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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

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Cell Research 2001年第1期11卷 74-80页

作者： YANG ZHI XING GUO YONG AN, ZHI PING ZHU (shanghai Institute of Plant Physiology, Chinese Academy of Sciences, shanghai 200032, China) (department of biology, Henan university, Kaifeng 475001, China) 1shanghai Institute of Plant Physiology Chinese Academy of Sciences Shanghai 200032 China 2department of biology Henan University Kaifeng 475001 China 1shanghai Institute of Plant Physiology Chinese Academy of Sciences Shanghai 200032 China

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

关键词： Rice bicoid-related DNA polar distribution embryogenesis.

The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

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Cell Research 2004年第1期14卷 81-85页

作者： Fei GAO~1, Jing YI~1, Jing Qi YUAN~2, Gui Ying SHI~1, Xue Ming TANG~(1,*) ~1department of Cell biology, shanghai Second Medical university, shanghai 200025, China ~2department of Automation, shanghai Jiaotong university, shanghai 200031, China. Tang XM Shanghai Med Univ 2 Dept Cell Biol Shanghai 200025 Peoples R China

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate, to detect the level fluctuation of reactive oxygen species （ROS） during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry, with TdT mediated dUTP nick end labeling （Tunel） and propidium iodide （PI）, indicated that As2O3 （2μM） could induce apoptosis in NB4 cells prevailingly from G2/M phase, and this efficacy was enhanced upon co-administration of 2, 3-dimethoxy-1, 4-naphthoquinone （DMNQ）（2.5μM） which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells’ susceptibility to apoptosis by As2O3.

关键词： arsenic trioxide apoptosis cell cycle reactive oxygen species (ROS).

LINEAR TRANSPORT EQUATION WITH INDEFINITE COLLISION OPERATOR

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Chinese Annals of Mathematics,Series B 1991年第3期12卷 318-334页

作者：褚轶 department of Applied Mathematics Shanghai Jiao Dong University Shanghai China.

The operator theory on indefinite inner product spaces is used to discuss the halfrange problem of linear transport equation with indefinite collision operator. A counterexample to [1] is given and a relation between ... 详细信息

关键词： collision kerA diagonal lemma assumed hough 五正昭衍二七 maximal

Study on intracellular trafficking of Mac-1 by direct visualization

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Science China(Life Sciences) 2004年第6期47卷 521-529页

作者： YAN Ming1, MAO Jifang2, WEI Yi3, ZHONG Jigen1, YANG Shengsheng2 & XU Renbao1 1. department of Pathophysiology, Second Military Medical university, shanghai 200433, China 2. department of Biochemistry and Molecular biology, Second Military Medical university, shanghai 200433, China 3. Biological Science School, fudan university, shanghai 200433, China Correspondence should be addressed to Yan Ming 1. department of Pathophysiology Second Military Medical University 200433 Shanghai China 2. department of Biochemistry and Molecular biology Second Military Medical University 200433 Shanghai China 3. Biological Science School Fudan University 200433 Shanghai China

Previously, we constructed DNA vectors containing cDNA of Mac-1 subunits (CD11b or CD18b) fused with fluorescence protein (FP). cDNA fragments and the DNA constructs were then transfected into CHO cells (as CHO-Mac-1-FP). The structure and function of Mac-1-FP obtained from the CHO-Mac-1-FP cells are nearly identical to that expressed in wild type leuko- cytes. In the present study, the intracellular trafficking of Mac-1 was visualized directly by moni- toring the fluorescent intensities of YFP-CD18 and PE-conjugated monoclonal antibody against CD11b under a confocal microscope in CHO-Mac-1-FP cells. The results indicate that: (ⅰ) al- though Mac-1 was not detected in the cell membrane at resting state, it had been translocated and clustered into the cell membrane by 1 h and internalized 2 h after PMA stimulation, at which point the fluorescence intensity began to diminish gradually, probably due to partial degradation of Mac-1. The fluorescence of CD18 and CD11b reappeared on the cell membrane 1 h after re-treatment with PMA, suggesting the recycling of non-degraded Mac-1. (ⅱ) The adhesion rate of CHO-Mac-1-FP to magnetic beads coupled ICAM-1 increased within 4 h after their initial in- teraction, accompanied by the clustering of Mac-1-FP. After 8 h,the adhesion rate declined and fluorescence also decreased simultaneously. The pattern of change in fluorescence in CHO-Mac-1-FP cells elicited by ICAM-1 beads was similar to that elicited by PMA, suggesting that endocytosis and degradation of Mac-1 occurred after the interaction with ICAM-1. Thus, we conclude that the intracellular trafficking of Mac-1 after activation is associated with membrane translocation, endocytosis, degradation and recycling. These changes are in parallel with the adhesion of CHO-Mac-1-FP cells with ICAM-1, and may be involved in the adhesion and de- tachment of leukocytes. The detachment of leukocytes may be caused by endocytosis of Mac-1.

关键词： Mac-1 (macrophage differentiation antigen associated with complement three receptor function), PMA, ICAM-1, trafficking.

Effects of free sterols on the fluidity of polar lipids in rice dry embryo

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Chinese Science Bulletin 1999年第10期44卷 933-937页

作者： YANG Ling and SU Weiai biology department of Zhejiang Normal university, Jinhua 321004, China shanghai Institute of Plant Physiology , Chinese Academy of Sciences , shanghai 200032 , China biology department of Zhejiang Normal university Jinhua China shanghai Institute of Plant Physiology Chinese Academy of Sciences Shanghai China

Free sterols from rice dry embryo reduce the onset phase transition temperature and widen the transition range of total polar lipids and phosphatidylglycerol, which increases the membrane fluidity below their phase transition temperature and decreases the fluidity of membranes above their phase transition temperature. And 50% of free sterols result in a complete disappearance of phase transition. The fluidity of other main components of polar lipids is decreased by free sterols above 0℃. The results confirm that free sterols can modulate membrane lipid fluidity.

关键词： free sterol membrane lipids fluidity rice embryo.

Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus *** Strain 10-22

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 519-528页

作者： Lei QIN , Heng-An WANG1 , Zhong-Qin WU, Xiao-Feng ZHANG, Mei-Lei JIN,Zi-Xin deng2, and Guo-Ping ZHAOshanghaiResearch Center of Biotechnology, shanghai Institute for Biological Sciences, Chinese Academy of Sciences,Graduate School of the Chinese Academy of Sciences, shanghai 200233, China 1School of Agriculture and biology, shanghai Jiaotong university, shanghai 201101, China 2School of Life Science and Biotechnology, shanghai Jiaotong university, shanghai 200030, China shanghaiResearch Center of Biotechnology Shanghai Institute for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences School of Agriculture and biology Shanghai Jiaotong University

The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10- 22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane α-helical spanners, belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5–20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m- chlorophenylhydrazone, the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

关键词： multidrug resistance (MDR) gene replacement (knockout) Streptomyces Streptomyces hygroscopicus subsp. yingchengensis strain 10-22

Calculation of the molecular exchanging energy of binary surfactants system on the surface monolayer of aqueous solution

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Science China Chemistry 2007年第4期50卷 468-475页

作者： WANG ZhengWu1 & YI XiZhang2 1 department of Food Sciences & Technology, School of Agriculture and biology, shanghai Jiao Tong university, shanghai 200240, China 2 Institute of Theoretical Chemistry, Shandong university, Jinan 250100, China 1. department of Food Sciences & Technology School of Agriculture and Biology Shanghai Jiao Tong University Shanghai 200240 China 2. Institute of Theoretical Chemistry Shandong University Jinan 250100 China

By using the binary anionic/cationic surfactants system CH3(CH2)nOSO_3/CH3(CH2)nN+(CH3)3 as an ex-ample, the molecular exchanging energy (ε) of adsorption on the surface monolayer of aqueous solu-tion has been studied. ε can be obtained with two methods. One is from the relationship between ε and the molecule interaction parameter (β). This relationship is founded by considering that the adsorption of mixed surfactants on the surface monolayer of solution satisfies the dimensional crystal model condition under which β can be obtained by testing the surface tension of solution. The other is directly from the molecular structure of surfactants with the Lennard-Jones formula. The results for the studied system show that these two methods coincide well.

关键词： surfactant molecule exchanging energy dimensional crystal model

Detection of distribution of copper inside and outside of lysosomes in cultured hepatolenticular degeneration fibroblasts by electron probe X-ray microanalysis

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Hepatobiliary & Pancreatic Diseases International 2003年第2期2卷 586-589页

作者： Wen Liu Jin-Yan Li Ji Jin Ji Zuo the department of Medical Genetics, Scholl of Medicine, fudan university, shanghai 200032, China department of biology, Zhenjiang Medical College, Zhenjiang 212000, China department of Medical Genetics School of Medicine Fudan University Shanghai 200032 China.

OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were clutured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu^(2+) inside lysosomes of HLD cells (14.6±2.1 mmol/kg) and of heterozygote cells (11.6±0.6 mmol/kg) was higher than that of normal cells (4.5±1.2 mmol/kg) (P<0.01). The content of Cu^(2+) outside lysosomes of HLD cells (17.5±4.2 mmol/kg) and of heterozygote cells (12.0±0.9 mmol/kg) was higher than that of normal cells (4.7±1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.

关键词： hepatolenticular degeneration fibroblast lysosome electron probe X-ray microanalysis copper-binding protein