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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

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

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

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Expression of Hepatitis C Virus E2 Ectodomain in *** and Its Application in the Detection of Anti-E2 Antibodies in Human Sera

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Acta Biochimica et Biophysica Sinica 2004年第1期36卷 57-63页

作者： Jing liu, Xin-Xin zhang, Shen-Ying zhang, Min LU, Yu-Ying KONG, Yuan WANG, and Guang-Di LI ( 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 2 Department of Infectious Diseases, Ruijin Hospital, Second Medical University of shanghai, shanghai 200025, 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 Chin

The second envelope glycoprotein （E2） of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment （aa 385-730） with a four-amino-acid mutation （aa 568-571 PCNI to RVTS） was expressed as hexa-histidine-tagged full length protein [E2N730（m）] in E. coli and purified to over 85% purity. Purified E2N730（m） was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2specific antigenicity. Rabbit antiserum raised against E2N730（m） recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730（m） was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previousl

关键词： hepatitis C virus envelope protein E2 expression and purification Escherichia coll

Gene expression profile favoring phenotypic reversion: a clue for mechanism of tumor suppression by NF-IL6 3'UTR

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cell Research 2003年第6期13卷 509-514页

作者： DING GAN liu, QIU HONG JIANG, YUN Yl WEI, LI SUN, BEI BEI FU, Fu KUN ZHAO, QIONG ZHOU ’ state key laboratory of molecular biology and key laboratory of Proteomics, institute of biochemistryand cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai200031, china. E-mail: dgliu@*** state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China. dgliu@***

Transfection of cDNA in 3’untranslated region of human nuclear factor for interleukin-6 (NF-IL6 3’UTR) induced tumor suppression in a human hepatoma cell line. cDNA array analysis was used to reveal changes in gene expression profile leading to tumor suppression The results indicate that this suppression was not due to activation of dsRNA-dependent protein kinase, nor to inactivation of oncogenes; rather, all the changes in expression of known genes, induced by NF-IL6 3’UTR cDNA may be ascribed to the suppression of cellular malignancy. Therefore, our results imply that this 3’untranslated region may have played role of a regulator of gene expression profile.

关键词： cDNA array NF-IL6 hepatoma 3 ’UTR tumor suppression.

Systemic delivery of full-length C/EBPβ / liposome complex suppresses growth of human colon cancer in nude mice

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cell Research 2005年第10期 24-30页

作者： Li SUN, Bei Bei FU, Ding Gan 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

C/EBPβ (CCAAT/enhancer-binding protein β) is an important transcription factor involved in cellular proliferation and differentiation. Overexpression of the full-length C/EBPβ protein results in cellular growth arrest and apoptosis. Using a nonviral liposome as carrier, we delivered the full-length C/EBPβ expression plasmid, pCN, into nude mice bearing CW-2 human colon cancer tumors via tail vein. Southern blots revealed that the major organs and tumors were transfected. Experimental gene therapy showed that a strong suppression of tumor growth was observed in the pCN- treated mice, and such suppression was due to the overexpression of C/EBPβ, leading to the increased apoptosis in tumors of pCN-treated mice. No apparent toxic effects of pCN/liposome complex were observed in the animals. Thus, C/EBPβ has tumor suppression effect in vivo and may be used in gene therapy for cancers.

关键词： tumor supression C/EBPβ colon cancer apoptosis.

Studies on the Temperature Effect on Bacteriorhodopsin of Purple and Blue Membrane by Fluorescence and Absorption Spectroscopy

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 691-696页

作者： lan-Ying CHENG~(1,2) Yue zhang~3 Shi-Gui liu~1 Kun-Sheng HU~3 Kang-Cheng RUAN~2* 1 College of Life Science,Sichuan University,Chengdu 610064,china 2 state key laboratory of molecular biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china 3 institute of Biophysics,Academia Sinica,Beijing 100101,china state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of *** temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp *** binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to *** results suggest that significant structural difference existed between bRband *** was also found that both kinds of bR are highly thermal *** maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm *** transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high *** phenomenon has not been reported before.

关键词： bacteriorhodopsin purple membrane blue membrane fluorescence and absorption spectroscopy

Fibroblast growth factor 7 acts as a modulator of nociception

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Fibroblast growth factor 7 acts as a modulator of nociceptio...