자초(紫草) 열수추출물이 각질형성세포 HaCaT의 세포 연접 관련 유전자의 발현에 미치는 영향 연구

조남준 ( Namjoon Cho ),이병권 ( Byeongkwon Lee ),이웅희 ( Woonghee Lee ),김기광 ( Keekwang Kim ),김균언 ( Kyoon Eon Kim ),한효상 ( Hyosang Han ) 대한본초학회 2017 大韓本草學會誌 Vol.32 No.3

Objectives : The aim of this research was to determine the diverse effects of Lithospermi Radix Water Extract (LR) on human keratinocyte HaCaT cells, and to examine whether those effects could be applied to the human skin. Methods : We examined effect of LR on the cell viability of using the MTS assay in human keratinocyte HaCaT cells. The antioxidation effect of LR was analyzed relative to the well-known antioxidant resveratrol, using an ABTS assay. Quantitative RT-PCR analysis revealed that, in HaCaT cells, LR influenced the mRNA expression of tight-junction genes associated with skin moisturization. Furthermore, a wound-healing assay demonstrated altered cell migration in LR-treated HaCaT cells. Result : The cytotoxicity was confirmed to be higher in LR at a concentration of 800 ㎍/㎖ using the MTS assay in HaCaT cells. In comparison to 100 μM resveratrol, 1,600 ㎍/㎖ LR showed either a similar or superior antioxidation effect. LR treatment in HaCaT cells reduced the mRNA expression levels of claudin 3, claudin 4, claudin 6, claudin 8, and ZO-2 to less than 0.80-fold, whereas JAM-A and Tricellulin mRNA expression level increased more than 1.33- fold. In addition, HaCaT cells migration was decreased to 83.9% by LR treatment. Conclusions : LR of antioxidation activity will have an anti-aging effect on the skin by reducing oxidative stress. Further studies are required to address the implications for human skin, given LR`s effects of altering mRNA expression of tight junction-related gene and decreasing cell migration of HaCaT cells.

蘇木과 그 지표물질인 brazilin이 인간 유래 각질 형성 세포의 tight junction 유전자 발현에 미치는 영향

천성혜(Seong Hye Cheon),최선경(Sun Kyung Choi),조남준(Nam Joon Cho),김기광(Kee Kwang Kim),이웅희(Woong Hee Lee),황형서(Hyung Seo Hwang),김균언(Kyoon Eon Kim),한효상(Hyosang Han) 한의병리학회 2018 동의생리병리학회지 Vol.32 No.2

Zebrafish 발생 과정에서 chk1 의 기능 분석

김동선,김균언,장미숙,이명철 한국유전학회 2000 Genes & Genomics Vol.22 No.3

As an initial approach to examine the function of cell cycle checkpoint kinase 1(chk1) in embryonic development, a 1.461 Kb long cDNA encoding a zebrafish chk1(Zchk1) was isolated from the 12-day-old zebrafish cDNA library. DNA sequences of the cDNA shares 87% homology with human chk7 (hchk1). However, the cDNA contains an incomplete open reading frame lacking approximately 100 by at the 3'-end region. RT-PCR analysis showed that the level of Zchk1 mRNAs was present from 1 cell stage throughout the embryonic development. Whole mount in situ hybridization demonstrated that the mRNA persisted at low level through 50% epiboly, and became restricted in the anterior part of antero-posterior axis in 3 somites, and only in the tail region of primodia 11. Inhibition of the Zchk1 expression by microinjection of Zchk1 anti-sense RNA into 1-4 cell stage embryos caused retardation of embryonic development at late somite stage, which in turn resulted in dorsally rolled tail. Taken together, these results indicate that Zchk1 may be involved in the regulation of cell cycle during embryogenesis as well as in the formation of the dorsoventral axis in the posterior region.

LIM-homeobox 2 (Lhx-2)에 의한 갑상선자극호르몬 β-subunit 유전자의 전사조절

김기광,송석빈,이진욱,박진수,김용은,이재훈,김균언 충남대학교 생물공학연구소 2002 생물공학연구지 Vol.8 No.2

Thyrotropin (TSH) β is a subunit of TSH, the expression of which is limited to the thyrotrope cells of the anterior pituitary gland. LIM homeodomain factors appear to play a role in expression of the glycoprotein hormone α-subunit gene. Studies demonstrated that LIM homeodomain factor-2 (Lhx2) can bind to a pituitary-specific enhancer element designated as PGBE in the mouse α-subunit gene. Lhx3 (pLIM) can also enhance α-subunit gene expression. However, the mode of Lhx2 action is not yet characterized in the TSH β-subunit gene expression. In this study, deletion analysis was employed to delineate the sequence of the rTSH β-subunit gene which are inolved in Lhx2 regulation. Transient transfection experiment is αTSH and GH_3 cells demonstrated that the -170 / -79 region of the rTSH β-subunit gene, when fused to a luciferase reporter, was sufficient for the activation of this gene by Lhx2. Furthermore, gel mobility shift assay showed that homeodomain of Lhx2 binds to the -170 / -79 region of the rTSH β-subunit promoter. Taken together, these data suggest a model, in which the Lhx2 activates transcription of the TSH β-subunit gene.

사람의 성산자극 호르몬 α-Subunit 유전자와 결합하는 Trans-Acting Factor의 확인 및 분석

송석민,백상기,김균언 충남대학교 생물공학연구소 1999 생물공학연구지 Vol.7 No.-

The 5' flanking region of the α-subunit gene of human chorionic gonadotropin was tested for the binding with the nuclear extracts of the JAR choriocarcinoma cells. Gel mobility shift assay has demonstrated that two BstNI restriction fragments, 150 bp and 340 bp respectively, have binding activities with the nuclear extracts. The 150 bp fragment was chosen to examine in detail, since several transcription factor binding sites have already been reported within the 340 bp fragment. Band competition assay showed that the binding of 150 bp fragment is highly sequence-specific. The binding activity was further narrowed down to a 55 bp fragment, which was generated by Nsi1 digestion of the 150 bp fragment. DNasel footprinting assay revealed the binding sites on the nucleotide levels from -511 to -499. The binding sites were again confirmed by a oligonucleotide-directed mutagenesis of the 150 bp fragment followed by a gel mobility shift assay. Functional aspect of this binding was evaluated with the DNA-mediated gene transfer techniques. However, in several appoaches tried so far, no effect on the transcription efficiency was observed, implicating a structural role of the binding. In this line, it is worth to note that the binding activity of the 150 bp fragment was observed in every cell or tissue nuclear extracts tested so far.

LIM domain과 상호작용 하는 transcription cofactor hTAiL-1의 기능 분석

박경숙,송석빈,김균언 충남대학교 자연과학연구소 1999 忠南科學硏究誌 Vol.26 No.1

The LIM homeodomain (LIM-HD) proteins, which contain two tandem LIM domains followed by a homeodomain, are critical transcription factors for embryonic development. The LIM domain is a conserved cysteine-rich zinc-binding motif and mediate protein-protein interactions. We have isolated two transcription activator interacting with LIM domain (hTAiL-1 and -2) from human brain cDNA library on the basis of its ability to interact with the LIM-HD protein Lh-2 (or Lhx-2). Here we report on the function of the hTAiL-1 protein. Northern blot analysis using human tissue blot revealed that hTAiL-1 is very abundantly expressed in the heart and muscle. In order to confirm that hTAL-1 interacts with LIM domain, β-galactosidase assay using yeast two-hybrid system was firstly employed, followed by an in vitro protein interaction assay and a mammalian two-hybrid assay. The results of these assay demonstrated that hTAiL-1 interact with LIM domain in a very weak mode. In vivo functional data using transfection experiments in SL-2 cells suggested that Sp1 transcription factor may be involved in the interaction of hTAiL-1 with Lh-2. In fact, hTAiL-1 stimulates the promoter activity deriven by Sp1 more efficiently than by Lh-2. In addition, effect of hTAiL-1 can be observed in a series of transfection experiment with differentiated L6 cells. Taken together, these results indicated that hTAiL-1 may mediate specific protein-protein interactions between LIM-HD and additional Sp1 basal transcription factor.

흰쥐 μ(mu)opioid Receptor 유전자의 클로닝 및 발현 조절

설은영,송석빈,김균언 한국유전학회 1998 Genes & Genomics Vol.20 No.3

Chromosomal gene encoding a μ(mu) opioid receptor (μORc) was cloned from a rat genomic library using μORc cDNA as a probe. Sequence analysis of the clone identified a transcription start site, four AP-1 sites, two Oct-1 sites and one CREBP1 site in the immediate 5' flanking region as well as a translation initiation codon ATG in exon I. However, canonical TATA box was not found in the rat μORc promoter region. Northern blot analysis demonstrated that μORc gene is expressed in the hypothalamus-derived GT1-1 cells and pituitary gonadotrope-derived αT3-1 cells but neither in pituitary lactotrope-derived GH₃ cells nor in pituitary thyrotrope-derived αTSH cells. In addition, expression of the gene was induced in the cells treated with cAMP for 24 hrs. A series of deletion mutants of the 5'-flanking region were subcloned into the luciferase reporter vector, and tested for their transcriptional activities by transfecting into αT3-1 cells. The construct containing up to -2967 bp region exhibited maximum transcriptional activity among the tested constructs, whereas the region below -1705 bp conferred significantly low levels of activity. Therefore, it is conceivable that the region between -2967 bp and -1705 bp is responsible for the basal expression of the μORc gene.

사람의 성선자극 호르몬 α-Subunit 유전자와 결합하는 Trans - Acting Factor 의 확인 및 분석

백상기,송석빈,김균언 한국유전학회 1998 Genes & Genomics Vol.20 No.1

The 5' flanking region of the α-subunit gene of human chorionic gonadotropin was tested for the binding with the nuclear extracts of the JAR choriocarcinoma cells. Gel mobility shift assay has demonstrated that two BstNI restriction fragments, 150 bp and 340 bp respectively, have binding activities with the nuclear extracts. The 150 bp fragment was chosen to examine in detail, since several transcription factor binding sites have already been reported within the 340 bp fragment. Band competition assay showed that the binding of 150 bp fragment is highly sequence-specific. The binding activity was further narrowed down to a 55 bp fragment, which was generated by Nsil digestion of the 150 bp fragment. DNasel footprinting assay revealed the binding sites on the nucleotide levels from -511 to -499. The binding sites were again confirmed by a oligonucleotide-directed mutagenesis of the 150 bp fragment followed by a gel mobility shift assay. Functional aspect of this binding was evaluated with the DNA-mediated gene transfer techniques. However, in several approaches tried so far, no effect on the transcription efficiency was observed, implicating a structural role of the binding. In this line, it is worth to note that the binding activity of the 150 bp fragment was observed in every cell or tissue nuclear extracts tested so far.