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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 {3. and cell biology, shanghai institutes for Biological sciences, chinese academy of sciences, Graduate School of the chinese academy of sciences, shanghai 20003., 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 {3. biology, shanghai institutes for Biological sciences, chinese academy of sciences, Graduate School of the chinese academy of sciences, shanghai 20003., 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 (–3.28 to –2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at –3.04 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

Identification of γ1 subunit of GABAA receptor in rat testis

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cell Research 2002年第1期12卷 33-37页

作者： JING HUA HU, YUAN CHANG YAN Lab of Molecular cell biology, institute of {3. and cell biology, shanghai institutes for Biological sciences, chinese academy of sciences, shanghai {13., china 1Lab of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 1Lab of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

The isoform type ofγsubunits of GABAA receptor is a molecular determinant of its pharmacological characteristics. At present, the existence of GABAA receptor in mammalian sperm is still a controversy. By using degenerate primers designed according to highly conserved region in all threeγ(γ1, γ2 andγ3. subunits cloned in rat brain, we performed reverse transcription polymerase chain reaction (RT-PCR) to examine the expression pattern of -γsubunits of GABAA receptor in rat testis. Only one 3.0 bp fragment was obtained from RT-PCR in rat testis and sequencing results showed that it representedγ1 subunit, but notγ2 orγ3.subunit. Using the cloned fragment as probe, a 3.8 kb transcript which in size as same asγ1 ubunit in rat brain was detected in rat testis mRNA by performing Northern blot assay. Furthermore, results of in situ hybridization assay confirmed thatγ1 subunit was expressed in round spermatids and spermatozoa, maybe also in secondary spermatocyte. These evidences proved that 71 subunit of GABAA receptor is exclusively expressed in rat testis and this feature may be the structural basis of the specific function of GABAA receptors in sperm acrosome reaction.

关键词： GABAA receptor rat testis γ1 subunit.

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 {3. for Biological sciences,chinese academy of sciences,shanghai 20003.,china Laboratory of Proteomics,institute of biochemistry and {13. biology,shanghai {3. for Biological sciences,chinese academy of sciences,shanghai 20003.,china College of sciences,Zhejiang University.Hangzhou 3.0029,china Graduate School of the chinese academy of sciences,shanghai 20003.,china institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China Graduate School of the chinese academy of sciences{3. 20003. 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

Directional migration of leukocytes: their pathological roles in inflammation and strategies for development of anti-inflammatory therapies

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cell Research 2001年第2期11卷 85-88页

作者： GENG JIAN Guo institute of biochemistry and cell {3., shanghai institutes for Biological sciences, chinese academy of sciences, 3.0 Yue Yang Road, shanghai 20003., china institute of biochemistry and cell {3. Chinese Academy of Sciences Shanghai. jggeng@***

Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury the constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial present tation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines.

关键词： Directional cell migration leukocytes inflammation therapy.

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 {3. biology,shanghai institutes for Biological sciences,chinese academy of sciences,shanghai 20003.,{13. 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

维普期刊数据库

Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

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cell Research 2004年第3期14卷 227-233页

作者： Yan REN, Jia Rui WU Laboratory of Molecular cell biology, institute of {3. and cell biology, shanghai institutes for Biological sciences, chinese academy of sciences, 3.0 Yueyang Road, shanghai {13., china. Laboratory of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chkl checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern II was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly dete

关键词： intra-S-phase checkpoint DNA replication DNA damage tripchlorolide.

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 {3. biology, shanghai institutes for Biological sciences,chinese academy of sciences, shanghai 20003., {13. 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

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

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cell Research 2001年第2期11卷 101-106页

作者： GUO BEN CHANG, YONG HUA XU institute of biochemistry and cell {3., shanghai institutes for Biological sciences, chinese academy of sciences, 3.0 Yue-yang Road, shanghai 20003., china 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-yang Road Shanghai 200031 China 1institute of biochemistry and cell {3. Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-yang Road Shanghai 200031 China

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains nuclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in BcL-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13.acetate), a PKC activator, suppressed nail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

关键词： trail Jurkat T cells apoptosis Bcl-2 PEA PKC Bis.