning된 유전자이다. 최근에는 FHIT발현 이상의 빈도가 폐암, 유방암, 소화기암, 두경부암 등 여러 암조직에서 높게 보고되어 아마도 이 유전자가 암억제유전자인 것으로 추정되고 있다. 그리고 FHIT 유전자 부위에는 HPV 16 DNA의 염색체 융합 부위로 추정되는 FRA3B 유전자가 포함되어 있어 있고 이에 대한 연구가 활발히 진행되고 있다. 본 연구에서는 자궁경부암 발생에 있어 FHIT의 역할을 규명하기 위하여 정상, 전암병변 및 침윤성 자궁경부암 조직에서 FHIT 단백질 발현 양상을 면역조직화학염색을 통하여 관찰하고, FHIT 유전자내의 D3S1300, D3S1234와 3′쪽의 D3S1295의 microsatellite에 대하여 이형성소실(loss of heterozygosity)를 PCR을 통해 분석하여 다음과 같은 결과를 얻었다. FHIT 단백의 발현은 정상 자궁경부상피의 기저층과 자궁경부내구의 선상피세포에서 강하게 발현되었다. 자궁경부내구의 편평화생과 전암 병변에서 FHIT의 발현은 유지되었으나, 침윤암에서는 소실되었다. 스푼형 편평상피세포에서는 FHIT의 발현이 감소되었다. 자궁경부암에서 FHIT 유전자 영역의 microsatellite 분석상, D3S1300 및 D3S1234에서 LOH가 일부 관찰되었으나, D3S1295에서는 관찰되지 않았다. 본 연구결과 FHIT는 전암 병변에서는 발현이 있지만 침윤성암으로 발전하게 되면서 소실되는 특성을 보인다. 이러한 사실로 FHIT는 자궁경부암에서 암억제유전자의 가능성이 높다. 또한 HPV 감염이 의심되는 스푼형 편평상피세포에서 FHIT발현이 감소하였고, FHIT유전자에 HPV 융합부위인 FRA3B가 포함되어 있다는 점, 그리고 FHIT의 LOH가 FRA3B 영역에 가까운 부위에서 많이 일어난다는 점으로 보아 HPV의 감염이 FHIT의 발현을 억제하는 것으로 추정되고, 이것이 자궁경부암의 침윤성 획득에 있어서 중요한 유전적 변이인 것으로 생각된다. Allelic deletions involving the short arm of chromosome 3(3p13-21.1) have been observed frequently in cervical carcinomas. Recently the fragile histidine triad(FHIT) gene was cloned and mapped to this chromosomal region(3p14.2). From various studies involving tumor cell lines and primary cancers, the FHIT gene has been presumed to be a candidate for tumor suppressor gene involving various tumors. In FHIT gene, the most common aphidicolin-inducible fragile site, FRA3B exists and the FRA3B has been considered as a region of the spontaneous integration site of HPV 16. In order to elucidate the role of the FHIT in carcinogenesis of cervical cancer, this study was designed to investigate both the expression of FHIT protein in normal, preinvasive and invasive cancer samples employing immunohistochemical study and allelic loss of FHIT gene locus against several microsatellite markers employing the PCR analysis. Immunohistochemical studies of FHIT protein revealed following features. In normal ectocervical squamous epithelium, the expression of FHIT was relatively weak and confined to the basal layer, but in normal endocervical glandular epithelium it was very strong. The expression of FHIT was reduced as the tumor progressed from early lesion to invasive cancer. The koilocytosis was associated with diminished expression of FHIT protein. The study of allelic loss of FHIT gene locus was undertaken against two intragenic (D3S1300, D3S1234) and one extragenic (D3S1295) microsatellite markers. The 5th intron, D3S1300, showed allelic change in 6 of 15 assays and 7th intron, D3S1234 showed allelic change in 10 of 29 assays. There was no apparent LOH from 29 assays in D3S1295. In conclusion, the expression of FHIT protein was markedly reduced or absent in cervical squamous cell carcinoma and the chromosome breakage in FHIT region might be related to the diminished expression of FHIT. On the basis of the reduced expression of FHIT and its encompassment of FRA3B region, it is suggested that disruption of FHIT, a putative tumor suppressor gene, might be the mechanism by which HPV infection enhances cervical tumorigenesis and clonal outgrowth.

백서 방광 출구 부분폐색에서 방광의 Inducible Nitric Oxide Synthase (iNOS)의 발현 변화

이안기,성연선,곽상준,서성일,최황,김시황 大韓泌尿器科學會 1997 Korean Journal of Urology Vol.38 No.7

2-Aminoethoxydiphenyl Borate(2-APB)를 이용한 IP3 수용체억제가 와우유모세포의 Gentamicin에 의한 이독성에 미치는 영향

문정환,정재윤,안진철,성연선,임은석,이정구 대한이비인후과학회 2007 대한이비인후과학회지 두경부외과학 Vol.50 No.5

Background and Objectives:Elevated intracellular calcium level is known to play important roles in the apoptotic pathway. IP3 receptor (ligand-gated channels that release Ca2+ from intracellular stores) is emerging as a key site for regulation of apo-ptosis. 2-Aminoethoxydiphenyl borate (2-APB)APB on gentamicin ototoxicity in vitro, using the HEI-OC1 cell line. Materials and Method:HEI-OC1 cells were treated with 100μM gentamicin. Using a CaspACE assay, we measured the caspases-3 activity in the gentamicin treated hair cells with and without 2-APB pre-incubation. We also observed intra-cellular calcium concentrations in HEI-OC1 cels using a confocal micro-scopy (calcium gren-1 stain). Live cel imaging was performed by using fluorescence video-time lapse system. Results:Cytosolic calcium elevation by gentamicin was remarkably inhibited by 2-APB. Caspases-3 activities of gentamicin treated cells APB, caspases-3 activities and cel death of gentamicin treated cels were shown to decrease. Conclusion:2-APB reduces Caspases-3 activity in the gentamicin treated HEI-OC1 cells by inhibi-tion of cytosolic calcium increase. (Korean J Otolaryngol 2007 ;50 :385-90)

Ectopic Overexpression of Coiled-Coil Domain Containing 110 Delays G2/M Entry in U2-OS Cells

Sue Nyoung Lee,홍경만,성연선,곽상준 한국발생생물학회 2020 발생과 생식 Vol.24 No.2

Coiled-coil domain containing 110 (CCDC110, KM-HN-1) is a protein containing C-terminal coiled-coil domain (CCD) which was previously discovered as a member of the human cancer/testis antigen (CTA). In addition, CCDC110 has both nuclear localization signal sequence and the leucine zipper motif. Although the functional role of CCDC110 has yet to be fully identified, the mRNA expression levels of CCDC110 are known to be highly elevated in various cancer types including testis, implying its relevance to cancer pathogenesis. In this study, we first developed several monoclonal antibody (mAb) hybridoma clones targeting CCDC110 and further isolated clone by characterizing for its specificity using immunoblotting and immunoprecipitation approaches with basal parenchymal sperm cells in testis tissue. Next, using these mAbs, we showed that the Tet-inducible overexpression of CCDC110 protein delayed the entry of G2/M phase in U2-OS osteosarcoma cells. Based on these results, we propose that CCDC110 plays a crucial role in cell cycle progression.

2-aminoethoxydiphenyl borate(2-APB)를 이용한 IP3 수용체 억제가 와우유모세포의 gentamicin에 의한 이독성에 미치는 영향 /

문정환,정재윤,안진철,성연선,임은석,이정구 한국레이저가공학회 2007 한국레이저가공학회 학술대회 논문집 Vol. No.

Genomewide transcription profiles altered by BMI-1026 and Roscovitine and its implication in cellular senescence

이지혜,민창희,곽상준,성연선 한국바이오칩학회 2012 BioChip Journal Vol.6 No.4

Cyclin-dependent kinases (CDKs) are highly conserved over evolutionary path and function as master regulators of the cell division cycle. Thus CDK inhibitors have been focused in the drug development for cancer and other cell proliferative disorders. Among these inhibitors Roscovitine is well known compound and under clinical trial currently while far less has been reported on another CDK inhibitor - BMI-1026, designed as a drug candidate also targeting the cell cycle. In this study we tried to define biological effects and possible mechanisms of the two chemicals by analyzing the altered transcriptome profiles when BMI-1026 and Roscovitine are applied on WI-38 cells. Our result revealed both BMI-1026 and Roscovitine produced similar gene expression profiles, where E2F target genes were suppressed in common, including cyclin A, MCM3 and PCNA along with activated p53 pathway. This finding strongly indicates that molecular machineries responsible for other established senescence models are also employed in the changes in WI-38 cells induced by the two CDK inhibitors.