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Expression and purification of lipoprotein-associated phospholipase A2,a key enzyme involved in atherosclerosis

Acta Pharmacologica Sinica 2006年第6期27卷 679-684页

作者： Fu-jun ZHANG~(2,4)Mao-jun CAI~3.Jing-kang SHEN~3.Yi-ping WANG~(2,5)2 State Key Laboratory of Drug Research.3.Department of Medicinal Chemistry,shanghai Institute of Materia Medica,shanghai Institutes for Biological Sciences,Chinese Academy of Sciences.shanghai 201203.{3. 4 Graduate school of the Chinese Academy of Sciences.Chinese Academy of Sciences,shanghai 201203.{3. State Key Laboratory of Drug Research Chinese Academy of Sciences Shanghai China Graduate school of the Chinese Academy of Sciences Chinese Academy of Sciences Shanghai China Department of Medicinal Chemistry Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

Aim:To express and purify lipoprotein-associated phospholipase A2（Lp-PLA2）,and to establish a screening model for Lp-PLA2inhibitors using the expressed Lp-PLA2,Methods:We cloned the full-length cDNA of Lp-PLA2 from differentiatedTHP-I cells,and subcloned the cDNA into the baculovirus transfer *** addition,we introduced an N-terminal Kozak sequence for high-level translation initiation and a C-terminal sequence of 6 histidine residues forpurification,The fusion enzyme was expressed in Sf9 insect cells and purified byNi2+affinity *** Lp-PLA2activity was measured us-ing[3.]PAF as a substrate,and we examined the enzyme activity of recombinantLp-PLA2pretreated with the known Lp-PLA2inhibitor ***:Wesuccessfully cloned the full-length Lp-PLA2gene from differentiated THP-1 *** fusion enzyme was expressed in Sf9 insect cells at a high level and purifiedefficiently through a 2-step *** recombinant Lp-PLA2activity wasmeasured using[3.]PAF as a substrate,and proved to be enzymatically ***-PLA2inhibitor SB43.495 produced a good inhibition curve for inhibition ofrecombinant Lp-PLA2with an IC50of 57±1μmol/***:We expressed andpurified Lp-PLA2at a high level in insect cell-baculovirus expression *** ratio was much greater than that obtained from human plasma and we estab-lished a screening model for Lp-PLA2inhibitors using the expressed Lp-PLA2.

关键词： lipoprotein-associated phospholipase A2 atherosclerosis coronary heart diseases cloning baculovirus purification

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Induction of liver cytochrome P450 1A2 expression by flutamide in rats

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Acta Pharmacologica Sinica 2005年第11期26卷 1382-1386页

作者： Hai-xue WANG, Xiao LIU, Chang-jiang XU, Xiao-chao MA~3. Jian-er LONG~4, Duan LI~(4,5) ~2 Department of Pharmacology, school of Pharmacy, Fudan University, shanghai 20003., china ~3.State Key Laboratory of Drug Research, {3. Institute of Biological Sciences, {3. Institute of Materia Medica, Chinese Academy of Sciences, shanghai 20003., china ~4 Institute of Molecular Virus, school of Basic Medical Science, Fudan University, shanghai 20003., china Department of Pharmacology School of Pharmacy Fudan University Shanghai China State Key Laboratory of Drug Research Shanghai Institute of Biological Sciences Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai China Institute of Molecular Virus School of Basic Medical Science Fudan University Shanghai China

Aim: To investigate the modulation of liver cytochrome P450 1A2 (CYP1A2) expression by giving flutamide to adult rats. Methods: Rats were given 50, 100, and 200 mg/kg po of flutamide for 2 weeks. Liver CYP 1A2 mRNA was measured using reverse transcription-polymerase chain reaction. CYP1A2 protein was detected using immunoblotting. CYPl A2 activity was assayed using high performance liquid chromatography, with caffeine as the CYPl A2 substrate. Results: CYP1A2 mRNA levels after flutamide treatment at 100 mg/kg and 200 mg/kg were, respectively, 1.86 and 3.11-fold higher than those of the control. Correspondingly, CYP1A2 protein increased 1.78 and 2.89-fold and CYPl A2 activity increased approximately 1.65 and 2.83.fold, respectively, relative to controls. Flutamide treatment at 50 mg/kg had no significant effect on CYP1A2 mRNA, protein, or enzyme activity. Conclusion: Giving rats flutamide induced liver CYP1A2 expression in a dose-dependent manner.

关键词： flutamide cytochrome P450 1A2 induction

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Chimney Effect Assessment of the Double-skin Facade

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湖南大学学报（自然科学版） 2009年第S1期36卷 146-150页

作者： QIU Zhong-zhu1,LI Peng2,CHOW Tin-tai3.REN Jian-xing1,WANG Wen-huan1(1.school of Energy & Environment Engineering,shanghai University of Electric Power,shanghai 20090,{3. 2.school of Mechanical Engineering,Tongji University,shanghai 200092,{3. 3.Division of Building Science & Technology,City University of Hong Kong,Hong Kong SAR,{3.) school of Energy and Environment Engineering Shanghai University of Electric Power Shanghai 200090 China school of Mechanical Engineering Tongji University Shanghai 200092 China Division of Building Science and Technology City University of Hong Kong Hong Kong Hong Kong

The mathematic model of heat transfer through ventilated double glazing was verified with the measured data,which were from a test chamber equipped with glass face temperature,solar radiation,ambient temperature,and wind speed measurement *** the model validation,the double-skin facade assessment was carried out through simulation with ESP-r software integrating thermal simulation and air low net work *** air flow situation in the air gap was analyzed on the basis of the hourly air velocity simulation data within typical winter week,summer week,spring week and autumn *** differences of chimney effect in different seasons were discussed,and the thermal loads resulted from the ventilated and unventilated double skin facade were presented.

关键词： chimney effect assessment double-skin facade

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Development of a quantitative,cell-based,high-content screening assay for epidermal growth factor receptor modulators

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Acta Pharmacologica Sinica 2007年第10期28卷 1698-1704页

作者： Jue WANG~2,Xin XIE~(2,3.~2 The National Center for Drug Screening,shanghai Institute of Materia Medica,Chinese Academy of Sciences Graduate school of Chinese Academyof Sciences shanghai 201203.china The National Center for Drug Screening Shanghai Institute of Materia Medica Chinese Academy of Sciences Graduate school of Chinese Academy of Sciences Shanghai China

Aim:To develop a robust,cell-based,high-content screening （HCS） assay basedon receptor internalization for the identification of novel modulators of the epi-dermal growth factor receptor （EGFR）.Methods:Agonist-induced receptorinternalization is part of the signaling cascade of ***-taggedepidermal growth factor （EGF） was used to visualize the internalized receptor-ligand *** fluorescent intracellular spots were detected and mea-sured with an ArrayScan HCS *** that can competitively bind toEGFR or interfere with EGFR internalization process would result in a reducednumber and intensity of intracellular fluorescent *** assay was validated,optimized,and applied to a large-scale screening of a library containing 48 000synthetic ***:The competition between fluorescent EGF andunlabeled EGF reveals the IC50of unlabeled EGF is approximately 0.2 nmol/L,which is comparable with other published *** compounds with arelatively high degree of interference with EGFR internalization were *** of the compounds was proven to be agonist of the EGFR since it inducedphosphorylation of the receptor and extracellular signal-regulated protein ki-nase （ERK）.Conclusion:This automated,objective,and easy-to-use assay pro-vided abundant information,quantitative results,and demonstrated the potentialuse of HCS methods in searching membrane receptor modulators.

关键词： high-content screening epidermal growth factor receptor receptor internalization competitive binding

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Wrinkled petals and stamens 1,is required for the morphogenesis of petals and stamens in Lotus japonicus

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Cell Research 2006年第5期16卷 499-506页

作者： Jiang Hua Chen~1 Ji Liang Pang~2 Li Lin Wang~2 Yong Hai Luo~1 Xin Li~3.Xiang Ling Cao~1 Kui Lin~3.Wei Ma~3.Xiao He Hu~1 Da Luo~(1,3. ~1National Laboratory of Plant Molecular Genetics,Institute of Plant Physiology and Ecology,shanghai Institutes for Biological Sciences,and Graduate school of the Chinese Academy of Sciences,Chinese Academy of Sciences,3.0 Fenglin Road,{3.,china ~2school of Life Science,Hangzhou Normal College,16 Xuelin Road,Hangzhou 3.0018,china ~3.2} of Life Science and Biotechnology,shanghai Jiao Tong University,1954 Huashan Road,{3.,china National Laboratory of Plant Molecular Genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences and Graduate School of the Chinese Academy of Sciences Chinese Academy of Sciences Shanghai China school of Life Science Hangzhou Normal College Hangzhou China school of Life Science and Biotechnology Shanghai Jiao Tong University Shanghai China

Although much progress has been made in understanding how floral organ identity is determined during the floraldevelopment,less is known about how floral organ is elaborated in the late floral developmental *** we de-scribe a novel floral mutant,wrinkledt petals and stamensl (wps1),which shows defects in the development of petalsand *** analysis indicates that wps1 mutant is corresponding to a single recessive locus at the long arm ofchromosome *** early development of floral organs in wps1 mutant is similar to that in wild type,and the malfunctionof the mutant commences in late developmental stages,displaying a defect on the appearance of petals and *** mature flower,petals and stamen filaments in the mutant are wrinkled or folded,and the cellular morphology underL1 layer of petals and stamen filaments is *** is found that the expression patterns of floral organ identity genesare not affected in wps1 mutants compared with that of wild type,consistent with the unaltered development of allfloral ***,the identities of epidermal cells in different type of petals are *** histologicalanalysis shows that in wps1 flowers all petals are irregularly folded,and there are knotted structures in the petals,whilethe shape and arrangement of inner cells are malformed and *** on these results,we propose that Wps1acts downstream to the class B floral organ identity genes,and functions to modulate the cellular differentiation duringthe late flower developmental stages.

关键词： Lotus japonicus wps1 mutant ABC model floral organ differentiation

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Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca,Ampullaria crossean

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 702-708页

作者： Yan-Hong LI~(1#) Rui GUO~(3.) Qiu-Yu YIN~1 Ming DING~1 Si-Liang ZHANG~3.Gen-Jun XU~(1,2) Fu-Kun ZHAO~(1,2*) 1 Key Laboratory of Proteomics,Institute of Biochemistry and Cell Biology,shanghai Institutes for Biological Sciences.Graduate school of the Chinese Academy of Sciences,Chinese Academy of Sciences,{3.,china 2 College of Life Science,Zhejiang Sci-Tech University,Hangzhou 3.0018,china 3.Bioengineering Institute,East china University of Science and Technology,shanghai 20023.,china Key Laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Graduate School of the Chinese Academy of Sciences Chinese Academy of SciencesShanghai 200031 China Bioengineering Institute East China University of Science and TechnologyShanghai 200233 China College of Life Science Zhejiang Sci-Tech UniversityHangzhou 310018 China

Two novel endo-β-1,4-glucanases,EG45 and EG27,were isolated from the gastric juice ofmollusca,Ampullaria crossean,by anion exchange,hydrophobic interaction,gel filtration and a secondround of anion exchange *** purified proteins EG45 and EG27 appeared as a single bandon sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa,*** optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for *** optimumtemperature range for EG27 was broad,between 50℃ and 60℃;for EG45 it was 50℃.The analysis on thestability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 evenwhen the incubation time was prolonged to 24 h at 3.℃,whereas EG45 remained relatively stable at *** 85% of the activity of EG27 could be retained upon incubation at 60℃ for 24 ***,less than 10% residual activity of EG45 was detected at 50℃.Among different kinds of substrates,bothenzymes showed a high preference for carboxymethyl ***45,in particular,showed a carboxymethylcellulose hydrolytic activity of 146.5 IU/mg *** enzymes showed low activities to xylan (from oatspelt) and Sigmacell 101,and they were inactive to p-nitrophenyl-β-D-cellobioside,salicin and starch.

关键词： cellulase endo-β-1,4-glucanase Ampullaria crossean purification substrate specificity

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Phorbol-induced surface expression of NR2A subunit homologues in HEK293.cells

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Acta Pharmacologica Sinica 2006年第12期27卷 1580-1585页

作者： Chan-ying ZHENG~2 Xiu-juan YANG~3.Zhan-yan FU~4 Jian-hong LUO~(2,5)2 Department of Neurobiology,Zhejiang University school of Medicine,Hangzhou 3.003.,china 3.Institute of Neuroscience,shanghai Institutes for Biological Sciences,the Chinese Academy of Sciences,{3.,china 4 Department of Physiology and Biophysics,Georgetown University school of Medicine Washington 20057,USA Department of Neurobiology Zhejiang University School of Medicine Hangzhou China Institute of Neuroscience Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai China Department of Physiology and Biophysics Georgetown University School of Medicine Washington USA

Aim:N-methyl-D-aspartate receptors(NMDAR)are heteromeric complexes pri-marily assembled from NR1 and NR2 *** normal conditions,NR2 sub-units assemble into homodimers in the endoplasmic reticulum(ER).Thesehomodimers remain in the ER until they coassemble with NR1 dimers and aretrafficked to the cell ***,it still remains unclear whether functionalhomomeric NMDAR exist in physiological or pathological ***:We transfected GFP-NR2A alone into HEK293.cells,treated the cells with PKCactivator 12-myristate-13.acetate(PMA),and then detected surface NR2A sub-units with a live cell immunostaining *** also used a series of NR2Amutants with a partial deletion of its C-terminus to identify the regions that areinvolved in the PMA-mediated surface expression of NR2A ***:NR2A subunits were expressed on the cell membrane after incubation with PMA(200 nmol/L,3. min),although no functional NMDA channels were detected afterPMA-induced membrane *** with an ER marker alsorevealed that NR2A subunits were exported from the ER after PMA ***,the deletion of amino acids between 1149-13.7 or 13.4-1464 ofNR2A inhibited PMA-induced surface expression ofNR2A ***:First,our data suggests that PMA treatment can induce the surface expression ofhomomeric NR2A ***,this process is probably mediated bythe NR2A C-terminal region between positions 1149 and 1464.

关键词： NMDA receptor NR2A subunit PMA surface expression HEK293.cells

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Generalized two-dimensional correlation spectroscopy——Theory and applications in analytical field

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Science china Chemistry 2004年第3期47卷 257-266页

作者： DOU Xiaoming1, YUAN Bo1, ZHAO Haiying1, YIN Guangzhong2 & Yukihiro Ozaki3.1. Molecular and Photonics Laboratory, Physical Department, shanghai Jiao Tong University, shanghai 20003., china 2. Suzhou No.2 People抯 Hospital, Suzhou 215000, china 3. Chemical Department, school of Science and Technology, Kwansei-Gakuin University, Sanda 669-13.7, Japan Molecular and Photonics Lab. Phys. Dept. Shanghai Jiaotong Univ. Shanghai 200030 China Suzhou No. 2 People's Hospital Suzhou 215000 China Chem. Dept. Sch. of Sci. and Technol. Kwansei-Gakuin Univ. Sanda 669-1337 Japan

Since the theory of generalized two-dimensional (2-D) correlation spectroscopy was proposed, it has been keenly concerned in scientific research and its analytical method has been widely applied in various analytical fields. The mathematical process to construct generalized 2-D correlation spectroscopy and the physical meaning of 2-D correlation spectral map are described, and three examples in the fields of chemical analysis and molecular biology are provided, such as the component analysis of organic solvent, the analysis of biological molecules in the solvent with different pH values and structural analysis of protein. The theory and analytical method of generalized 2-D correlation spectroscopy are also detailedly commented.

关键词： generalized two-dimensional correlation spectroscopy, analytical method.

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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, shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate school of the Chinese Academy of Sciences, {3., china school of Life Science and Biotechnology, 2 shanghai Jiaotong University, {3., china Institute of Neurobiology, Fudan University, shanghai 20043., 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 167 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 293. 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

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Prevention of pathological change and cognitive degeneration of Tg2576 mice by inoculating Aβ_(1-15) vaccine

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Science china(Life Sciences) 2008年第8期51卷 743-750页

作者： HU JinJia1, LI GuoYing2, WANG HuaQiao3. LIN Xian3.& YAO ZhiBin3.1 Department of Anatomy, Medical College of shanghai Jiao Tong University, shanghai 200025, china 2 Department of Anatomy, Preclinical College, Guangdong University of Pharmacy, Guangzhou 510006, china 3.Department of Anatomy, Zhongshan school of Medicine, Sun Yat-sen University, Guangzhou 510080, china Department of Anatomy Medical College of Shanghai Jiao Tong University Shanghai China Department of Anatomy Preclinical College Guangdong University of Pharmacy Guangzhou China Department of Anatomy Zhongshan School of Medicine Sun Yat-sen University Guangzhou China

This study aims to discuss the effect of preventing pathological changes and cognitive degeneration of Tg2576 mice by inoculating the subunit fragment of Aβ ***-two Tg2576 mice were randomly divided into four groups, each having eight mice:Group I, the control group, inoculated with adjuvants;Group II, the Aβ42 group, inoculated with Aβ42 vaccine;Group Ⅲ, the Aβ1―15 group, inoculated with Aβ1―15 vaccine;and Group IV, the Aβ3.―42 group, inoculated with Aβ3.―42 *** titer of the serum anti-body against Aβ42(Group II) was significantly higher than that of the control group(Group I), and a low level of antibodies could be detected in the brain homogenate in the three vaccine-inoculated *** water maze test showed that the Aβ42 group, Aβ1―15 group and Aβ3.―42 group were obviously im-proved compared with the control *** cultured splenocytes sampled from each group were induced by Con A or their respective antigens, and the cell proliferation of the three vaccine-inoculated groups was significantly higher than that of the control *** the Aβ42 group, IL2 and IFN-γ were relatively low and IL4 and IL10 were relatively *** contrast, IL4 and IL10 were much higher in the Aβ1―15 group and IL2 and IFN-γ were much higher in the Aβ3.―42 *** immunohistochemical test showed a large number of senile plaques in the brain cortex and hippocampus of the mice in the con-trol group, no senile plaque in the brain of the Aβ1―15 group and Aβ42 group mice, and a small number of senile plaques in the brain of the Aβ3.―42 group *** results suggest that the subunit fragment of Aβ1―15 vaccine could prevent not only cognitive and behavioral degeneration but also Aβ deposition and formation of senile plaques in Tg2576 mice.

关键词： Alzheimer’s disease β-amyloid peptide vaccine Tg2576 mice senile plaque

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