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Establishment of liver specific glucokinase gene knockout mice: a new animal model for screening anti-diabetic drugs

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Acta Pharmacologica Sinica 2004年第12期25卷 93-99页

作者： Ya-li ZHANG, Xiao-hong TAN2, Mei-fang XIAO, Hui LI, Yi-qing Mao, Xiao YANG2, Huan-ran TAN3 Department of Pharmacology, School of Basic medical science, Peking University Health science Center, beijing 100083 2institute of biotechnology academy ofmilitary medical science, beijing 100071, china Department of Pharmacology School of Basic Medical Science Peking University Health Science Center Beijing 100083 China

AIM: To characterize the liver-specific role of glucokinase in maintaining glucose homeostasis and to create an animal model for diabetes. METHODS: We performed hepatocyte-specific gene knockout of glucokinase in mice using Cre-loxP gene targeting strategy. First, two directly repeated loxP sequences were inserted to flank the exon 9 and exon 10 of glucokinase in genomic DNA. To achieve this, linearized targeting vector was electroporated into ES cells. Then G418- and Gancyclovir-double-resistant clones were picked and screened by PCR analysis and the positives identified by PCR were confirmed by Southern blot. A targeted clone was selected for microinjection into C57BL/6J blastocysts and implanted into pseudopregnant FVB recipient. Chimeric mice and their offspring were analyzed by Southern blot. Then by intercrossing the Alb-Cre transgenic mice with mice containing a conditional gk allele, we obtained mice with liver-specific glucokinase gene knockout. RESULTS: Among 161 double resistant clones 4 were positive to PCR and Southern blot and only one was used for further experiments. Eventually we generated the liver specific glucokinase knockout mice. These mice showed increased glucose level with age and at the age of 6 weeks fasting blood glucose level was significantly higher than control and they also displayed impaired glucose tolerance. CONCLUSION: Our studies indicate that hepatic glucokinase plays an important role in glucose homeostasis and its deficiencies contribute to the development of diabetes. The liver glucokinase knock- out mouse is an ideal animal model for MODY2, and it also can be applied for screening anti-diabetic drugs.

关键词： animal disease model non insulin dependent diabetes mellitus type 2 diabetes mellitus glucokinase knockout mice

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Development of Cell Lines Stably Expressing Staphylococcal Nuclease Fused to Dengue 2 Virus Capsid Protein for CTVI

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 577-582页

作者： Cheng-Feng QIN and E-De QIN institute of Microbiology and Epidemiology, academy of Military medical sciences, beijing 100071, china institute of Microbiology and Epidemiology Academy of Military Medical Sciences

To explore the potential application of capsid-targeted viral inactivation (CTVI) strategy in prophylactic model against dengue virus (DV) infection, here we fused a Ca2+-dependent nuclease, staphylococcal nuclease (SN), to the capsid protein of dengue 2 virus (D2C) at the carboxyl terminal, and constructed the desired expression plasmid pc/D2C-SN and control plasmids pc/D2C-SN* and pc/D2C. A mammalian cell line BHK-21 was transfected by electroporation with those plasmids and thereafter selected by 5 μg/ml blasticidin. The resistant cell clones were then expanding cultured and screened by RT-PCR and Western Blot assays. The nuclease activity of the expressed fusion protein D2C-SN was analyzed by in vitro DNA digestion assay. It was confirmed cell lines stably expressing D2C-SN and control constructs were obtained. The intracellular expressed fusion protein D2C-SN had ideal nuclease activity and no cytotoxicity on mammalian cells. Those engineered cell lines provided the experimental system for CTVI application in prophylactic model and paved the new road for combating DV infection with CTVI.

关键词： dengue 2 virus capsid protein staphylococcal nuclease CTVI stable expression

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Structure and regulatory function of plant transcription factors

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Chinese science Bulletin 2001年第4期46卷 271-278页

作者： LIU Qiang, ZHANG Guiyou & CHEN ShouyiDepartment of Biological science and biotechnology, Tsinghua Univorsity, beijing 100084, china Department of Biological science and biotechnology, State Laboratory of Biomembrane and Membrane biotechnology, Tsinghua University, beijing 100084, china institute of Genetics, Chinese academy of sciences, beijing 100101, china Department of Biological science and biotechnology Tsinghua University Beijing China Department of Biological science and biotechnology State Laboratory of Biomembrane and Membrane Biotechnology Tsinghua University Beijing China institute of Genetics Chinese Academy of Sciences Beijing China

The expression of inducible genes in plants is regulated by specific transcription factors at the transcrip-tional level. A typical transcription factor usually contains a DNA-binding domain, a transcription regulation domain, a dimerization site and a nuclear localization domain. These functional domains define the characteristic, localization and regulatory role of a transcription factor. Transcription factors recognize and bind to specific cis-acting elements or interact with other proteins, and then activate or repress the transcription of target genes by their functional domains. In recent years, elucidation on the structure and function of transcription factors has become an important subject in plant molecular biology.

关键词： transcription factor DNA-binding domain cis-acting element.

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Molecular pharmacognosy

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science china(Life sciences) 2010年第6期53卷 643-652页

作者： HUANG LuQi1,XIAO PeiGen2,GUO LanPing1 & GAO WenYuan3 1institute of Chinese Materia Medica,china academy of Chinese medical sciences,beijing 100700,china 2institute of Medicinal Plant Development,Chinese academy of medical sciences,Peking Union medical College,beijing 100094,china 3School of Pharmaceutical science and Technology,Tianjin University,Tianjin 300072,china 1. institute of Chinese Materia Medica China Academy of Chinese Medical Sciences Beijing 100700 China2. Institute of Medicinal Plant Development Chinese Academy of Medical Sciences Peking Union Medical College Beijing 100094 China3. School of Pharmaceutical Science and Technology Tianjin University Tianjin 300072 China

This article analyzes the background and significance of molecular pharmacognosy,including the molecular identification of medicinal raw materials,phylogenetic evolution of medicinal plants and animals,evaluation and preservation of germplasm resources for medicinal plants and animals,etiology of endangerment and protection of endangered medicinal plants and animals,biosynthesis and bioregulation of active components in medicinal plants,and characteristics and the molecular bases of top-geoherbs.

关键词： molecular pharmacognosy pharmacognosy borderline subject

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Production of human liver prolidase by Saccharomyces cerevisiae as host cells

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Acta Pharmacologica Sinica 2004年第6期25卷 92-98页

作者： Shu-hao WANG, Min LIU, Mu-gen CHI, Qing-ding WANG, Man-ji SUN2 institute of Pharmacologyand Toxicology, academy of Military medical science, beijing 100850, china institute of Pharmacology and Toxicology Academy of Military Medical Science Beijing 100850 Chin

AIM: To clone and express the recombinant human liver prolidase in yeast and explore the activities of both dipeptidaseand organophosphoric acid anhydrolase(OPAA). METHODS:The cDNAencodinghuman liver prolidase derived from healthy adult liver was cloned into the pYES2, an expression vector of S cerevisiae, and then trans- formed into S cerevisiae INVSc1 by electroporation. The transformant with the highest enzymatic activity was induced by galactose for expression. The optimal induction conditions (temperature, induction time, and the initial amount of inoculation cells) were estimated by orthogonal experimental design. The recombinant prolidase and OPAA activities were assayed by spectrocolorimetric methods. RESULTS: The recombinant enzyme catalyzed the hydrolysis of organophosphorous compound soman as well as the hydrolysis of dipeptide Gly-Pro. Under the optimal induction conditions (20 h, 25 oC, initial OD600=0.4), the maximum activities of prolidase and OPAA came to 226.5 and 578 μmol·min-1·g-1 protein in cell lysate supernatants, respectively. SDS-PAGE of the recombinant enzyme in disrupted cell supernatants showed a molecular weight of 56 kDa. Intensity scanning of the SDS-PAGE gel revealed that the enzyme accounted for 3.16 % of the total protein in the supernatant. One liter incubation medium produced 7 g of wet yeast cell containing 4.56 mg of the recombination protein. CONCLUSION: The recombinant human liver prolidase produced by yeast cell (S cerevisiae) exhibited both dipeptidase and OPAA activities.

关键词： prolidase liver human S Cerevisiae organophosphorous compounds

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The Effect of CD3-Specific Monoclonal Antibody on Treating Experimental Autoimmune Myasthenia Gravis

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Cellular & Molecular Immunology 2005年第6期2卷 461-465页

作者： Ruonan Xu~1 Jianan Wang~1 Guojiang Chen~1 Gencheng Han~1 Renxi Wang~1 Beifen Shen~1 Yan Li~(1,2) ~1institute of Basic medical science,academy of military medical science,beijing 100850,china ~2Department of Molecular Immunology,institute of Basic medical science,27 Taiping Road,beijing 100850,china. institute of Basic medical science Academy of Military Medical Science Beijing 100850 China.

CD3-specific monoclonal antibody was the first one used for clinical practice in field of transplantation. Recently, renewed interests have elicited in its capacity to prevent autoimmune diabetes by inducing immune tolerance. In this study, we tested whether this antibody can also be used to treat another kind of autoimmune disease myasthenia gravis （MG） and explored the possible mechanisms. MG is caused by an autoimmune damage mediated by antibody- and complement-mediated destruction of AChR at the neuromuscular junction. We found that administration of CD3-specific antibody （Fab）2 to an animal model with experimental autoimmune myasthenia gravis （EAMG）（B6 mice received 3 times of AChR/CFA immunization） could not significantly improve the clinical signs and clinical score. When the possible mechanisms were tested, we found that CD3 antibody treatment slightly down-regulated the T-cell response to AChR, modestly up-regulation the muscle strength. And no significant difference in the titers of IgG2b was found between CD3 antibody treated and control groups. These data indicated that CD3-specific antibody was not suitable for treating MG, an antibody- and complementmediated autoimmune disease, after this disease has been established. The role of CD3-specific antibody in treating this kind of disease remains to be determined. Cellular ＆ Molecular Immunology. 2005;2（6）：461-465.

关键词： CD3-specific monoclonal antibody immune tolerance EAMG

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A novel aberrant splicing of G_(sa) transcript in human leukemia cell lines

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Chinese science Bulletin 1999年第4期44卷 340-344页

作者： YE Qinong, YAO Xiao, WANG Hengliang, ZHANG Shu, LIU Huaitian, SU Guofu, HUANG Cuifen and ZHOU Tingchong beijing institute of biotechnology, beijing 100071, china Department of Biology, Shaanxi Normal College, Xi’an 710062, china beijing medical University, School of Oncology, beijing 100036, china beijing institute of Microbiology and Epidemiology, beijing 100071, china beijing institute of Basic Medicine sciences , beijing 100850, china beijing institute of biotechnology Beijing China Department of Biology Shaanxi Normal College Xi’an China School of Oncology Beijing Medical University Beijing China beijing institute of Microbiology and Epidemiology Beijing China beijing institute of Basic Medicine sciences Beijing China

The α subunit of the stimulatory G protein, Gsα, is involved in the stimulation of adenylate cyclase pathway of signal transduction. The various Gsα mRNAs are generated either by alternative splicing or by using alternative promoter. A novel aberrant splicing of Gsα transcript in human leukemia cell lines is reported. The entire coding region of Gsα gene obtained, referred to as GsαLeu, was amplified by PCR with cDNA from Jurkat human leukemia cDNA library or the reverse-transcribed first strand cDNA from human leukemia cell line HL-60 as a template. The result of DNA sequencing indicated that,compared with originally reported Gsα, GsαLeu has two in-frame deletions, one in amino acid residues 23—249 and the other in amino acid residues 254—260, suggesting that G（sαLeu） may play an important role in leukemia. The complete coding region of GsαLu was inserted downstream of the thrombin site of pGEX-2T fusion protein expression vector and expressed in the form of GST/G(

关键词： Gsa splicing leukemia cloning and expression.

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Plant protein kinase genes induced by drought, high salt and cold stresses

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Chinese science Bulletin 2000年第13期45卷 1153-1157页

作者： LIU Qiang , ZHANG Yong & Chen Shouyi1. Deparlmenl of Biological science and biotechnology, Tsinghua University, beijing 100084, china 2. Department of Biological science and biotechnology, Slate Key Laboratory of Biomembrane and Membrane biotechnology, Tsinghua University, beijing 100084, china 3. institute of Genetics, Chinese academy of sciences, beijing 100101, china Department of Biological science and biotechnology Tsinghua University Beijing China Department of Biological science and biotechnology State Key Laboratory of Biomembrane and Membrane Biotechnology Tsinghua University Beijing China institute of Genetics Chinese Academy of Sciences Beijing China

Drought, high salt and cold are three different kinds of environment stresses that severely influence the growth, development and productivity of crops. They all decrease the water state of plant cells, and consequently result in the harm of plant from water deficit. Several genes encoding protein kinases and induced by drought, high salt and low temperature have been isolated from Arabidopsis. These protein kinases include receptor protein kinase (RPK), MAP kinases, ribosomal-protein kinases and transcription-regulation protein kinase. The expression features of these genes and the regulatory roles of these protein kinases in stress response and signal transduction are discussed.

关键词： protein kinase environment stress signal transduction gene expression.

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Inhibition of the activating signals in NK92 cells by recombinant GST-sHLAG1α chain

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Cell Research 2004年第2期14卷 155-160页

作者： Ai Yu YAO~(1,2), Hai Yang TANG~1, Yun WANG~1, Mei Fu FENG~(1,*), Rou Li ZHOU~(2,*) ~1State Key Laboratory of Biomembrane and Membrane biotechnology, institute of Zoology, Chinese academy of science, beijing 100080, china ~2Department of Cell Biology and medical Genetics, School of Basic medical sciences, Health science Center, Peking University, beijing 100083, china. State Key Laboratory of Biomembrane and Membrane biotechnology Institute of Zoology Chinese Academy of Science Beijing 100080 China Department of Cell Biology and medical Genetics School of Basic Medical Sciences Health Science Center Peking University Beijing 100083 China

The soluble HLA-G1 (sHLA-G1) isoform was found to be secreted by trophoblast cells at the matemo-fetal interface, which suggests that it may act as an immunomodulator during pregnancy. In this paper, we reported that GST-sHLA-G1α chain could bind to its receptor ILT-2 on NK92 cells and then the latter recruited Src homology 2 domaincontaining tyrosine phosphatase-1 (SHP-1), which consequently dephosphorylated some important protein tyrosine kinases and blocked the activation of downstream molecules such as MEK and ERK so that the cytotoxicity of natural killer (NK) cells was inhibited. These results indicated that GST-sHLA-G1α chain might be exploited in new immunotherapy strategies aiming at inducing immunotolerance during allograft, xenograft and autoimmune situations. In addition, we found that modification of O-linked β-N-acetylglucosamine (O-GlcNAc) was involved in NK cells’ activating and inhibitory signals. This may provide a novel molecular target for inducing immunotolerance but needs furthe

关键词： NK92 GST-sHLA-G1α chain signal transduction SHP-1 O-G1cNAc.

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IL-8-induced L-selectin shedding regulates its binding kinetics to PSGL-1

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Chinese science Bulletin 2009年第16期54卷 2786-2793页

作者： JIA XiaoLing1,2,3, CHEN Juan1,2 & LONG Mian1,2 1 National Microgravity Laboratory, institute of Mechanics, Chinese academy of sciences, beijing 100190, china 2 Center of Biomechanics and Bioengineering, institute of Mechanics, Chinese academy of sciences, beijing 100190, china 3 School of Biological science and medical Engineering, Beihang University, beijing 100083, china National Microgravity Laboratory Institute of Mechanics Chinese Academy of Sciences Beijing 100190 China Center of Biomechanics and Bioengineering Institute of Mechanics Chinese Academy of Sciences Beijing 100190 China School of Biological science and medical Engineering Beihang University Beijing 100083 China

L-selectin plays a crucial role in inflammation cascade by initiating the tethering and rolling of leukocytes on endothelium wall. While many L-selectin molecules are rapidly shed from the cell surface upon activation, the remaining membrane-anchored L-selectin may still play an important role in regulating leukocyte rolling and adhesion with different binding kinetics. Here we developed an in vitro model to activate Jurkat cells via interlukin-8 (IL-8) and quantified the two-dimensional (2D) binding kinetics, using a micropipette aspiration assay, of membrane-anchored L-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) ligand coupled onto human red blood cells (RBCs). The data indicated that L-selectin shedding reduced the amount of membrane-anchored L-selectin and lowered both its reverse and forward rates. These results suggested that the rolling dynamics of activated leukocytes was determined by two opposite impacts: reducing the surface presentation would enhance the rolling but lowering the kinetic rates would decrease the rolling. This finding provides a new insight into under-standing how L-selectin shedding regulates leukocyte rolling and adhesion.

关键词：结合动力学选择素配体白细胞介素8 Jurkat细胞诱导细胞表面微管吸吮

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