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Sphingosine Kinase Assay System with Fluorescent Detection in High Performance Liquid Chromatography
You-Xun Jin,유환수,Akio Kihara,Chang-Hwan Choi,Seikwan Oh,문동철,Yasuyuki Igarashi,이용문 대한약학회 2006 Archives of Pharmacal Research Vol.29 No.11
Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; 100 μM of C17-Sph and 30 μg protein of F9-12 cells lysate in 20 min. Sphingosine analog C17-Sph was efficiently phosphorylated by Sphk activity (Km :67.08 μM, Vmax :1507.5 pmol/min/mg). New product C17-S1P was separated from S1P in reversedphase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while 20 μM of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.
Sphingosine Kinase Assay System with Fluorescent Detection in High Performance Liquid Chromatography
Jin, You-Xun,Yoo, Hwan-Soo,Kihara, Akio,Choi, Chang-Hwan,Oh, Seik-Wan,Moon, Dong-Cheul,Igarashi, Yasuyuki,Lee, Yong-Moon The Pharmaceutical Society of Korea 2006 Archives of Pharmacal Research Vol.29 No.11
Activation of Sphingosine kinase (Sphk) increases a bioactive lipid, sphingosine 1-phosphate (S1P) and has been observed in a variety of cancer cells. Therefore, inhibition of Sphk activity was an important target for the development of anticancer drugs. As a searching tool for Sphk inhibitor, we developed fluorescent Sphk activity assay combined with high performance liquid chromatography (HPLC). Previously we established murine teraticarcinoma mutant F9-12 cells which lack S1P lyase and stably express Sphk1. By using F9-12 cells, optimal assay conditions were established as follows; $100\;{\mu}M\;of\;C_{17}-Sph\;and\;30\;{\mu}g$ protein of F9-12 cells lysate in 20 min. Sphingosine analog $C_{17}-Sph$ was efficiently phosphorylated by Sphk activity ($K_{m}:67.08\;{\mu}M,\;V_{max}\;:1507.5\;pmol/min/mg$). New product $C_{17}-S1P$ was separated from S1P in reversed-phase HPLC. In optimized conditions, 300 nM of phorbol 12-myristate 13-acetate (PMA) increased Sphk activity approximately twice while $20\;{\mu}M$ of N,N-dimethylsphingosine (DMS) reduced 70% of Sphk activity in F9-12 cells lysate. In conclusion, we established non-radioactive but convenient Sphk assay system by using HPLC and F9-12 cells.
Jin, Xun,Lee, Joong-Seub,Kwak, Sungwook,Jung, Ji-Eun,Kim, Tae-Kyung,Xu, Chenxiong,Hong, Zhongshan,Li, Zhehu,Kim, Sun-Myoung,Whang, Kwang Youn,Hong, Ki-Chang,You, Seungkwon,Choi, Yun-Jaie,Kim, Hyunggee Korean Society for Molecular Biology 2006 Molecules and cells Vol.21 No.2
<P>We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and p16(INK4a) functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and p16(INK4a) pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.</P>
Joo, Hyoe-Jin,Yim, Yong-Hyeon,Jeong, Pan-Young,Jin, You-Xun,Lee, Jeong-Eui,Kim, Heekyeong,Jeong, Seul-Ki,Chitwood, David J,Paik, Young-Ki Biochemical Society 2009 The Biochemical journal Vol.422 No.1
<P>Caenorhabditis elegans excretes a dauer pheromone or daumone composed of ascarylose and a fatty acid side chain, the perception of which enables worms to enter the dauer state for long-term survival in an adverse environment. During the course of elucidation of the daumone biosynthetic pathway in which DHS-28 and DAF-22 are involved in peroxisomal beta-oxidation of VLCFAs (very long-chain fatty acids), we sought to investigate the physiological consequences of a deficiency in daumone biosynthesis in C. elegans. Our results revealed that two mutants, dhs-28(tm2581) and daf-22(ok693), lacked daumones and thus were dauer defective; this coincided with massive accumulation of fatty acyl-CoAs (up to 100-fold) inside worm bodies compared with levels in wild-type N2 worms. Furthermore, the deficiency in daumone biosynthesis and the massive accumulation of fatty acids and their acyl-CoAs caused severe developmental defects with reduced life spans (up to 30%), suggesting that daumone biosynthesis is be an essential part of C. elegans homoeostasis, affecting survival and maintenance of optimal physiological conditions by metabolizing some of the toxic non-permissible peroxisomal VLCFAs from the worm body in the form of readily excretable daumones.</P>
Ze Chen,You-quan Li,Qiao-Yun Ren,Jin Luo,Yonghong Hu,Kai Li,Guang-Yuan Liu,Jian-xun Luo,Jingze Liu,Hong Yin 대한기생충학열대의학회 2015 The Korean Journal of Parasitology Vol.53 No.3
Gynandromorphic ticks are extremely rare, and often attract parasitologists’ attention. During our examination of tick specimens, an engorged gynandromorph of Hyalomma asiaticum was noticed. This is the first record of gynandromorphic ticks from China. In this study, several important morphological structures of normal and gynandromorphic H. asiaticum were analyzed. Comparing to the normal H. asiaticum, the gynandromorphic specimen was a typical bipartite protogynander. Its right side showed normal female characteristics, whereas the left side had normal male traits. Different from other gynandromorphic ticks containing 1 anus, this tick reported here had 2 complete anuses, and the anus of the male part had a single adanal plate.
A sheathless CE/ESI-MS interface with an ionophore membrane-packed electro-conduction channel
Shi, Lian-Hua,Jin, You-Xun,Moon, Dong-Cheul,Kim, Sook-Kyung,Park, Sang-Ryoul WILEY-VCH Verlag 2009 Electrophoresis Vol.30 No.10
<P>A robust and convenient sheathless CE/ESI-MS interface realized with an ionophore membrane-packed electro-conduction channel is described. Sheathless interfaces that may provide higher sensitivity for MS detection than sheath flow-supported interfaces generally show instability and short lifetimes due to their imperfection in making an electrical contact with the emitter tip. In this work, we designed a sheathless interface based on an ionophore membrane-packed electro-conduction channel. At the joining point of the CE capillary and the emitter capillary, the conduction channel was implemented toward the exterior of the interface body, where a platinum wire electrode was placed. The conduction channel transferred the electric field from the external Pt electrode to the joining point, but prevented the effluent of CE from leaking. The interface body was designed to have receptacles for standard capillary tubing with finger-tight fittings, which allowed easy replacement of capillary tubing. Stable electrospray was observed for an extended time period without any signs of bubbling or damage to the emitter tip. No significant increment of dead-volume at the interface was observed for well-aligned capillaries. Sensitive and stable CE-MS detection of the model compound of creatinine and uric acid was demonstrated.</P>
백설매(Xue-Mei Bai),이금희(Jin-Ji Li),김미훈(Mei-Xun Jin),정유진(You-Jin Cheng),이종혁(Jong-Hyeok Lee) 한국정보과학회 2005 한국정보과학회 학술발표논문집 Vol.32 No.1
일반적으로 중국어의 명사구는 최단명사구, 기본명사구, 최장명사구로 분류된다. 최장명사구에 대한 정확한 식별은 문장의 전체적인 구조를 파악하고 문장의 정확한 지배용언을 찾아내는데 중요한 역할을 한다. 본 논문에서는 특성에 따라 5개의 클래스로 세분화된 문장부호를 학습자질로 사용하여 최장명사구 자동식별을 진행한다. 제안된 기법은 평균길이가 4인 최장명사구의 식별실험에서 기본모델(baseline)보다 4.5% 향상된 평균 85.1%의 우수한 F-measure 성능을 보인다.
문장부호 정보와 확장된 청크에 기반한 중국어 최장명사구 식별
백설매(Xue-Mei Bai),김미훈(Mei-Xun Jin),이금희(Jin-Ji Li),정유진(You-Jin Chung),이종혁(Jong-Hyeok Lee) 한국정보과학회 언어공학연구회 2005 한국정보과학회 언어공학연구회 학술발표 논문집 Vol.2005 No.10
명사구는 기본명사구와 최장명사구로 분류된다. 최장명사구에 대한 정확한 식별은 문장의 전체적인 구문구조를 파악하고 문장의 정확한 지배용언을 찾아내는데 중요한 역할을 수행한다. 본 논문에서는 확장된 청크(chunk) 개념과 다섯 개의 클래스로 세분화된 문장부호 정보를 사용한 최장명사구 식별 기법을 제안한다. 제안된 기법은 기본모델(baseline)보다 4.05% 향상된 평균 88.63%의 우수한 F-measure 성능을 보인다.