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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.china 4 graduate school of the chinese academy of sciences.chinese academy of sciences,shanghai 201203.china 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[3H]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[3H]PAF as a substrate,and proved to be enzymatically ***-PLA2inhibitor SB435495 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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Comparative studies of huperzine A,donepezil,and rivastigmine on brain acetylcholine,dopamine,norepinephrine,and 5-hydroxytryptamine lev-els in freely-moving rats

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Acta Pharmacologica Sinica 2006年第9期27卷 1127-1136页

作者： Yan-qi LIANG Xi-can TANG~2 State Key Laboratory of Drug Research,shanghai Institute of Materia Medica,shanghai Instituties for Biological sciences,graduate school of the chinese academy of sciences,chinese academy of sciences,shanghai 201203.china State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Instituties for Biological Sciences Graduate School of the Chinese Academy of Sciences Chinese Academy of Sciences Shanghai China

Aim:To assess the effects of cholinesterase inhibitors huperzine A,donepeziland rivastigmine on cerebral neurotransmitters in the cortex and hippocampus infreely-moving ***:Double-probe cerebral microdialysis and HPLC withelectrochemical detection were used to detect ***:Ourresults showed that huperzine A(0.***.0.5,and 0.75μmol/kg,po)dose-depen-dently elevated extracellular acetylcholine(ACh)levels in the medial prefrontalcortex(mPFC)and *** administration of donepezil(5.4μmol/kg)orrivastigmine(1μmol/kg)also elicited significant increases in ACh in the mPFC *** time course of cortical acetylcholinesterase(ACHE)inhibitionwith the 3 inhibitors minored the increases of ACh at the same *** markedelevation of ACh after oral administration of huperzine A(0.5μmol/kg)anddonepezil(5.4μmol/kg)was associated with a significantly increased release ofdopamine(DA)in the mPFC or *** of the 3 inhibitors affectednorepinephrine(NE)and 5-hydroxytryptamine(5-HT)levels in the mPFC *** effects of huperzine A and rivastigmine did not depend on theroute of administration,but donepezil was less efficacious by the oral route thanby ip *** ability of huperzine A to increase ACh levels was unchangedwhen tests were performed after multiple oral administration of the drug at 0.5μmol/kg,once per day for 30 ***:The present findings showed that,inmolar terms,huperzine A had similar potency on increasing mPFC ACh and DAlevels as compared to the 11-and 2-fold dosages of donepezil and rivastigmine,respectively,and had longer lasting effects after oral dosing.

关键词： huperzine A donepezil rivastigmine acetylcholine dopamine norepinephrine 5-hydroxy tryptamine Alzheimer’s disease rats

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Development of a complex scintillation proximity assay for highthroughput screening of PPAR7 modulators

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Acta Pharmacologica Sinica 2005年第3期26卷 339-344页

作者： Bin WU Jie GAO Ming-wei WANG~2 The National Center for Drug Screening,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,chinese academy of sciences graduate school of chinese academy of sciences,shanghai 201203.china The National Center for Drug Screening Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences graduate school of chinese academy of sciences Shanghai China

Aim:To develop a complex high-throughput screening （HTS） assay based onscintillation proximity assay （SPA） technology for identification of novel peroxi-some proliferator-activated receptor gamma （PPARγ） ***:Full-length PPARγ and retinoid X receptor alpha （RXRα）,biotinylated PPAR responseelement （PPRE）,[3H]BRL49653 and streptavidin-coated FlashPlate or microbeadwere used to develop an HTS assay based on SPA ***‘ABCDE’method was validated against conventional hydroxyapatite （HA） assay and ap-plied to large-scale screening of 16 000 synthetic compounds and natural ***:（1） IC50values of positive control compounds （BRL49653 andtroglitazone） obtained from the‘ABCDE’method and HA assay were compa-rable and consistent with those reported elsewhere;（2） Approximately 178compounds,showing more than 70% competitive inhibition on BRL49653 bind-ing to PPARγ,were identified initially by the‘ABCDE’method （microbead）;（3）Secondary screening using FlashPlate and cross-reactivity studies with RARα,β,γ and RXRα,β,γ confirmed that 12 compounds possessed specific PPARγ bind-ing properties including 2 with IC50values less than 0.5 μmol/L and novel chemi-cal ***:The‘ABCDE’method using either FlashPlate ormicrobead,is a highly efficient,automatable,and robust tool to screen potentialPPARγ modulators in HTS *** application may be expanded to othernuclear receptors that form heterodimers upon activation.

关键词： peroxisome proliferator-activated receptor gamma retinoid X receptor alpha scintillation proximity assay high-throughput screening

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Cloning and Analyzing of Xenopus Mespo Promoter in Retinoic Acid Regulated Mespo Expression

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Acta Biochimica et Biophysica Sinica 2006年第11期38卷 759-764页

作者： Jin-Hu WANG Xiao-Yan DING* Institute of Biochemistry and Cell Biology,shanghai Institutes for Biological sciences,chinese academy of sciences,graduate school of the chinese academy of sciences,shanghai 200031,china 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

During vertebrate embryogenesis,presomitic mesoderm cells enter a segmental program togenerate somite,a process termed ***,a member of the bHLH transcription factor family,plays important roles in this ***,how Mespo expression is regulated remains *** this question,we isolated a genomic DNA sequence containing 4317 bp of Mespo 5’ flanking regionin *** assays show that this upstream sequence has transcription *** assayshows that this genomic contig is sufficient to recapitulate the dynamic stage-and tissue-specific expressionpattern of endogenous Mespo from the gastrula to the tailbud *** further mapped a 326 bp DNAsequence responding to retinoic acid *** results shed light on how Mespo expression is regulated,and suggest that retinoic acid signaling pathways play roles in somitogenesis through regulating Mespo.

关键词： bHLH paraxial presomitic segmental tailbud Xenopus laevis

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Apoptosis initiated by carbon tetrachloride in mitochondria of rat primary cultured hepatocytes

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Acta Pharmacologica Sinica 2005年第8期26卷 969-975页

作者： Yan CAI~(1,2), Li-kun GONG~(1,2), Xin-ming QI~(1,2), Xiang-hong LI~1, Jin REN~(1,3) ~1State Key Laboratory of Drug Researeh, shanghai Institute of Materia Medica, shanghai Institutes for Biological sciences, chinese academy of sciences, shanghai 201203, china ~2graduate school of the chinese academy of sciences, shanghai 201203, china State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China graduate school of the chinese academy of sciences Shanghai China

Aim: To investigate the mitochondria-initiated apoptosis pathway involved in Carbon tetrachloride （CCl4） hepatotoxicity in vitro. Methods: Several cytotoxic-ity endpoints, including WST-8 metabolism, lactate dehydrogenase leakage and morphological changes, were examined. The 5,5'-dithio-bis（2-nitrobenzoic acid） reaction was used to measure reduced glutathione level, and the malondialdehyde level was determined using the thiobarbituric acid assay. The release of cyto-chrome c and Bcl-XLwas detected by Western blot. Caspase-3 activity was mea sured using the fluorogenic substrate Ac-DEVD-AMC. DNA fragmentation wasused to evaluate cell apoptosis. Results: A time-and dose-dependent decrease in cellular glutathione content was observed, along with a concomitant increase in malondialdehyde levels following the application of CCl4. Caspase 3 activity was stimulated at all doses of CCl4, with the most significant activation at 3 mmol/L. Cytochrome c was released obviously after CCl4treatment. A time-dependent decrease in BcI-XLexpression was observed. DNA fragmentation results revealed apoptosis and necrosis following CCl4treatment. Conclusion: Oxidative damage is one of the essential mechanisms of CCl4hepatotoxicity, which triggers apoptosis via the mitochondria-initiated pathway.

关键词： carbon tetrachloride hepatocytes mitochondria caspase 3 cytochrome c

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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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Prevention of lipopolysaccharide-induced injury by 3,5-dicaffeoylquinic acid in endothelial cells

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Acta Pharmacologica Sinica 2007年第8期28卷 1143-1148页

作者： Ruo-peng ZHA~(1,2) Wei XU~(1,2) Wen-yi WANG~(1,2) Li DONG~1 Yi-ping WANG~(1,3) ~1State Key Laboratory of Drug Research,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,chinese academy of Seiences,shanghai 201203.china ~2graduate school of the chinese academy of sciences,shanghai 201203.china State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China graduate school of the chinese academy of sciences Shanghai China

Aim:To investigate the effect of 3,5-dicaffeoylquinic acid（3,5-diCQA）onlipopolysaccharide（LPS）-induced injury in human dermal microvascular endothe-lial cells（HMEC-I）.Methods:The anti-oxidant effect was detected using themalondialdehyde（MDA）assay in a rat liver microsome model of lipid *** viability was analyzed using the 3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide *** lipid peroxide injury was measured bylactate dehydrogenase（LDH）*** cells were detected by flowcytometry,and confirmed by DNA fragmentation ***-3 activitywas measured using a specific assay *** level of intracellular reactive oxygenspecies（ROS）was determined by flow cytometry with a 2,7-dichlorodihydro-fluorescein diacetate fluorescence ***:The exposure of microsomesto ascorbate-Fe2+resulted in lipoperoxidation according to an increase in the levelof *** formation decreased in a dose-dependent manner on treatmentwith 5,10,or 50μmol/L 3,*** with LPS for 16 h resulted in a 60%decrease in cell viability and an increase in LDH release from 47.6% to 61.5%.DNA laddering was observed by agarose gel *** level ofapoptotic cells peaked at 27% after treatment with LPS for 12 *** treat-ment with LPS for 12 h,intracellular ROS and caspase-3 activity ***-treatment with 3,5-diCQA at 5,10,or 50μmol/L for 1 h attenuated LPS-mediatedendothelial cell *** anti-apoptotic action of 3,5-diCQA was partiallydependent on its capacity for anti-oxidation and the suppression of ***:3,5-diCQA displays anti-oxidative and anti-apoptotic activ-ity in HMEC-1 due to scavenging of intracellular ROS induced by LPS,and thesuppression of caspase-3 activity.

关键词： 3 5-dicaffeoylquinic acid lipopolysaccharide human dermal microvascular endo-thelial cells reactive oxygen species apoptosis caspase-3

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In Vitro Reassembly of Tobacco Ribulose-1,5-bisphosphate Carboxylase/Oxygenase from Fully Denatured Subunits

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 737-745页

作者： Zhen-Hua YONG Gen-Yun CHEN* Jiao-Nai SHI Da-Quan XU Institute of Plant Physiology and Ecology,shanghai Institutes for Biological sciences,chinese academy of sciences,and graduate school of the chinese academy of sciences,shanghai 200032,china Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences and Graduate School of the Chinese Academy of SciencesShanghai 200032 China

It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase （Rubisco） from fully denatured subunits in vitro in higher plant,because large subunit of fullydenatured Rubisco is liable to precipitate when the denaturant is removed by common methods of directdilution and one-step *** our experiment,the problem of precipitation was resolved by an improvedgradual dialysis method,which gradually decreased the concentration of ***,fully denaturedRubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was *** restored activity of reassembled Rubisco was approximately 8% of natural *** of reassembled Rubisco increased greatly when heat shock protein 70 was present in the *** and Mg2+ were unnecessary for in vitro reassembly of Rubisco,and Mg2+ inhibited the *** reassembly was weakened when ATP,Mg2+ and K+ existed together in the reassembly process.

关键词： Rubisco reassembly gradual dialysis chaperonin 60 Hsp70

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The cDNA and Genomic DNA Organization of a Novel Toxin SHT-I from Spider Ornithoctonus huwena

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 656-660页

作者： Peng QIAO, Xiao-Pan ZUO, Zhi-Fang CHAI, and Yong-Hua JI* Institute of Physiology, shanghai Institutes for Biological sciences, chinese academy of sciences, graduate school of the chinese academy of sciences, shanghai 200031, china Institute of Physiology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China

In present study the full-length cDNA of a novel toxin from the venom gland of spider Ornithoctonus huwena, named as SHT-I, has been cloned using the strategy of rapid amplification of cDNA ends, and then the whole genomic sequence of SHT-I (Selenocosmia huwena toxin-I) was determined using sequence-specific primers synthesized based on the acquired 3’ and 5’ ends of SHT-I cDNA sequence. It is unexpectedly found that intron was lacking in the genomic sequence of SHT-I. The result might evoke an interesting question whether the gene code of inhibitor cystine-knot peptides from spider venom is distinct from that of those known toxic peptides of scorpion and cone snail.

关键词： Ornithoctonus huwena spider cDNA genomic DNA peptide toxin ICK motif

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Neuroprotective effects of cyclooxygenase-2 inhibitor celecoxib against toxicity of LPS-stimulated macrophages toward motor neurons

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Acta Pharmacologica Sinica 2005年第8期26卷 952-958页

作者： Yong HUANG, Jing LIU, Li-zhen WANG, Wei-yu ZHANG, Xing-zu ZHU~1 Department of Pharmacology, shanghai Institute of Materia Medica, shanghai Institutes for Biological sciences, chinese academy of sciences, graduate school of chinese academy of sciences, shanghai 201203, china Department of Pharmacology Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of Chinese Academy of Sciences Shanghai China

Aim: To establish an in vitro injured motor neuronal model and investigate the neuroprotective effects and possible mechanism of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on this model. Methods: After macrophages were stimulated with lipopolysaccharide (LPS)+interferon-γ(IFN-γ) in the pre-sence or absence of celecoxib for 24 h, the cell-free supernatant of LPS-stimulated macrophages was transferred to the culture of NSC34 cells. Viability of NSC34 cells was assessed by MTT assay after a further 24 h and 72 h incubation. After macrophages were stimulated by LPS+IFN-γ for 12 h or 24h, the release of pros-taglandin E2(PGE2), nitric oxide (NO), reactive oxygen species (ROS), tumor necro-sis factor α(TNF-α) and interleukin-1β (IL-1β) from macrophages was measured by radioimmunoassay, Griess assay, fluorescence assay and enzyme-linked immunosorbent assay, respectively. The mRNA levels of COX-2, inducible nitric oxide synthase (iNOS), TNF-α and IL-1β in macrophages were determined by reverse transcription-polymerase chain reaction after macrophages were stimu-lated for 6 h and 12 h. Results: The supernatant of LPS-stimulated mouse mac-rophages induced the death of NSC34 cells and celecoxib protected the NSC34cells against this toxicity. The LPS-induced increases in the release of PGE2, NO,TNF-α and IL-1β from macrophages were attenuated by pre-treatment with celecoxib. However, celecoxib showed no effect on the ROS levels upregulated by LPS+IFN-γin the macrophage supernatant. The mRNA levels of COX-2, iNOS, TNF-α and IL-1β were increased in LPS-activated macrophages and, except COX-2, reduced by pre-treatment with celecoxib. Conclusion: An in vitro injured motor neuronal model was established by using the toxicity of LPS-stimulated mouse macrophages toward motor neuronal NSC34 cells. In this model, celecoxib exerted neuroprotective effects on motor neurons via an inhibition of the neurotoxic secretions from activated macrophages.

关键词： amyotrophic lateral sclerosis celecoxib macrophage NSC34 cell lipopolysaccharide cyclooxygenase-2 prostaglandin E2 nitric oxide reactive oxygen species

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