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Effect of leukemia inhibitory factor on embryonic stem cell differentiation:implications for supporting neuronal differentiation

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Acta Pharmacologica Sinica 2006年第1期27卷 80-90页

作者： Zhao HE~(2,3) Jing-jing LI~(2,3) Chang-hong ZHEN~(3,4) Lin-ying FENG~3 Xiao-yan DING~(2,5)2 Laboratory of Molecular and Cell Biology and Laboratory of Stem Cell Biology,Institute of Biochemistry and Cell Biology:shanghai Institutes for Biological sciences,chinese academy of sciences,shanghai 200031,china.3 shanghai Institute of Materia Medica.chinese academy of sciences.shanghai 201203,china.4 graduate school of the chinese academy of sciences,shanghai 200031,china. Laboratory of Molecular and Cell Biology and Laboratory of Stem Cell Biology Institute of Biochemistry and Cell Biology shanghai Institutes for Biological sciences Chinese Academy of Sciences Shanghai China shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai China graduate school of the chinese academy of sciences Shanghai China

Aim:Leukemia inhibitory factor (LIF),a pleiotropic cytokine,has been used ex-tensively in the maintenance of mouse embryonic stem cell *** thiscurrent work,we examined the effect of the LIF signaling pathway in embryonicstem (ES) cell differentiation to a neural ***:In the presence of LIF(1000U/mL),the production of neuronal cells derived from embryoid bodies (EB)was tested under various culture *** of the LIF pathway wasexamined with specific *** effects of cell apoptosis and proliferationon neural differentiation were *** cell differentiation into three-germlayers was ***:Under various culture conditions,neuronal differ-entiation was increased in the presence of *** the LIF-activated STAT3signaling pathway with specific inhibitors abolished the neuronal differentiationof ES cells,whereas inhibition of the LIF-activated MEK signaling pathway im-paired the differentiation of ES cells toward a glial *** suppressed cellapoptosis and promoted cell proliferation during ES cell *** inhib-ited the differentiation of ES cells to both mesoderm and extraembryonic endo-derm fates,but enhanced the determination of neural ***:These results suggest that LIF plays a positive role during the differentiation ofES cells into neuronal cells.

关键词： leukemia inhibitory factor embryonic stem cells neural fate embryoid bodies

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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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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

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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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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

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Novel Tumor-associated Antigen of Hepatocellular Carcinoma Defined by Monoclonal Antibody E4-65

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Acta Biochimica et Biophysica Sinica 2007年第5期39卷 359-365页

作者： Ke ZOU~(1,2), Jihang JU~(1,2), and Hong XIE~~(1*)1 Institute of Biochemist13, and Cell Biology, shanghai Institutes for Biological sciences, chinese academy of sciences, shanghai 200031, china. 2 graduate school of the chinese academy of sciences Beijing 100049, china Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China graduate school of the chinese academy of sciencesBeijing 100049 China

A monoclonal antibody, E4-65, produced by immunizing mice with SMMC-7721 cells, a human hepatocellular carcinoma (HCC) cell line, was used to identify and characterize an unreported HCC-associated antigen. Indirect immunofluorescence studies showed that E4-65 antibody reacted with five out of eight HCC cell lines, but not with 10 non-HCC tumor cell lines or a normal liver cell line. Using immunohistochemical examination, E4-65 antigen was detected on the cell membranes and in the cytoplasm of human liver tumor tissues, but was not found in most other tumors, or normal adult or fetal tissues, except for a weakly positive reaction in tissues of the digestive system. Western blot analysis showed that E4-65 antibody bound to a 45 kDa protein in the human HCC cell line and tissue lysates. Enzyme treatment and lectin blotting did not detect the carbohydrate chain in E4-65 antigen. This HCC-associated protein represents a potentially useful target for diagnoses and immunotherapy of human HCC.

关键词： hepatocellular carcinoma immunohistochemistry monoclonal antibody neoplasm antigen

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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

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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

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Cloning and Characterization of a New Isoform of Mouse Interleukin-18

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Acta Biochimica et Biophysica Sinica 2005年第12期37卷 826-834页

作者： Yong-Jie YANG Zhao-Yuan WANG Song-Hua CHEN Xi-Rui GEInstitute of Biochemistry and Cell Biology,shanghai Inatitute for Bological sciences,chinese academy of sciences graduate school of the chinese academy of Science.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

Interleukin-18 (IL-18) is a novel proinflammatory cytokine with potent interferon (IFN)-yinducing activity that plays an important biological role in the enhancement of the activity of natural killer cellsand cytotoxic T *** this study,we have identified a novel short form of IL-18 in mouse,***-18s might be an alternative splicing variant of IL-18 and its cDNA contains a 57 bp *** IL-18,IL-18s is also widely expressed in mouse tissues,It was suggested that IL-18s mighthave a caspase-1-dependent mechanism for maturation and secretion similar to that of IL-18:when transfectedin COS-7 cells,pro-IL-18s (22 kDa) could be detected,and the mature IL-18s (16 kDa) could also be detectedwhen combined with *** observed that recombinant mouse IL-18s did not show any IL-18-likefunction,and IL-18s could enhance the ability of IL-18 to increase IFN-γ production by approximately 40%in mouse *** effect was observed primarily at relative low concentrations of *** IL-18s might regulate the activity of IL-18 in the physiological conditions.

关键词： IL-18 isoform IL-18s IFN-γ caspase-1 factor Xa

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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 200032,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 200032,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 200032 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

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