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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

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

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

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

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

维普期刊数据库

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

Mechanisms of microRNA-mediated gene regulation

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Science china(Life sciences. 2009年第12期52卷 1111-1116页

作者： ZHAO Shuang & LIU Mo-Fang· state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences. 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 the Chinese Academy of Sciences Shanghai 200031 China

microRNAs (miRNAs) are identified as a class of non-protein regulators and a new source for broad control of gene expression in eukaryotes. The past years have witnessed substantial progress in understanding miRNA functions and mechanisms, although a few controversies remain. Various hypotheses and models have been suggested for the mechanisms of miRNA repression, including translational inhibition at the level of initiation or elongation, rapid degradation of the nascent peptide, mRNA degradation, and mRNA sequestration into P bodies (processing bodies) and SGs (stress granules) for degradation or/and storage. Recently, some noncanonical miRNA regulation, such as miRNA activation and de-repression of miRNA inhibition, have been uncovered. This review discusses some recent advances about how miRNAs regulate their targets and various modes of miRNA function.

关键词： miRNA repression activation de-repression processing bodies stress granules

维普期刊数据库

Effects of Huangqi (Hex) on Inducing cell Differentiation and cell Death in K562 and HEL cells

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Acta Biochimica et Biophysica Sinica 2004年第3期36卷 211-217页

作者： Xiao-Dong CHENG1, Chun-Hui HOU1, Xue-Jun ZHANG2, Heng-Yue XIE1, Wei-Ying ZHOU1, Lei YANG2, Shu-Bing ZHANG1, and Ruo-Lan QIAN1 state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences. the chinese academy of sciences. 320 Yueyang Road, shanghai 200031, china laboratory of molecular cell biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences. the chinese academy of sciences.320 Yueyang Road shanghai 200031,china the chinese academy of sciences. 320 Yueyang Road, shanghai 200031, china state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China laboratory of molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Huangqi (Astragalus membranaceus), a traditional chinese medicine, has been used to ameliorate side effects of cancer chemotherapy in china. However, little is known about its molecular mechanisms. Here we show that induction of K562 or HEL cells with 1.5 mg/ml of Huangqi (Hex) (Components extracted from Huangqi) for 3–5 d results in the expression of β-globin gene in both cell lines and leads to terminal differentiation. Moreover, the apoptosis in HEL cells can be induced by increasing concentration of Huangqi (Hex) to 4.5 mg/ml for 3–5 d. Upregulation of Apaf-1, caspase-3 and acetylcholinesterase (AChE) in HEL cells may play a crucial role in the process of apoptosis. The prospect of inducing expression of adult (β) globin gene and apoptosis selectively in cancer cells is obviously attractive from a therapeutic point of view.

关键词： cell death and differentiation Huangqi (Hex) human erythroleukemia cell lines

Overexpression of a novel gene , Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor

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cell Research 2001年第4期11卷 285-291页

作者： WANG JI WU, JIA RUI WU state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai Research Center of Life Science, shanghai institutes for biological sciences. chinese academy of sciences. 320 Yue- Yang Road, shanghai 1state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Research Center of Life Science Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 200031 China 1state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Research Center of Life Science Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 200031 China

MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutan (mcm10-1) of budding yeast shows a growth arrest at 37 C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 C. Interestingly, this mcm10-1 suppressor undergoes cell Cycle arrest at 14 C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of trans- formation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.

关键词： Saccharomyces cerevisiae DNA replication Mcm10 Cms1 cell cycle.