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Functional analysis of schistosomes EF-hand domain-containing tegument proteins

chinese Science Bulletin 2007年第15期52卷 2100-2107页

作者： YU FuDong1,2,3,KANG Bin1,3,LI YuanYuan3,4 & LI YiXue1,3 1 Bioinformatics Center,key laboratory of systems biology,shanghai institutes for biological sciences,chinese academy of Sci-ences,shanghai 200031,china 2 Graduate University of chinese academy of sciences,shanghai 200031,china 3 shanghai Center for Bioinformation Technology,shanghai 200235,china 4 key laboratory of systems biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china Bioinformatics Center Key Laboratory of Systems Biology Shanghai Institutesfor Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

Schistosomes cause schistosomiasis disease which severely threatens human health. Little is known about the functions of EF-hand domain containing schistosomes tegument proteins other than as an-tigens. More possible functions of these tegument proteins were investigated with in silico analyses including protein-protein functional interaction,site-specific variation and glycosylation modification. The analysis results suggested that schistosomes could actively modulate host immune responses for its own favor through functional interactions with host proteins with immunomodulatory function,and passively regulate host immune responses through sequence variation under positive selection and glycosylating the recognition sites of host immune attack. In addition,the analysis of the C-terminal domain of these tegument proteins indicated that they could assist schistosomes in escaping host immune attacks through inhibiting chemotaxis and non-complement fixing antibody (IgG4) responses. In summary,our results suggested that these tegument antigen proteins could assist schistosomes in escaping and modulating host immune responses for self-protection during the process of host-para-site interaction.

关键词：寄生虫血吸虫皮蛋白质健康

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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 Sciences

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 Sciences

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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Regulation of mammalian pre-mRNA splicing

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Science china(Life sciences) 2009年第3期52卷 253-260页

作者： HUI JingYi State key laboratory of Molecular biology,Institute of Biochemistry and Cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china 1. State key laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

In eukaryotes,most protein-coding genes contain introns which are removed by precursor messenger RNA(pre-mRNA) *** splicing is a process by which multiple messenger RNAs(mRNAs) are generated from a single pre-mRNA,resulting in functionally distinct *** genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene ***-regulation of splicing causes a wide range of human *** review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA *** also discusses emerging directions in the field of alternative splicing.

关键词： pre-mRNA splicing alternative splicing splicing regulation signal transduction microRNA

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Cleavage of Supercoiled Circular Double-stranded DNA Induced by a Eukaryotic Cambialistic Superoxide Dismutase from Cinnamomum camphora

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Acta Biochimica et Biophysica Sinica 2004年第9期36卷 609-617页

作者： Bao-Zhong WANG, Xu-Bin WEI&, and Wang-Yi LIU* State key laboratory of Molecular biology, Institute of Biochemistry and Cell biology, shanghai institutes for biological sciences,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

A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity re- quires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA- cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

关键词： cambialistic SOD Fenton-type reaction hydroxyl radical relaxed circular duplex DNA supercoiled DNA cleavage

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Cinnamomin——a Versatile Type Ⅱ Ribosome-inactivating Protein

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Acta Biochimica et Biophysica Sinica 2004年第3期36卷 169-176页

作者： Hong XU and Wang-Yi LIU State key laboratory of Molecular biology, the Institute of Biochemistry and Cell biology, shanghai institutes for biological sciences, the chinese academy of sciences, 320 Yueyang Road, shanghai 200031, china State key laboratory of Molecular biology the Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.

关键词： cinnamomin cinphorin ribosome-inactivating protein

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Detection of Tumor Marker CA125 in Ovarian Carcinoma Using Quantum Dots

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 681-686页

作者： Hui-Zhi WANG, Hai-Yan WANG, Ru-Qiang LIANG, and Kang-Cheng RUAN* key laboratory of Proteomics, 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 key laboratory of Proteomics Institute of Biochemistry and Cell BiologyShanghai Institutes for Biological SciencesChinese Academy of SciencesGraduate School of the Chinese Academy of SciencesShanghai 200031China

Semiconductor quantum dots (QDs) offer several advantages over organic dyes in fluores- cence-imaging applications, such as higher quantum yield, exceptional photostability, and a narrow, tunable, and symmetric emission spectrum. To explore whether QDs could specifically and effectively label tumor markers and be used in immunohistochemistry as a novel type of fluorescent probe, we used quantum dots with maximum emission wavelength 605 nm (QD605) to detect the ovarian carcinoma marker CA125 in specimens of different types (fixed cells, tissue sections, and xenograft piece). Additionally, we compared the photostability of QD signals with that of a conventional organic dye, FITC. All labeling signals of QDs were found to be more specific and brighter than those of FITC. Moreover, the QDs exhibited exceptional photostability during continuous illumination for 1 h by a high-intensity laser (Ar laser power 100 mW) at 488 nm, while the FITC signals faded very quickly and became undetectable after 24 min of illumination. These results indicate that QD-based probes can offer substantial advantages over existing fluorophores in many applications, and can be used effectively in immunohistochemistry as a novel class of fluorescent probes.

关键词： quantum dots detection of tumor marker CA125 ovarian carcinoma

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Rational redesign of inhibitors of furin/kexin processing proteases by electrostatic mutations

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Acta Pharmacologica Sinica 2004年第12期25卷 146-152页

作者： Xiao-hui CAI, Qing ZHANG, Da-fu DING2 key laboratory of Proteomics, 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 key laboratory of Proteomics 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

AIM: To model the three-dimensional structure and investigatethe interaction mechanism ofthe proprotein convertase furin/kexin and their inhibitors (eglin c mutants). METHODS: The three-dimensional complex structures of furin/ kexin with its inhibitors, eglin c mutants, were generated by modeller program using the newly published X-ray crystallographical structures of mouse furin and yeast kexin as templates. The electrostatic interaction energy of each complex was calculated and the results were compared with the experimentally determined inhibition con- stants to find the correlation between them. RESULTS: High quality models of furin/kexin-eglin c mutants were obtained and used for calculation of the electrostatic interaction energies between the proteases and their inhibitors. The calculated electrostaticenergies of interaction showeda linear correlation to the experimental inhibition constants. CONCLUSION: The modeled structures give good explanations of the specificity of eglin c mutants to furin/ kexin. The electrostatic interactions play important roles in inhibitory activity of eglin c mutants to furin/kexin. The results presented here provided quantitative structural and functional information concerning the role of the charge- charge interactions in the binding of furin/kexin and their inhibitors.

关键词： furin kexin eglin c drug design electrostatic mutation

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MicroRNA in cell differentiation and development

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Science china(Life sciences) 2009年第3期52卷 205-211页

作者： SHI Yi & JIN YouXin State key laboratory of Molecular biology,shanghai Institute of Biochemistry and Cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china State key laboratory of Molecular biology Shanghai Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

The regulation of gene expression by microRNAs(miRNAs) is a recently discovered pattern of gene regulation in animals and *** have been implicated in various aspects of animal development and cell differentiation,such as early embryonic development,neuronal development,muscle development,and lymphocyte development,by the analysis of genetic deletions of individual miRNAs in *** studies show that miRNAs are key regulators in animal development and are potential causes of human *** we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

关键词： miRNA embryonic development neuronal development muscle development lymphocyte development

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Multiple Signaling Pathways Control Tbx6 Expression during Xenopus Myogenesis

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 390-396页

作者： Pan-Feng FANG, Rui-Ying HU, Xing-Yue HE, and Xiao-Yan DING* laboratory of Molecular and 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, shanghai 200031, china laboratory of Molecular and 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

Tbx6 is critical for somite specification and myogenesis initiation. It has been shown that Activin/Nodal, VegT/Nodal, FGF, and BMP signaling pathways are involved early in specifying mesoderm or later in patterning mesoderm, and Xnot plays roles in setting up the boundary between notochord and paraxial mesoderm. In this study, we introduce the dominant negative form of above genes into embryos to evaluate if they are responsible for regulating Tbx6 expression. The results show that: (1) Activin/Nodal and VegT/Nodal signals are necessary for both initiation and maintenance of Tbx6 expression, and Nodal is sufficient to induce ectopic Tbx6 expression; (2) FGF signal is necessary for the initiation and maintenance of Tbx6, but it is not sufficient to induce Tbx6 expression; (3) BMP is also necessary for the expression of Tbx6, and the induction of Tbx6 expression by BMP is dose dependent; (4) Xnot has no effect on the expression of Tbx6. Our results suggest that several signaling pathways are involved in regulating Tbx6 expression, and pave the route to reveal the molecular mechanism of initiating myogenesis.

关键词： Tbx6 FGF BMP Activin/Nodal VegT/Nodal

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