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Recombinant Cl - amylase 의 세포내 수송에 있어서 Signal sequence 의 역할
김철호,권석태,이대실 ( Cheorl Ho Kim,Suk Tae Kwon,Dae Sil Lee ) 생화학분자생물학회 1991 BMB Reports Vol.24 No.4
The possible signal sequence capable of transporting the Cl-amylase from B. circulans has been analyzed with in vitro mutagenesis techniques. A residue in the NH₂-terminal region near to the postulated cleavase site was changed by site-directed mutagenesis from a serine into proline and threonine. Comparison of Cl-amylase acitivity outside and inside the cell in strains containing the cloned wild type and mutagenised genes showed that this single amino acid prevents largely the translocation of the enzyme in the periplasmic space: in transformed E. coli the proline -mutant Cl-amylase showed 5% secretion of wild type Cl-amylase and threonine-mutant Cl-amylase.
김철호,권석태,이대실,Kim, Cheorl-Ho,Kwon, Suk-Tae,Lee, Dae-Sil Korean Society for Biochemistry and Molecular Biol 1991 한국생화학회지 Vol.24 No.4
B. circulans 유래의 Cl-amylase는 전형적인 분비효소 단백질이다. 본 효소 단백질의 세포내 수송기구를 해명하고 분비에 필요로 하는 signal sequence 의 기능을 확인하기 위하여 signal sequence의 16번째 아미노산 잔기(serine)를 proline(${\alpha}$-helix breaker)과 threonine으로 In vitro mutagenesis를 통해 바꾸었다. Recombinant wild형(serine)과 mutation된 Cl-amylase(proline과 threonine) 유전자 각각을 대장균에 도입하여 세포내의 단백질 수송을 효소활성과 변역학적 방법으로 검토한 결과, proline-mutant형 이 wild형의 약 5% 정도로 periplasmic space에서 검출되었으며, 분자량에 있어서는 약 3,000 dalton의 차를 보였다. 그러나, threonine-mutant는 wild(serine) type의 경우와는 차이가 검출되지 않았다. 상기의 결과들로, 대장균에서의 이종유전자 산물의 성공적인 processing과 protein transportation을 확인, 해명하게 되였다. The possible signal sequence capable of transporting the Cl-amylase from B. circulans has been analyzed with in vitro mutagenesis techniques. A residue in the $NH_2$-terminal region near to the postulated cleavase site was changed by site-directed mutagenesis from a serine into proline and threonine. Comparison of Cl-amylase acitivity outside and inside the cell in strains containing the cloned wild type and mutagenised genes showed that this single amino acid prevents largely the translocation of the enzyme in the periplasmic space: in transformed E. coli the proline -mutant Cl-amylase showed 5% secretion of wild type Cl-amylase and threonine-mutant Cl-amylase.
Ames 및 umu assay를 이용한 감궁탕의 안전성평가
손윤희,김철호,남경수,Shon Yun Hee,Kim Cheorl Ho,Nam Kyung Soo 한국생명과학회 2005 생명과학회지 Vol.15 No.2
감궁탕의 돌연변이원성을 유무를 알아보기 위해 Salmonella typhimurium TA 98 및 TA100을 이용한 돌연변이 원성 실험에서도 감궁탕은 어느 균에서도 돌연변이원성을 나타내지 않았으며, 이는 S-9 mixture 의해 감궁탕이 대사가 된 후에도 이와 유사한 경향을 나타내었다. 또한, SOS umu test의 경우에서도 $\beta-galactosidase$활성에는 별다른 영향을 미치지 않는 것으로 보아 감궁탕은 돌연변이원성을 일으키지 않는 것으로 판정되었으며 S-9 mixture처리에 의한 대사 후에도 이와 유사한 실험결과가 나타났다. 따라서 감궁탕은 그 자체 및 대사 후에도 DNA에 별다른 영향을 미치지 못하는 비교적 안전한 생약처방으로 여겨진다. Gamgung-tang (GGT) that is included in Gamdu-tang (consists of Glycyrrhizae Radix, black beans) and Gunggui-tang(consists of Angelicae Radix and Cnidii Rhizoma) showed therapeutic effect of autoimmume thyroiditis in the previous reports. GGT was tested for the safety using Ames and umu gene expression mutagenicity tests. In Ames test, Salmonella typhimurium TA98 and TA100 were used to identify mutagenic property, and the number of histidine revertants was measured. In SOS umu test, Salmonella typhimurium TA1535 containing plasmid pSK1002 was used as a test strain, and we monitored the levels of umu operon expression by measuring the $\beta-galactosidase$ activity. Mutagenic activity in any assays we tested was not found. After treating S-9 mixture with GGT, mutagenic activity was also not found. The results of this study suggested that there was no DNA damage and mutagenicity of GGT.
Genomic Structure Analyses of Five Kinds of Human Sialyltransferase Gene
강남영,김상완,김철호,이영춘,Kang Nam-Young,Kim Sang-Wan,Kim Cheorl-Ho,Lee Young-Choon Korean Society of Life Science 2004 생명과학회지 Vol.14 No.6
인간유래 시알산전이효소 유전자들의 특이적 발현과 그들의 mRNA isoform의 생성에 대한 조절기구를 이해하기 위하여 5종류의 human 시알산전이효소 유전자(hST3Cal II, hST8Sia II, hST8Sia III, hSTS8Sia IV, hST8Sia V)들의 게놈구조를 분석하였다. hST3Gal II 유전자는 17 kb이상의 게놈상에 46 bp에서 1017 bp의 길이를 가진 exon이 6개로 이루어져 있고, hST8Sia III유전자는 10 kb이상의 게놈상에 125bp에서 2023bp의 길이를 가진 exon이 4개로 이루어져 있어 다른 human 시알산전이효소 유전자들보다 짧고 단순한 구조를 가지고 있었다. 반면에 다른 3종류의 유전자(hST8Sia II, hST8Sia IV, hST8Sia V)들은 70 kb이상의 게놈상에 5개이상의 exon으로 이루어져 있으며, 5종류 모두 exon-intron boundary는 GT-AG rule을 나타내고 있었다. 특히 모든 시알산전이효소에 고도로 보존되어 있는 sialylmotif L은 hST8Sia III유전자에서는 하나의 exon에 존재하는 반면에, 다른 시알산전이효소 유전자에서는 분리된 exon에 존재하여 exon의 구조적 다양성을 나타내고 있다. 또한, 본 연구에서는 5'-RACE와 cap site hunting법에 의해 hST3Gal II 유전자의 전사개시점을 결정하였다. Sialyltransferases cloned so far show the remarkable tissue-specific expression, which is correlated with the existence of cell type-specific sialylated sugar structure in glycoconjugates. In the previous studies, we found various mRNA isoforms of human sialyltransferases generated by alternative splicing and alternative promoter utilization. To understand the regulatory mechanisms for specific expression of human sialyltransferase genes and for production of their mRNA isoforms, in this study, we have isolated and characterized five kinds of human sialyltransferase genes: hST3Gal II, hST8Sia II, hST8Sia III, hST8Sia IV, and hST8Sia V. The hST3Gal II gene is composed of six exons, which span over 17kb, with exons ranging in size from 46 to over 1017 bp. The hST8Sia III gene comprises over 10 kb, and consists of only four exons, which is much smaller and simpler than other human sialyltransferase genes. In contrast, three genes (hST8Sia II, hST8Sia IV and hST8Sia V) span more than 70 kb, and comprise five or more exons. All exon-intron boundaries follow the GT-AG rule. In particular, the sialylmotif L, which is a highly conserved region in all cloned sialyltransferases, was found in one exon of hST8Sia III, whereas this motif is encoded by discrete exons in the other human sialyltransferases. Exon structures of these sialyltransferase genes show the structural diversity, as found in other human sialyltransferase genes reported so far. We determined the transcription start site of hST3Gal II gene by the 5'-RACE and cap site hunting experiments.
인진호(茵蔯蒿)가 Fas-FasL 매개형 간세포 Apoptosis에 미치는 영향
김형환,안중환,김종대,김철호,김선강,Kim, Hyeong-Hwan,An, Joong-Hwan,Kim, Jong-Dae,Kim, Cheorl-Ho,Kim, Seon-Kang 대한한방내과학회 2001 大韓韓方內科學會誌 Vol.22 No.3
Objectives: Recently, it was known that the major cause of hepatitis is apoptosis reaction mediated by Fas-FasL. Since Artemisia Capillaris Fructus has long been applied to cure the jaundice in oriental medicine. Therefore, this study was carried out to examine the effect of fractions of Artemisia Capillaris Fructus on Fas-FasL-mediated apoptosis in hepatocytes. Methods: This study employed propidium iodide negative cell count assay and some the other biochemical assays. Results : This study confirms that hepatitis has been occured by apoptosis mediated by Fas-FasL in cultured hepatocyte and fractions of Artemisia Capillaris Fructus restrain apoptosis induced Fas-FasL. Conclusions : Water-extracted fraction, methanol extracts, ether-soluble fraction, and buthanol-soluble fractions of Artemisia Capillaris Fructus restrain Fas-FasL-mediated apoptosis in hepatocyte. Silica gel chromatograph of Buthanol-soluble fraction of Artemisia Capillaris Fructus restrain Fas-FasL-mediated apoptosis in hepatocyte. Artemisia Capillaris Fructus could be applied to cure hepatitis.