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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 2.0032.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

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

Hyaluronan Promotes Tumor Lymphangiogenesis and Intralymphantic Tumor Growth in Xenografts

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Acta Biochimica et Biophysica Sinica 2005年第9期37卷 601-606页

作者： Li-Xia GUO Ke ZOU Ji-Hang JW Hong XIE Laboratory of Biotherapy,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 Laboratory of Biotherapy 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

Hyaluronan (HA),a high molecular weight glycosaminoglycan in the extracellular matrix,hasbeen implicated in the promotion of malignant phenotypes,including tumor ***,little isknown about the effect of HA on tumor-associated *** this study,mouse hepatocellularcarcinoma Hca-F cells combined with or without HA were injected subcutaneously into C3H/Hej mice,thenangiogenesis and lymphangiogenesis of implanted tumors were examined by immunostaining for platelet-endothelial cell adhesion molecule-1 and lymphatic vascular endothelial hyaluronan receptor-1 ***,we found HA promotes tumor lymphangiogenesis and the occurrence of intratumoral lymphaticvessels,but has little effect on tumor ***,HA also promotes intralymphatic tumorgrowth,although it is not sufficient to potentiate lymphatic *** results suggest that HA,which is elevated in most malignant tumor stroma,may also play a role in tumor progression by promotinglymphangiogenesis.

关键词： hyaluronan tumor lymphangiogenesis intralymphatic tumor growth

Expression of Monomeric Insulin Precursor in Pichia pastoris and Its Conversion into Monomeric B27 Lys Destripeptide Insulin by Tryptic Hydrolysis

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 234-240页

作者： Jin-Guo DING~(1,3) Jian FEI~2 Da-Fu CUI~1 You-Shang ZHANG~1 ~1Institute of Biochemistry and Cell Biology,and ~2Model Organism Research Center,shanghai Institutes for Biologicol sciences, chinese academy of sciences,shanghai 200031,china ~3graduate 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 Shanghai 200031 China graduate school of the chinese academy of sciences shanghai 200031 China Model Organism Research Center Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from itsprecursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient *** monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium *** by a combination of hydrophobic,size-exclusion,and ion-exchange *** purifiedMIP was converted,by tryptic hydrolysis,to B27 Lys DTrI,which was then purified by ion-exchangechromatography to homogeneity as assessed by native gel electrophoresis,HPLC,amino acid composition,and electrospray mass-spectrometric analysis.B27 Lys DTrI exhibited superior monomeric properties insize-exclusion *** yield of MIP was 200 mg per liter of culture,and the overall yield ofpurified B27 Lys DTrI from the crude MIP was 70%.The in vivo biological activity of B27 Lys DTrI asdetermined by the mouse convulsion assay was 21 U/mg,identical to that obtained by semisynthesis.

关键词： monomeric B27 Lys destripeptide insulin tryptic hydrolysis Pichia pastoris

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

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Acta Biochimica et Biophysica Sinica 2005年第11期37卷 737-742页

作者： Jin-Peng GAO Zhen-Hua YONG Feng ZHANG Kang-Cheng RUAN Chun-He XU Gen-Yun CHEN Institute of Plant Physiology & Ecology.shanghai Institutes for Biological sciences,chinese academy of sciences,shanghai 2.0032.china Laboratory of Proteomics,Institute of Biochemistry and Cell Biology,shanghai Institutes for Biological sciences,chinese academy of sciences,shanghai 200031,china College of sciences,Zhejiang University.Hangzhou 310029,china graduate school of the chinese academy of sciences,shanghai 2.0032.china Institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China graduate school of the chinese academy of sciencesshanghai 2.0032 China College of sciences Zhejiang UniversityHangzhou 310029 China Laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China

To determine the contribution of charged amino acids to binding with the photosystem Ⅱcomplex(PSⅡ),the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N-succinimidyl propionate(NSP)or glycine methyl ester(GME)in the presence of a water-soluble carbodiimide,*** on isoelectric point shift,4-10 and 10-14 amino groups were modified in the presence of2 and 4 mM NSP,***,3-4 carboxyl groups were modified by reaction with *** of negatively charged carboxyl groups with GME did not alter the binding activity of theextrinsic 18 kDa ***,the NSP-modified 18 kDa protein,in which the positively charged aminogroups had been modified to uncharged methyl esters,failed to bind with the PSⅡ membrane in the presenceof the extrinsic 23 kDa *** defect can not be attributed to structural or conformational alterationsimposed by chemical modification,as the fluorescence and circular dichroism spectra among native,GME-and NSP-modified extrinsic 18 kDa proteins were ***,we have concluded that the positive chargesof lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSⅡ membranes in thepresence of the extrinsic 23 kDa ***,it was found that the negative charges of carboxylgroups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSⅡmembranes.

关键词： extrinsic 18 kDa protein circular dichroism electrostatic interaction chemical modification

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

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 673-680页

作者： Chun-Hui HOU, Jian HUANG, and Ruo-Lan QIAN* State Key Laboratory of Molecular Biology, 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 State Key Laboratory of Molecular 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

The developmental control of the human ε-globin gene expression is mediated by transcrip- tional regulatory elements in the 5’ flanking DNA of this gene. A previously identified negative regulatory element (–3028 to –2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at –3004 bp, –2975 bp and –2948 bp were characterized. Electrophoresis mobility shift assay (EMSA) showed that the putative NF-κB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (–177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at –2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-globin gene proximal promoter. However, the mutation of GATA site at –2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by ε-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at –2948 bp on SV40 promoter was not affected by the mutation of the putative NF-κB site, whereas it could be abolished by the mutation of GATA site at –2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by ε-globin gene proximal promoter. Those results suggested that ε- NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.

关键词： ε-globin gene regulatory element NF-κB GATA

An Effective Method for Raising Antisera Against β-defensins: Double-copy Protein Expression of mBin1b in E. coli

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 571-576页

作者： Li-Qing XIAO, Ai-Hua LIU, and Yong-Lian ZHANG State Key Laboratory of Molecular Biology, 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 State Key Laboratory of Molecular 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

Bin1b is a rat epididymis specific β-defensin which may have fertility related functions in addition to its antimicrobial activity. β-defensins are cysteine-rich cationic antimicrobial peptides that have their important implications in innate and adaptive immunity. Though considerable numbers of new β-defensins have been discovered, few corresponding antibodies have been reported. The small peptide with special structure and antimicrobial nature of β-defensins make them very difficult to express in prokaryotic system. Here we adopted a double-copy protein expression scheme based on which not only the mBin1b protein was successfully expressed but also the immunity of the antigen was enhanced. The validity of the antisera was verified by using Western blotting and immunohistochemical analyses. It will be a useful tool for deeply investigating the roles of Bin1b and also provide a simple but effective method in raising antisera against other members of the β-defensin gene family.

关键词： Bin1b epididymis β-defensin double-copy protein polyclonal antiserum

Transformations and non-degenerate maps

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Science china Mathematics 2005年第Z1期48卷 195-205页

作者： LI Baokui & WANG Yuefei Institute of Mathematics, academy of Mathematics and System sciences, chinese academy of sciences, Beijing 100080, china graduate school, chinese academy of sciences, Beijing 100080, china Institute of Mathematics Academy of Mathematics and System Sciences Chinese Academy of Sciences Beijing China graduate school Chinese Academy of Sciences Beijing China

We shall prove the equivalences of a non-degenerate circle-preserving map and a M(o)bius transformation in ^Rn, of a non-degenerate geodesic-preserving map and an isometry in Hn of a non-degenerate line-preserving map and an affine transformation in Rn. That a map is non-degenerate means that the image of the whole space under the map is not a circle, or geodesic or line respectively. These results hold without either injective or surjective, or even continuous assumptions, which are new and of a fundamental nature in geometry.

关键词： M(o)bius transformation, circle-preserving map, non-degenerate map.

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