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Cloning,Structural Organization and Chromosomal Mapping of Rat Costimulatory Molecule 4-1.BL

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 694-701页

作者： Qiu-Ming DONG~1.Li-Jie MA~2 Guang-Bo ZHANG~1.Ya-Fang WU~3 Jia-Yao SHEN~1.Ying CHEN~1.Yong-Jing CHEN~1.Xiang-Ke PU~1.Sai-yu HANG~1.Xue-Guang ZHANG~(1.) 1.Biotechnology {1.,Soochow {1.,Suzhou 21.007,china 2 Genetics {1.,fudan {1.,shanghai 2.0433,china 3 First Affiliated Hospital of Soochow {1.,Suzhou 21.006,china Biotechnology institute Soochow UniversitySuzhou 215007 China Genetics institute Fudan UniversityShanghai 200433 China First Affiliated Hospital of Soochow universitySuzhou 21.006 China

4-1.BL (TNFSF9) is a member of the tumor necrosis factor (TNF) ligand superfamily,whichis expressed on some activated antigen presenting cells and B *** isolated a rat cDNA clone encodingthe rat homologue of the human 4-1.BL (GenBank accession ***259541..The deduced rat 4-IBBLprotein,consisting of 308 amino acids with a molecular weight of 33,469 Da,was a typical type Ⅱtransmembrane glycoprotein,the same as human and routine 4-IBBL.“SDAA” in the cytoplasmic domain ofrat 4-1.BL was deduced to act as the phosphorylation site for casein kinase Ⅰ(“SXXS” motif),which ispresent in the cytoplasmic domains of human and murine 4-1.BL,and all other TNF ligand family membersknown to utilize reverse *** two introns of 4-1.BL were also cloned (GenBank accession ***332409).Rat 4-IBBL is much more homologous with murine 4-1.BL than with human 4-1.BL,in bothnucleotide and amino acid *** 4-1.BL was expressed in all tested tissues:brain,lung,colon,liver,thymus,testicle,kidney,adrenal,stomach,spleen and *** chromosomal location of rat 4-1.BL wasfirst identified by bioinformatics,then by fluorescence in situ hybridization at 9q1.(GenBank accessionUni Gene ***.46783).Rat,murine and human 4-1.BL genes are evolved from a common *** and characterization of the rat counterpart of human 4-1.BL will facilitate studies of the biologicalfunction of this molecule.

关键词： TNF superfamily rat 4-1.BL cloning mapping

A New Combination of Mutated loxPs in a Vector for Construction of Phage Antibody Libraries

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Acta Biochimica et Biophysica Sinica 2005年第7期37卷 495-500页

作者： yu GAN~(1.2) Xin-Tai ZHAO~2 1.{1. Medical College,{1. university,{1. 200032,china 2 Laboratory for Cellular and Molecular Immunology,Cancer institute,{1. Jiaotong university,{1. 200032,china shanghai Medical College Fudan UniversityShanghai 200032 China Laboratory for Cellular and Molecular Immunology Cancer Institute Shanghai Jiaotong UniversityShanghai 200032 China

In the construction of large antibody libraries by in vivo recombination,two non-homogeneousloxP sites are required for the exchange of V genes between phagemids to create many new *** mutated loxP51. was designed not to recombine with the wild-type loxP （loxPwt） in early studies anda combination of the two has been used to construct antibody *** recent reports have shown thatrecombination occurs between loxPwt and *** suggests that the combinational use of loxP51.and loxPwt might lead to the loss of the V gene diversity of antibody ***,it is necessary tofind a new combination of loxPs to avoid the excision recombination in the antibody *** this study,we found that the excision recombination between loxP51. and loxP2272,another mutated loxP sequence,was undetectable within one phagemid,while the excision recombination between loxP51. and loxPwtoccurred at a frequency of 40%.higher than that reported ***,the in vivo recombinationof different phagemids with loxP51. and loxP2272 showed that the V gene exchange was efficiently mediatedto produce new *** was concluded that the loxP51. and loxP2272 combination wasmore favorable for reducing the excision recombination and constructing large phage antibody libraries withhigh diversity.

关键词： mutated loxP excision recombination phage antibody library

Identification and Characterization of glnA Promoter and its Corresponding Trans-regulatory Protein GlnR in the Rifamycin SV Producing Actinomycete,Amycolatopsis mediterranei U32

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Acta Biochimica et Biophysica Sinica 2006年第12期38卷 831-843页

作者： Hao yu~(1.) Wen-Tao PENG~1.Yang LIU~1.Ting WU~2 yu-Feng YAO~1.Ming-Xue CUI~1.Wei-Hong JIANG~1.Guo-Ping ZHAO~(1.3,4*) 1.Laboratory of Molecular Microbiology,institute of Plant Physiology and Ecology,{1. institutes for Biological Sciences,Chinese Academy of Sciences,{1. 200032,china 2 Department of Medicinal Chemistry,School of Pharmacy,fudan university,{1. 200032,china 3 State Key Laboratory of Genetic Engineering & Department of Microbiology,School of Life Science,fudan university,{1. {1.},china 4 Laboratory of Disease and Health Genomics,Chinese National Human Genome Center at {1.,{1. 201.03,china Laboratory of Molecular Microbiology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China Department of Medicinal Chemistry School of Pharmacy Fudan University Shanghai 200032 China State Key Laboratory of Genetic Engineering & Department of Microbiology School of Life Science Fudan University Shanghai 200433 China Laboratory of Disease and Health Genomics Chinese National Human Genome Center at Shanghai Shanghai 201203 China

The genetic requirements for the transcription of glnA,encoding the major glutaminesynthetase in a rifamycin SV-producing Amycolatopsis mediterranei strain,U32,were *** experiments showed that the promoter of U32 glnA （pglnA） was likely to have two transcriptioninitiation sites:P1.nd P2,located 1.7 and 45 nucleotides （nt） upstream of the translational start codon,*** mobility shift and DNase I footprinting analyses revealed a 30 bp cis-element locatedat 45 to 75 nt downstream of P1.or 38 to 68 nt upstream of *** sequence of the cis-element displayedhigh similarity to the corresponding regions of pglnA from Streptomyces coelicolor and *** as a reporter gene,the expression levels of U32 pglnA and its deletion derivatives under differentnitrogen-source conditions were analyzed by detecting the catechol dioxygenase activities in ***54,*** J508 and *** FS1. （glnR mutant）.These in vivo studies showed that theactivation of U32 pglnA in *** required GlnR,and its binding to the U32 pglnA was furtherconfirmed by the gel mobility shift *** and heterologous expression of the U32 glnR allowed usto detect the in vitro interaction between the U32 GlnR and the corresponding pglnA *** shown by in vivo glnR inactivation and complementation indicated that GlnR is essential for theactive transcription of glnA in U32.

关键词： glnA promoter cis-element glnR Amycolatopsis mediterranei

Identification and Characterization of a Rat Novel Gene RSEP4 Expressed Specifically in Central Nervous System

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Acta Biochimica et Biophysica Sinica 2004年第7期36卷 501-507页

作者： Xi-Dao WANG1.2, Ling-Wei KONG1. Zhi-Qin XIE1. yu-Qiu ZHANG3, Zhi-Xin LIN2, Zhi-Qi ZHAO3,Lei yu4, and Nai-He JING1. 1.Laboratory of Molecular Cell Biology, Laboratory of Stem Cell Biology, institute of Biochemistry and Cell Biology, {1. institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, {1. 200031. china School of Life Science and Biotechnology, 2 {1. Jiaotong university, {1. 200030, china institute of Neurobiology,{1.},{1.}, china 3 4 Department of Cell Biology, Neurobiology and Anatomy, College of Medicine, university of Cincinnati, Cincinnati, Ohio, USA Laboratory of Molecular Cell Biology Laboratory of Stem Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China School of Life Science and Biotechnology Shanghai Jiaotong UniversityShanghai 200030 China institute of Neurobiology Fudan UniversityShanghai 200433 China Department of Cell Biology Neurobiology and Anatomy College of Medicine University of CincinnatiCincinnati Ohio USA

The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length of RSEP4 cDNA is 2006 bp, with a 501.nucleotide open reading frame (ORF) that encodes a 1.7 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1. CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

关键词： central nervous system (CNS) rat Northern blot in situ hybridization

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

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World Journal of Gastroenterology 2001年第5期7卷 647-651页

作者： Ping-Sheng Chen~(1.2) Wei-Rong Zhai~1.Xiao-Mei Zhou~3 Jin-Sheng Zhang~1.yue-E Zhang~1.yu-Qin Ling~1.Ying-Hong Gu~1.1.Department of Pathology,School of Basic Medical Sciences,fudan {1.,shanghai 200032,china2 Ping-Sheng Chen now works in the Department of Pathology,School of Basic Medical Sciences the (former Nanjing Railway Medical College),Southeast {1.,Nanjing 21.009,china3 institute of Cancer Research,shanghai 200032,china Department of Pathology School of Basic Medical Sciences Fudan University 138 Yixueyuan Road Shanghai 200032 China.

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1.(MT1.MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 1.h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=1.; control: 3.2 +/- 1.0, n = 7; P1.; control: 3.6 +/- 1.0, n = 7; P 334 +/- 1.922, n = 9; control: 1..277 +/- 7.424, n = 1.; P 1.. Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 1.h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.01.4, n = 1.; control: 0.021. +/- 0.0098, n = 1.; P 1., and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771. n = 1.; control: 4.304 +/- 1.083, n = 1.; P 1.MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

关键词： Animals Cell Division Cell Hypoxia Cells, Cultured Gelatinase A Gene Expression Regulation, Enzymologic Hepatocytes Hyperoxia Metalloendopeptidases RNA, Messenger Rats Rats, Sprague-Dawley Research Support, Non-U.S. Gov't Tissue Inhibitor of Metalloproteinase-2

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter Ⅰ (GAT1.

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Cell Research 2004年第1期14卷 54-59页

作者： Jia Hua HU~(1.*), Jin Fu ZHANG~(2,*), Ying Hua MA~1. Jie JIANG~1. Na YANG~1. Xin Bo LI~3, Zhi Guang yu CHI~4, Jian FEI~(1.5,**), Li He GUO~(1.**) ~1.aboratory of Molecular Cell Biology, {1. of Biochemistry and Cell Biology, {1. {1.s for Biological Sciences, Chinese Academy of Sciences, {1. 200031. china. ~2Department of Urology, Tongji Hospital, Tongji university, 389 Xincun Road, {1. 200065, china. ~3New Drug R & D Center, North china Pharmaceutical Group Corporation, 388 Heping East Road, Shijia Zhuang 05001., Hebei, china. ~4School of life sciences,{1.}, 220 Handan Road,{1.}, china. ~5{1. Research Center for Biomodel Organism, {1. 201.03, china. Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China shanghai Research Center for Biomodel Organism Shanghai 201203 China Department of Urology Tongji Hospital Tongji University 389 Xincun Road Shanghai 200065 China New Drug R & D Center North China Pharmaceutical Group Corporation 388 Heping East Road Shijia Zhuang 050015 Hebei China School of life sciences Fudan University 220 Handan Road Shanghai 200433 China

It is well documented that γ-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABA_A and GABA_B receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter Ⅰ (GAT1. also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1.overexpressing mice to explore GAT1.function in male reproductive system. We found that the expression level of GAT1.continuously increased in wild-type mouse testis from 1.month to 2 months after birth. GAT1.overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1.overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wi

关键词： GABA GAT1 testes sperm reproduction transgenic.

Jackknife and Bootstrap Tests of the Composition Vector Trees

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Genomics, Proteomics & Bioinformatics 2010年第4期8卷 262-267页

作者： Guanghong Zuo1.2, Zhao Xu1.3, Hongjie yu1.4, and Bailin Hao1.5,6 1.-Life Research Center & Department of Physics,{1.,{1., china 2shanghai institute of Applied Physics, Chinese Acadamy of Sciences, shanghai 201.00, china 3Applied Biosystems, Inc., Beijing 1.0027, china 4fudan-VARI Center for Genetic Epidemiology,{1.,{1., china 5institute of Theoretical Physics, Chinese Acadamy of Sciences, Beijing 1.01.0, china 6Santa Fe institute, Santa Fe, NM 87505, USA. T-Life Research Center & Department of Physics Fudan University Shanghai 200433 China.

Composition vector trees （CVTrees） are inferred from whole-genome data by an alignment-free and parameter-free method. The agreement of these trees with the corresponding taxonomy provides an objective justification of the inferred phylogeny. In this work, we show the stability and self-consistency of CVTrees by performing bootstrap and jackknife re-sampling tests adapted to this alignment-free approach. Our ultimate goal is to advocate the viewpoint that time-consuming statistical re-sampling tests can be avoided at all in using this alignment-free approach. Agreement with taxonomy should be taken as a major criterion to estimate prokaryotic phylogenetic trees.

关键词： composition vector phylogeny topological distance bootstrap jackknife CVTree

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Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor

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Acta Pharmacologica Sinica 2007年第1期28卷 111-117页

作者： Hua JIANG~2 Jie ZHANG~(2,3) Bi-zhi SHI~(2,3) yu-hong XU~3 Zong-hai LI~(2,3,4) Jian-ren GU~(2,3,4)2 State Key Laboratory of Oncogenes and Related Genes,{1. Cancer {1.,Medical College of fudan university 3 School of Pharmacy,{1. Jiaotong university,{1. 200032,china State Key Laboratory of Oncogenes and Related Genes Shanghai Cancer Institute Medical College of Fudan University Shanghai China School of Pharmacy Shanghai Jiaotong University Shanghai China

Aim:To develop a simple method for monitoring protein localization of epidermalgrowth factor(EGF)in living ***:Enhanced green fluorescent protein(EGFP)was used as an autofluorescent tag to label EGF ***-PAGE andWestern blot analysis were used to detect the expression of the EGFP-tagged EGF(EGFP-EGF)*** cell-binding and internalization activity of EGFP-EGFwere analyzed by fluorescence-activated cell sorting(FACS)and confocal ***:EGFP-EGF protein was expressed in Escherichia coli and purified.A cell-binding assay demonstrated that the EGFP-EGF protein could bind effi-ciently to the cells expressing *** binding and internalization of EGFP-EGFcan be visualized even at a very low concentration under confocal *** FACS-based assay for internalization activity indicated the accumulation ofinternalized EGFP-EGF over ***,the results of the competitionassay indicated its EGFR binding *** such a method,it does notneed to label EGF with chemicals and avoid light in the experimental ***:The fusion protein EGFP-EGF has several characters including highsensitivity,stability and convenience for manipulation,and is a powerful tool forthe study of EGF endocytosis.

关键词： receptor epidermal growth factor endocytosis purification expression

Electrophysiological actions of cyclosporin A and tacrolimus on rat hippocampal CA1.pyramidal neurons

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Acta Pharmacologica Sinica 2007年第12期28卷 1891-1897页

作者： Yong yu~2,Xue-qin CHEN~3,Yao-yuan CUI~(2,4),Guo-yuan HU~(3,4)~2 Department of Neurosurgery,Zhongshan Hospital,fudan {1.,shanghai 200032,china ~3 State Key Laboratory of Drug Research,shanghai institute of Materia Medica,Chinese Academy of Sciences,shanghai 201.03,china Department of Neurosurgery Zhongshan Hospital Fudan University Shanghai China State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai China

Aim:The aim of the present study was to investigate the electrophysiologicalactions of cyclosporin A （CsA） and tacrolimus （FK506） on neurons in the brain,and to elucidate the relevant ***:Whole-cell current-clamprecording was made in CA1.pyramidal neurons in rat hippocampal slices;whole-cell voltage-clamp recording was made in dissociated hippocampal CA1.pyrami-dal neurons of ***:CsA （1.0 μmol/L） and FK506 （50 μmol/L） did notsignificantly alter the passive electrical properties of hippocampal CA 1.pyramidalneurons,but slowed down the repolarizing phase of the action ***（1.-1.0 μmol/L） selectively inhibited the delayed rectifier K+current （IK） in aconcentration-dependent *** did not affect the kinetic properties of *** dialysis of CsA （1.0 μmol/L） had no effect on *** inhibition of IKby CsA （1.0 μmol/L） persisted under the low Ca2+conditions that blocked thebasal activity of ***:CsA exerted calcineurin-independentinhibition on the IKin rat hippocampal pyramidal *** together withour previous finding with FK506,it is conceivable that the spike broadening causedby the immunosuppressant drugs is due to direct inhibition on the IK.

关键词： action potential calcineurin cyclosporin A delayed rectifier K~+ channel hippocampus tacrolimus