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Interaction between Plasminogen Activator Inhibitor Type-2.and Pre-mRNA Processing Factor 8

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Acta Biochimica et Biophysica Sinica 2004年第9期36卷 623-628页

作者： Jing FAN, Yu-Qing ZHANG, Ping LI, Chang TONG, Li TAN, and Yun-Song ZHU*Department of molecular Genetics, shanghai Medical School of Fudan University, {2. {2. of molecular medicine.ministry of education, shanghai 2.0032. china Department of molecular Genetics Shanghai Medical School ofFudan UniversityKey Laboratory of Molecular MedicineMinistry of EducationShanghai 200032China

The plasminogen activator inhibitor type-2.(PAI-2. dependent apoptosis protection is due to the 33 amino acids fragment located between helix C and D of PAI-2. this fragment may interact with some unknown intracellular proteins. In this study we used the fragment between helix C and D of PAI-2.as a bait to perform a yeast two-hybrid screen using a cDNA library constructed with HeLa cells during apoptosis, and retrieved a clone encoding 94 amino acid residues of C-terminus of pre-mRNA processing factor 8 (PRPF8). Co-immunoprecipitation experiments confirmed that PAI-2.could interact with PRPF8 in vivo. PAI-2.could bind PRPF8 C-terminal in both the inside and outside of nuclear. These results suggested that the interaction between these two proteins might not be involved in the apoptosis process.

关键词： plasminogen activator inhibitor type-2 pre-mRNA processing factor 8 (PRPF8) yeast two-hybrid system U5

Interaction of Plasminogen Activator Inhibitor-2.and Proteasome Subunit, Beta Type 1

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Acta Biochimica et Biophysica Sinica 2004年第1期36卷 42-46页

作者： Jing FAN, Yu-Qing ZHANG, Ping LI, Min HOU, Li TAN, Xia WANG, and Yun-Song ZHU( Department of molecular Genetics, shanghai Medical School of Fudan University, {2. {2. of molecular medicine. ministry of education, shanghai 2.0032. china ) Department of molecular Genetics Shanghai Medical School of Fudan University Key Laboratory of Molecular Medicine Ministry of Education Shanghai 200032 China

The apoptosis protection by plasminogen activator inhibitor-2.PAI-2. is dependent on a 33 amino acid fragment between helix C and D of PAI-2.which is probably due to the interaction of PAI-2.with unknown intracellular proteins. In this study, we used the fragment between helix C and D of PAI-2.as bait to screen a HeLa cell cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone encoding 2.1 amino acids of proteasome (prosome, macropain) subunit, beta type l(PSMβ1) which plays important roles in NF-jjjjjjjjB activation. GST-pulldown experiments confirmed the interaction between PAI-2.and PSMβ1 in vitro. These data suggest that the antiapoptosis activity of PAI-2.is probably related to its interation with PSMβ1.

关键词： plasminogen activator inhibitor type-2 proteasome (prosome, macropain) subunit, beta type 1 (PSMβ1) yeast two-hybrid system ubiquitin

A Novel Gene Delivery System Targeting Urokinase Receptor

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Acta Biochimica et Biophysica Sinica 2004年第7期36卷 485-491页

作者： Xing-Hui SUN, Li TAN, Chun-Yang LI, Chang TONG, Jin FAN, Ping LI, and Yun-Song ZHU*Department of molecular Genetics, key {2. of molecular medicine.of ministry education,shanghai Medical College of Fudan University, shanghai 2.0032. china Department of molecular Genetics Key Laboratory of Molecular Medicine of Ministry Education Shanghai Medical College of Fudan University Shanghai 200032 China

Recombinant proteins that combine different functions required for cell targeting and intra- cellular delivery of DNA present an attractive approach for the development of nonviral gene delivery vectors. Here, we described a novel protein termed ATF-lys10 which facilitated cell-specific gene transfer via receptor-mediated endocytosis. ATF-lys10 was composed of the amino-terminal fragment of urokinase and ten lysines at the carboxyl terminus. Bacterially expressed ATF-lys10 protein existed in soluble form, and had antigenicity of human urokinase. Purified ATF-lys10 specifically bound to uPAR-expressing cells and formed protein-DNA complexes with plasmid pGL3-control. After neutralization of excess negative charge with poly-L-lysine, these complexes served as a specific gene delivery vector for uPAR-expressing cells. Lyso- somotropic compounds, such as chloroquine, drastically increased the ATF-lys10 mediated gene delivery efficiency. Our results suggest that the recombinant protein ATF-lys10 with the properties of DNA binding and tumor cell targeting represents a promising method for gene transfer and expression in tumor cells.

关键词： amino-terminal fragment urokinase gene transfer endocytosis urokinase receptor

Construction and Characterization of Novel Staphylokinase Variants with Antiplatelet Aggregation Activity and Reduced Immunogenecity

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Acta Biochimica et Biophysica Sinica 2004年第5期36卷 336-342页

作者： Hua-Bo SU, Yu-Gao ZHANG, Jin-Tian HE, Wei MO, Yan-Ling ZHANG, Xian-Mei TAO, and Hou-Yan SONG* Department of molecular Genetics, shanghai Medical School, Fudan University, {2. {2. of molecular medicine. ministry of education, shanghai 2.0032. china Department of molecular Genetics Shanghai Medical School Fudan University Key Laboratory of Molecular Medicine Ministry of Education

To develop target thrombolytic agents with fibrinolytic activity, antiplatelet aggregation activity and reduced immunogenicity, two staphylokinase variants containing Arg-Gly-Asp (RGD) motif were constructed. Gene expression was induced in E. coli JF112. and the variants, designated DGR and RL1, were purified with gel filtration and ion-exchange chromatography and the purity was over 95%. The fibrinolytic activity and kinetic constants of the two variants were comparable to those of recombinant wild-type staphylokinase. Both the variants can inhibit the platelet aggregation at a final concentration of 2.μM. The titers of antibodies against variants were much lower than those against recombinant staphylokinase in guinea pigs, which indicated that the immunogenicity of the variants was greatly reduced. These results confirm that it is possible to design and produce a bifunctional protein that possesses fibrinolytic and antiplatelet aggrega- tion activities.

关键词： staphylokinase RGD antiplatelet aggregation immunogenicity

Leukemia,an effective model for chemical biology and target therapy

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Acta Pharmacologica Sinica 2007年第9期28卷 1316-1324页

作者： Guo-qiang CHEN~(2.3,4),Li-shun WANG~2.Ying-li WU~2.Yun YU~2.2.Department of Pathophysiology,key laboratory of Cell Differentiation and Apoptosis of Chinese {2. of {2.,shanghai Jiao TongUniversity School of medicine.SJTU-SM,formerly shanghai Second Medical University),shanghai 2.002.,china,~3 Institute of HealthScience,SJTU-SM/shanghai Institutes for Biological Sciences,shanghai 2.002.,china Department of Pathophysiology Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education Shanghai Jiao Tong University School of Medicine (SJTU-SM formerly Shanghai Second Medical University) Shanghai China Institute of Health Science SJTU-SM/Shanghai Institutes for Biological Sciences Shanghai China

The rapid rise of chemical biology aimed at studying signaling networks for basiccellular activities using specific,active small molecules as probes has greatlyaccelerated research on pathological mechanisms and target therapy of *** research is especially important for malignant tumors such as leukemia,aheterogeneous group of hematopoietic malignancies that occurs *** use of a chemical approach combined with genetic manipulation,great progresshas been achieved over the past few decades on the biological,molecular andcytogenetic aspects of leukemia,and in its diagnosis and *** particular,discoveries of the clinical effectiveness of all-trans retinoic acid and arsenictrioxide in the treatment of acute promyelocytic leukemia and the kinase inhibitorsImatinib and Dasatinib in the treatment of chronic myelogenous leukemia not onlymake target therapy of leukemia a reality,but also push mechanisms of leukemo-genesis and leukemic cell activities *** review will outline advances inchemical biology that help our understanding of the molecular mechanisms of celldifferentiation and apoptosis induction and target therapy of leukemia.

关键词： leukemia chemical biology target therapy retinoic acid arsenic trioxide differentia tion apoptosis

A pH-and temperature-sensitive macrocyclic graft copolymer composed of PEO ring and multi-poly(2.(dimethylamino) ethyl methacrylate) lateral chains

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Science china Chemistry 2010年第8期53卷 1653-1662页

作者： PANG XinChang,JING RongKuan,PAN MuGang & HUANG JunLian key laboratory of {2. Engineering of Polymers,{2. of education Department of Macro{2. Science,Fudan University,shanghai 2.0433,china 1. key laboratory of {2. Engineering of Polymers Ministry of Education Department of Macromolecular Science Fudan University Shanghai 200433 China

A novel method for the synthesis of macrocyclic graft copolymers was developed through combination of anionic ring-opening polymerization (AROP) and atom transfer radical polymerization (ATRP).A linear α,ω-dihydroxyl poly(ethylene oxide) with pendant acetal protected hydroxyl groups (l-poly(EO-co-EEGE)) was prepared first by the anionic copolymerization of ethylene oxide (EO) and ethoxyethyl glycidyl ether (EEGE).Then l-poly(EO-co-EEGE) was *** crude cyclized product containing the linear byproduct was hydrolyzed and purified by being treated with α-*** pure cyclic copolymer [c-poly(EO-co-Gly)] was esterified by reaction with 2.bromoisobutyryl bromide,and then used as ATRP macroinitiators to initiate polymerization of 2.(dimethylamino) ethyl methacrylate (DMAEMA),and a series of pH-and temperature-sensitive macrocyclic graft copolymers composed of a hydrophilic PEO as the ring and PDMAEMA as side chains (c-PEO-g-PDMAEMA) were *** behavior of pH-and temperature-sensitive macrocyclic copolymers was studied in aqueous solution by fluorescence and dynamic light scattering (DLS).The critical micellization pH values of macrocyclic graft copolymers and their corresponding linear graft copolymers (l-PEO-g-PDMAEMA) were *** the same conditions,the cyclic graft copolymer with the shorter side chains gave the higher critical micellization pH *** c-PEO-g-PDMAEMA showed the lower critical micellization pH value than the corresponding *** average hydrodynamic diameters (Dh) of the micelles were measured by DLS with the variation of the aqueous solution pH value and temperature.

关键词： macrocyclic PEO anionic ring-opening polymerization ATRP PDMAEMA pH-and temperature-sensitive

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Phylogenetic relationships of host insects of Cordyceps sinensis inferred from mitochondrial Cytochrome b sequences

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Progress in Natural Science:Materials International 2007年第7期17卷 789-797页

作者： Cheng Zhou~(1,2.*) Geng Yang~1 Liang Honghui~1 Yang Xiaoling~1 Li Shan~1 Zhu Yunguo~1 Guo Guangpu~1 Zhou Tongshui~2.Chen Jiakuan~2.(1.School of Life Science and Technology,Tongji University,shanghai 2.0092.china 2.{2. of education key laboratory for Biodiversity Science and Ecological Engineering,Institute of Biodiversity Science,Fudan University,shanghai 2.0433,china) School of Life Science and Technology Tongji University Shanghai 200092 China ministry of education {2. Lab. for Biodiversity Science and Ecological Engineering Institute of Biodiversity Science Fudan University Shanghai 200433 China

This study used the sequence of the mitochondrial Cytochrome b(Cytb)to estimate phylogenetic relationships among host Hepialidae insects of Cordyceps *** DNA of host insect was extracted from the dead larva head part of 18 cordyceps populations and 2.species of Hepialus,and the Cytb fragment of host insect was amplified with PCR *** nucleotide sequence alignments and their homologous sequences of 2. species host Hepialidae insects of Cordyceps sinensis were obtained from GenBank and were used to construct phylogenetic trees based on neighbor-joining *** results showed that genus Bipectilus diverged earlier than genus Hepialus and *** host insects of Cordyceps sinensis have multitudinous species with different morphological characteristics and geographical *** interspecific genetic differentiations are obvious in ***,the genus Hepialus might be considered as polyphyletic *** sequences have abundant variations among the host insects of Cordyceps sinensis on spe- cific and generic *** divergence rate of Cytb sequences among the species in Hepialus ranged from 0.2.% to 9.2.%,except that Hepialus pratensis and Hepialus jinshaensis have the same *** sequence can be used for species identification of host insects of Cordyceps sinensis,but further confirmation in more host insect species is *** obtain the Cytb sequence of host insect by ampli- fying DNA extracted from the head part of dead larva in cordyceps turns out to be an effective and accurate approach,which will be useful for studies on phylogeny and genetic structure of host insects of cordyceps populations,especially for analyzing relationships between C. sinensis and its host insects.

关键词： mitochondrial Cytochrome b Cordyceps sinensis Hepialus phylogeny

Spectroscopic Analysis on the Effect of Temperature on Kunitz Domain 1 of Human Tissue Factor Pathway Inhibitor-2

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Acta Biochimica et Biophysica Sinica 2007年第6期39卷 406-412页

作者： Chenqi ZHANG~(1#), Desheng KONG~(2.), Xingang LIU~1, Xiaomin YAN~1, Linsen DAI~(1*), and Duan MA~(2.) 1 Center of Analysis and Measurement, Fudan University, shanghai 2.0433, china {2., Fudan University, shanghai 2.0032. china Center of Analysis and Measurement Fudan UniversityShanghai 200433China keylaboratory of {2. {2. Ministry of EducationFudan UniversityShanghai 200032China

The conformation of Kunitz domain 1 of human tissue factor pathway inhibitor-2.(hTFPI-2.KD1) has been studied by Fourier transform infrared spectroscopy, circular dichroism, and Raman spectroscopy. It was found that hTFPI-2.KD1 contained approximately 17% s-helices, 2.% β-strands, 46% random coils, 13% β-turns, and two kinds of disulfide bonds (ggg and tgt) at 2.℃. The detailed conformational changes of the heated protein observed by Fourier transform infrared spectroscopy, circular dichroism and Raman spectroscopy revealed that hTFPI-2.KD1 was thermally stable. However, KD1 could form an intermediate form at high temperature, then return to its normal conformation when the temperature was lowered. Activity assays also showed that hTFPI-2.KD1 was able to keep its inhibitory activity on plasmin after being heated to 80℃ for 5min.

关键词： hTFPI-2.KD1 conformation disulfide bonds thermal stability structure and function

Transcriptional Regulation of Urokinase Receptor in High-(95D) and Low-metastatic (95C) Human Lung Cancer Cells

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 405-411页

作者： Chun-Yang LI, Li TAN, Guo-Jing ZHANG, Ping LI, Chang TONG, Jing FAN, and Yun-Song ZHU* key laboratory, the {2. {2., Department of Biochemistry and molecular Biology, shanghai Medical College,Fudan University, shanghai 2.0032. china key laboratory the Education Ministry Department of Biochemistry and Molecular Biology Shanghai Medical College Fudan UniversityShanghai 200032 China

To study the transcriptional regulation of urokinase receptor (uPAR) in high- (95D) and low-metastatic (95C) human lung cancer cells, we performed PCR to amplify 2.38 bp uPAR promoter from 95C and 95D cells. According to the results of sequencing, five different bases are found in uPAR promoter between 95C and 95D cells. The results of luciferase activity assay showed that these differences have no significant effect on the uPAR promoter activity. Based on a normal uPAR promoter, progressive truncated mutants were constructed. The transient transfection/reporter assay showed that the promoter region from –136 to +9 may interact with relevant nuclear factors, which result in different levels of uPAR expression between 95C and 95D cells.

关键词： uPAR promoter transcriptional regulation