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Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

World Journal of Gastroenterology 2002年第2期8卷 318-322页

作者： Ge-Jian Zhu Ying-Nian Yu,Department of Pathophysiology and Laboratory of Medical Molecular Biology,zhejiang University School of Medicine,hangzhou 310031,zhejiang Province,China Xin Li,Department of pharmaceutical analysis & drug metabolism,College of Pharmacology Science,zhejiang University,hangzhou 310031,zhejiang Province,China Yu-Li Qian, Present address:center of laboratory,Women’s hospital,School of Medicine,zhejiang University,hangzhou 310031,zhejiang Province,China Department of Pathophysiology and Laboratory of Medical Molecular Biology Zhejiang University School of Medicine Hangzhou 310031 Zhejiang Province China.

AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T>C, 114.C>T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 ***(-1).g(-1) S9 protein or 8.62 +/- ***(-1).mol(-1) CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.

关键词： Cloning, Molecular Animals Aryl Hydrocarbon Hydroxylases Cell Fractionation Cell Line China Gene Expression Humans Hypoglycemic Agents Liver Protein Isoforms Recombinant Proteins Research Support, Non-U.S. Gov't Tolbutamide

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Preparation and vibrational modes of C60·2CHBr3

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Chinese Science Bulletin 2003年第10期48卷 961-964页

作者： LI Hongnian, LI Haiyang, WU Taiquan, HE Shaolong, SUN Yangjun & XU Yabo Department of Physics, zhejiang University, hangzhou 310027, China Department of Physics Zhejiang University Hangzhou 310027 China

C60·2CHBr3 polycrystalline powder was prepared by the solution method. The vibrational modes of the sample were studied by Raman scattering and infrared adsorption techniques. The Raman spectra showed that the Ag modes were downshifted by 4-5 cm1, while the Hg(1) mode was unshifted. The analyses of the downshifts of the Ag modes revealed that a small amount of electrons were transferred from the hydrogen atoms to the C60 molecules. In the infrared spectra, the F1u modes of the pristine C60 were unshifted after the intercalation with CHBr3. However, the vibrational modes of CHBr3 changed remarkably. The C-Br stretching mode was downshifted by ～4 cm1. The adsorption of the C-H twisting mode was weakened obviously, and the adsorption of the C-H stretching mode was not observed. These results exhibited that there were nonnegligible interactions between CHBr3 and C60 molecules, and that the strongest interaction took place between the hydrogen atoms and the C60 molecules. The interaction should induce not only the changes of the vibrational modes reported here, but also those of the electronic states. So this work should offer important glues to the mechanism of the superconductivity at 117 K of the field-doped C60·2CHBr3.

关键词：制备振动模型 C60·2CHBr3 多晶粉末 Ag 银氢原子电子转移高温超导材料溴代甲烷富勒烯

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Effects of ELF magnetic fields on protein expression profile of human breast cancer cell MCF7

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Science China(Life Sciences) 2005年第5期48卷 506-514页

作者： LI Han1, ZENG Qunli1, 2, WENG Yu1, LU Deqiang1, JIANG Huai1 & XU Zhengping1, 2 1. Bioelectromagnetics Laboratory, zhejiang University , hangzhou 310031, China 2. Research center for Environmental Genomics, zhejiang University School of Medicine, hangzhou 310031, China 1. Bioelectromagnetics Laboratory Zhejiang University 310031 Hangzhou China 2. Research center for Environmental Genomics Zhejiang University School of Medicine 310031 Hangzhou China

Extremely Low Frequency Magnetic Fields (ELF MF) has been considered as a “possible human carcinogen” by International Agency for Research on Cancer (IARC) while credible mechanisms of its carcinogenicity remain unknown. In this study, a proteomics approach was employed to investigate the changes of protein expression profile induced by ELF MF in human breast cancer cell line MCF7, in order to determine ELF MF-responsive proteins. MCF7 cells were exposed to 50 Hz, 0.4 mT ELF MF for 24 h and the changes of protein profile were examined using two dimensional electrophoresis. Up to 6 spots have been statistically signifi-cantly altered (their expression levels were changed at least 5 fold up or down) compared with sham-exposed group. 19 ones were only detected in exposure group while 19 ones were missing. Three proteins were identified by LC-IT Tandem MS as RNA binding protein regulatory subunit、Proteasome subunit beta type 7 precursor and Translationally Controlled Tumor Protein. Our finding showed that 50 Hz, 0.4 mT ELF MF alternates the protein profile of MCF7 cell and may affect many physiological functions of normal cell and 2-DE coupled with MS is a promising ap-proach to elucidating cellular effects of electromagnetic fields.

关键词： ELF MF, MCF7, 2-DE, MS, protein expression profile.

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Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

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Cell Research 2007年第6期17卷 565-571页

作者： Hangping YaoXiajing TangXueting ShaoLei FengNanping WuKe YaoNational Key Laboratory of Infectious Diseases, Institute of Infectious Diseases, First Affiliated Hospital, School of Medicine, zhejiang University, hangzhou, ChinaEye center, Second Affiliated Hospital School of Medicine, zhejiang University, 88 Jiefang Road, hangzhou 310009, China NationalKeyLaboratoryofInfectiousDiseases InstituteofInfectiousDiseasesFirstAffiliatedHospitalSchoolofMedicineZhejiangUniversityHangzhouChina Eyecenter SecondAffiliatedHospitalSchoolofMedicineZhejiangUniversity

The apoptosis of lens epithelial cells has been proposed as the common basis of cataract formation, with oxidative stressas the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolideagainst oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanismsinvolved. HLE cells （SRA01-04） were incubated with 50μM H2O2 in the absence or presence of different doses ofparthenolide （10, 20 and 50μM）. To study apoptosis, the cells were assessed by morphologic examination and AnnexinV-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expressionof caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 andcaspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H2O2 for 18 h, a highfraction of HLE cells underwent apoptosis, while in the presence of parthenolide of different concentrations, dose-depen-dent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H2O2 in HLEcells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation of caspase-3 andcaspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLEcells from oxidative stress-induced apoptosis through inhibition of the activation of caspase-3 and caspase-9, suggestinga potential protective effect against cataract formation.

关键词： parthenolide human lens epithelial cells apoptosis caspase-3 and caspase-9

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rDNA ITS Sequence Analysis and Molecular Phylogeny of Four Fungal Strains Assigned to the Family Russulaceae

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食用菌学报 2007年第2期 29-32页

作者： FU Lizhong1, 2, LI Haibo1, 2, WEI Hailong1, 2, WU Qingqi1, 2, WU Dafeng2, WU Xueqian1, 21 Biotechnology Institute, zhejiang Academy of Forestry Science, hangzhou, zhejiang 310023,China 2Lishui Edible Fungi Research and Development center,zhejiang Essence Fungi DevelopmentCompany Ltd,Lishui,zhejiang 323000,China

Internal transcribed spacer (ITS) sequences of four edible and medicinal fungi belonging to the Russulaceae, collected from the Lishui mountainous area of zhejiang Province, were cloned and sequenced. Comparisons with sequence data in GenBank were undertaken using BLAST, and the four ITS sequences, together with 15 reference sequences obtained from GenBank, were used to construct a phylogenetic tree. rDNA ITS sequences of the four strains ranged from 673 to 766 bp in length and between 47.4% and 49.48% in GC content. Phylogenetic data indicated strain R57 to be Russula cyanoxantha. Differences in the size and GC content of ITS sequences from strains R32 and R57 (R. cyanoxantha) and strain L37 (Lactarius sanguifluus) were significant, whereas the corresponding differences between strains R32 and R57 were relatively small. A close kinship, with only small detectable differences in their respective ITS sequences, existed between R. mustelina, R. virescens and R. parazurea. A similar situation applied to strains of L. sanguifluus and L. salmonicolor.

关键词： Russulaceae rDNA Internal Transcribed Spacer Sequence analysis Phylogeny Macrofungi

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Linearization and general solution of （2＋1）-dimensional Broer-Kaup equation

Linearization and general solution of （2＋1）-dimensional B...

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中国数学力学物理学高新技术交叉研究会第8届学术研讨会

作者： Zhang Jiefang~(1 2) Wu Fengmin~1 Shi Jianqing~1 (~1 Research center of Engineering Science zhejiang University of Technology.hangzhou zhejiang 310032 China)(~2 Institute of Nonlinear physics zhejiang Normal University Jinhua 321004 zhejiang.China)

＜正＞THe linearization form of the（2＋1）-dimensional Broer-Kaup equation which is obtained from the constraints of the KP equation are *** from the their linear form. Three kinds of general solutions are *** solitary wave solution,multi-solitary wave solution and exponential localized solitary wave solution are some special cases of our results.

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Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

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Cell Research 2003年第5期13卷 361-367页

作者： JIA HUA HU, JIE JIANG, YING HUA MA, NA YANG, MAO HU ZHANG, MIN WU, JIAN FEI, LI HE GUO 1 Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 2 College of life science, zhejiang University, hangzhou 310027, China 3 Shanghai Research center for Biomodel Organism, Shanghai 201203, China 1Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 2College of life science Zhejiang University Hangzhou 310027 China 1Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 1Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 1Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@*** 2College of life science Zhejiang University Hangzhou 310027 China 3Shanghai Research center for Biomodel Organism Shanghai 201203 China 1Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. E-mail: mhzhang@***

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20％ v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITC-conjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germ cells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flow cytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes of epithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphology was

关键词： Fas ligand ethanol apoptosis testes transgenic mouse.

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我国教育领域的公私合作伙伴关系审视

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教育发展研究 2010年第8期30卷 49-54,59页

作者：高树昱吴华浙江大学科教发展战略研究中心博士研究生杭州310027 浙江大学民办教育研究中心主任教授杭州310028

本文分别对公私合作伙伴关系政策层面与项目层面的相关实践进行了梳理,探讨了公私合作伙伴关系实践中存在公私合作伙伴关系是一种有效动员、整合和优化社会教育资源的重要制度框架,在我国教育领域有着广泛实践,但相关的理论研究却比较... 详细信息

本文分别对公私合作伙伴关系政策层面与项目层面的相关实践进行了梳理,探讨了公私合作伙伴关系实践中存在公私合作伙伴关系是一种有效动员、整合和优化社会教育资源的重要制度框架,在我国教育领域有着广泛实践,但相关的理论研究却比较缺乏。观念束缚、政策歧视、规制失衡等问题,并提出了转变观念、消除歧视和建立有效的监管体系与评价体系等对策。

关键词：公私合作伙伴关系

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Low Concentration of BPDE Induces Alterations of Extracellular Protein Profile of Exposed Epithelial Cells

Low Concentration of BPDE Induces Alterations of Extracellul...

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2008年中国病理生理学会第十一届肿瘤和第十二届免疫专业委员会学术会议

作者： Hui Liu~(1,2,#,§) Jing Shen~(1,§) Lei Feng~(2,#) Yingnian Yu~1 1 Department of Pathology and Pathophysiology,zhejiang University School of Medicine,hangzhou 310058,China.2 Department of Toxicology and Nutriology,zhejiang University School of Medicine,hangzhou 310058,China. hangzhou center for Disease Control and Prevention,hangzhou 310006,China Department of Health Control,zhejiang Forestry University,Lin’an,hangzhou 311300,China

7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) exposure induces adduct formation and oxidative damage on DNA,and consequently triggers complicated stress responses,including such responses as signaling pathway activation,cell cycle arrest,DNA repair, translesion DNA synthesis and *** the present study,2-DE and MALDI-TOF MS were employed to analyze the differential extracellular protein patterns of human amniotic epithelial cells (FL cells) after exposure to 5 nM BPDE and *** a result,one protein spot that appeared in the culture medium of BPDE treatment group was successfully identified as 14.3-3ζ,and three up-regulated protein spots were identified as annexin A3,annexin V and hydroxypyruvate isomerase *** them,14.3-3ζwas further detected in the pleural fluid specimens of seven patients with lung adenocarcinoma or *** results demonstrate that BPDE exposure can induce alterations of extracellular protein profiles of exposed cells,which may be served as a starting point for searching candidate biomarkers for B[a]P exposure.

关键词： 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) Extracellular Protein Profile Human amniotic epithelial cells Stress Response

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A Review on the Episodic Mechanism of Multi-team Coordination

A Review on the Episodic Mechanism of Multi-team Coordinatio...

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第六届（2011）中国管理学年会——组织行为与人力资源管理分会场

作者： Wang Xiaochen1, 2, Wang Zhongming2, Xiao Yuchun3 (1,3 School of Business Administration, zhejiang Gong Shang University, hangzhou, China,310018) (2 Global Entrepreneurship Research center of zhejiang University, hangzhou, China,310018)

This paper aimed to find out the coordination mechanism from an episodic perspective and examined the problem of team coordination in multi-team system (MTS) based on a new unit-of-analysis. The author presented key findings on performance episode from relevant researches and reviewed the ones containing the special issue on the basis of the focal constructs and unit of analysis. The results showed that in model 1 the MTS transition processes could positively promote MTS action processes and performance, and then the team action processes could also improve MTS performance, but MTS transition processes exerted little impact on team action processes. And the model 2 illustrated that deliberate plans and contingency plans in transition processes could improve MTS coordination in the early stage of multi-team action, while the reactive strategy adjustment in action processes could positively facilitate MTS coordination in the later stage, both of which can improve MTS performance in the end. The previous studies on team coordination were fully based on the traditional IPO models, which did not match the complex situation in multi-team system. But this research is developed from a new and quite suitable perspective -episodic process- to study how the relevant component teams coordinate with each other to facilitate performance achievement, all of which can give us new inspirations about the operation process of multi-team (or multi-organization) systems in the real world.

关键词： performance episode multi-team system MTS coordination transition process action process

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