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The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv

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cell Research 2002年第1期12卷 47-54页

作者： LIANG CHEN, GANG LI, LEI TANG, JUE WANG, XI, RUI GE Laboratory of cellular Immunology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, Chinese Academy of sciences, 320 Yueyang Road, shanghai 200031, china Laboratory of cellular Immunology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors’ laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A -1 and SPC-A1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.

关键词： LC-1 LC-1 ScFv SPC-A-1 pDisplay lung cancer.

Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

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cell Research 2001年第1期11卷 61-67页

作者： MA YINGHUA, JIA HUA HU, WEN JUAN ZHAO, JIAN FEI, YUN YU, XIAO GANG ZHOU, ZHEN TONG MEI, LI HE GUO (institute of biochemistry and cell biology, shanghai institutes for biological sciences, Chinese Academy of sciences, shanghai 200031, china) (shanghai Inst institute of biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China shanghai institute of Physiology Chinese Academy of Sciences Shanghai China

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABAA receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

关键词： γ-aminobutyric acid transporter seizure susceptibility kainic acid electroencephalography transgenic mice

Transgenic mice overexpressingγ-aminobutyric acid transporter subtypeⅠ develop obesity

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cell Research 2000年第4期10卷 303-310页

作者： MA YING HUA JIA HUA HU XIAO GANG ZHOU RUO WANG ZENG ZHEN TONG MEI JIAN FEI LI HE GUO institute of biochemistry and cell biology, shanghai institutes for biological sciences, Chinese Academy of Science, shanghai 200031, china shanghai institute of institute of biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of Science Shanghai China shanghai institute of Physiology Chinese Academy of Science Shanghai China

Transgenic mice ubiquitously overexpressing murine γ aminobutyric acid transporter subtype Ⅰ were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgeinc mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. Tills preliminary finding indicates that the inappropriate level of γ-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.

关键词： γ-aminobutyric acid transporter subtype Ⅰ transgenic mouse obesit€

Structural Analysis of Fibroin Heavy Chain Signal Peptide of Silkworm Bombyx mori

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Acta Biochimica et Biophysica Sinica 2006年第7期38卷 507-513页

作者： Sheng-Peng WANG~(1,2)Ting-Qing GUO~1 Xiu-Yang GUO~1 Jun-Ting HUANG~2 Chang-De LU~(1*) 1 institute of biochemistry and cell biology,shanghai institutes for biological sciences,Chinese Academy of sciences,shanghai 200031,china 2 Sericultural Research institute,Chinese Academy of Agricultural sciences,Zhenjiang 212018,china institute of biochemistry and cell biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China Sericultural Research institute Chinese Academy of Agricultural SciencesZhenjiang 212018 China

To study the minimal length required for the secretion of recombinant proteins and silkproteins in posterior silk gland,the signal peptide(SP)of the fibroin heavy chain(FibH)of silkwormBombyx mori was systematically shortened from the *** effect on the secretion of protein wasobserved using enhanced green fluorescent protein(EGFP)as a *** of EGFP fusion proteinswas examined under fluorescence *** SPs with lengths of 20,18,16 and 12 *** directthe secretion of the reporter,yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP wasshortened to 12a.a., the secretion efficiency was decreased slightly and cleavage occurred within *** *** the FibH SP were used,the secretion of fusion protein was normal and the cleavage sitewas between the Gly-Ser linker and Met,the starting amino acid of EGFP. These findings are applicable forthe expression of foreign proteins in silkworm silk *** cleavage site of the SP is discussed andcompared with the predictive results of the SignalP 3.0 online prediction program.

关键词： signal peptide fibroin heavy chain silkworm Bombyx mori rAcMNPV

Nucleic Acid Binding Activity of Pns6 Encoded by Genome Segment 6 of Rice Ragged Stunt Oryzavirus

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Acta Biochimica et Biophysica Sinica 2004年第7期36卷 457-466页

作者： Chao-Gang SHAO#, Hui-Juan Lü#, Jian-Hua WU, and Zu-Xun GONG* Key Laboratory of Proteomics, institute of biochemistry and cell biology, shanghai institutes of Life sciences, Chinese Academy of sciences, Graduate School of the Chinese Academy of sciences, shanghai 200031, china Key Laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes of Life Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of SciencesShanghai 200031 China

The ORF of genome segment 6 (S6) of rice ragged stunt oryzavirus (RRSV) Philippines isolate was cloned and sequenced based on the S6 sequence of the Thailand isolate. Pns6, the 71 kD product of S6 expressed in E. coli, was demonstrated to be a viral non-structural protein of RRSV by Western blotting. The gel mobility shift assays showed that Pns6 had nucleic acid binding activity. Pns6 could interact with single- and double-stranded forms of DNA and RNA, showing a preference for single-stranded nucleic acid and a slight preference for RRSV ssRNA over the rice ssRNA, as demonstrated by both competition and displacement assays. The binding of Pns6 to nucleic acids is strong and sequence non-specific. By using five truncated derivatives of Pns6, it was found that the basic region from amino acid 201 to 273 of Pns6 was the unique nucleic acid binding domain. Subcellular fractionation of leaf tissues of RRSV-infected rice plants and subsequent Western blotting had shown that Pns6 accumulated predominately in the cytoplasmic membrane fraction. The possible role of RRSV Pns6 in virus replication and assembly is discussed.

关键词： rice ragged stunt oryzavirus (RRSV) nucleic acid binding non-structural protein S6

A Viral Protein Suppresses siRNA-directed Interference in Tobacco Mosaic Virus Infection

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 248-253页

作者： Ming-Min ZHAO De-Rong AN Guang-Hua HUANG~1 Zu-Hua HE~2 Jiang-Ye CHEN~1 College of Plant Protection,North West Science and Technology University of Agriculture and Forestry,Shaanxi 712100,china ~1State Key Laboratory of Molecular biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences. Chinese Academy of sciences,shanghai 200031,china ~2State Key Laboratory of Plant Molecular Genetics.institute of Plant Physiology and Ecology,shanghai institutes for biological sciences.Chinese Academy of sciences,shanghai 200032.china College of Plant Protection North West Science and Technology University of Agriculture and ForestryShaanxi 712100 China State Key Laboratory of Molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China State Key Laboratory of Plant Molecular Genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China

Plant viruses encode suppressors of post-transcriptional gene silencing (PTGS), an adaptivedefense response that limits virus replication and its spread in *** helper component proteinase (HC-Pro) of the potato virus A (PVA,genus Potyvirus) suppresses PTGS of silenced ***,the effectof HC-Pro on siRNA-directed interference in the tobacco mosaic virus (TMV) was examined by using atransient Agrobacterium tumefaciens-based delivery system in intact *** was shown that the interferenceeffect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic andhypersensitive *** the system host,all plants agro-infiltrated with HC-Pro plus siRNA constructsdisplayed the same symptoms as the negative ***,TMV RNA accumulation was found to beabundant in the upper leaves using reverse transcriptase-PCR (RT-PCR) and Northern blot *** thecontrary,plants agro-infiltrated with the siRNA construct alone were free of ***,ourstudy suggests that the transient expression of HC-Pro inhibited the siRNA-directed host defenses againstTMV infection.

关键词： siRNA HC-Pro tobacco mosaic virus suppressor

Developmental expression of Cyclin H and Cdk7 in zebrafish: the essential role of Cyclin H during early embryo development

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cell Research 2007年第2期17卷 163-173页

作者： Qing Yun Liu~1 Zhi Li Wu~1 Wen Jian Lv~1 Yuan Chang Yan~(1,2) Yi Ping Li~1 ~1Lab of Molecular cell biology,institute of biochemistry and cell biology,shanghai institutes for biological Science,Chinese Academy of sciences,Graduate School of the Chinese Academy of sciences,320 Yue Yang Road,shanghai 200031,china ~2Model Organism Division,E-institutes of shanghai University,shanghai 200025,china Lab of Molecular cell biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Science Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences Shanghai China Model Organism Division E-institutes of Shanghai University Shanghai China

Cyclin-dependent kinase 7(Cdk7)is the catalytic subunit of the metazoan Cdk-activating kinase(CAK).Activationof Cdk7 requires its association with a regulatory subunit,Cyclin *** the Cdk7/Cyclin H complex has beenimplicated in the regulation of RNA polymerase in several species,the precise function of their orthologs in zebrafish hasnot been fully *** this study,we isolated from zebrafish blastula embryos two cDNAs encoding the orthologsof human Cyclin H and Cdk7,and examined the role of Cdk7/Cvclin H in zebrafish *** analysisshowed that the zebrafish Cyclin H and Cdk7 cDNAs encode proteins with 65% and 86% identity to the respective hu-man ***-PCR and whole-mount in situ hybridization analyses of their expression in unfertilized eggs,embryosand organs of adult fish suggested that Cyclin H and Cdk7 messages are maternally *** data also showed thattheir transcripts were detected throughout *** of Cyclin H transcripts was found to be ubiquitousduring early stages of development and become restricted to the anterior neural tube,brain,eyes,procreate tissues,liverand heart by 5 days *** of a dominant-negative form of Cyclin H delayed the onset of zygotictranscription in the early embryo,resulting in apoptosis at 5 hours post-fertilization and leading to sever defects in tis-sues normally exhibiting high levels of Cyclin H *** results implicate Cyclin H in the regulation of thetranscriptional machinery during midblastula transition and suggest that it is an essential gene in early zebrafish larvaldevelopment.

关键词： Zebrafish Cyclin H Cdk7 whole-mount in situ hybridization embryonic development midblasmla transition

Molecular Cloning and Functional Analysis of ESGP,an Embryonic Stem cell and Germ cell Specific Protein

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Acta Biochimica et Biophysica Sinica 2005年第12期37卷 7+790-796页

作者： Yan-Mei CHEN Zhong-Wei DU Zhen YAO Laboratory of Molecular and cell biology.institute of biochemistry and cell biology shanghai institute for Biologicol sciences.Chinese Acadcmy of sciences.Graduate School of the Chinese Academy of sciences,shanghai 200031.china Laboratory of Molecular and 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

Several putative Oct-4 downstream genes from mouse embryonic stem (ES) cells have beenidentified using the suppression-subtractive hybridization *** this study,one of the novel genesencoding an ES cell and germ cell specific protein (ESGP) was cloned by rapid amplification of cDNA *** contains 801 bp encoding an 84 amino acid small protein and has no significant homology to anyknown *** is a signal peptide at the N-terminal of ESGP protein as predicted by SeqWeb (GCG)(SeqWeb version 2.0.2,http://***:8080/).The result of immunofluorescence assay suggestedthat ESGP might encode a secretory *** expression pattern of ESGP is consistent with the expres-sion of Oct-4 during embryonic *** protein was detected in fertilized oocyte,from 3.5 daypostcoital (dpc) blastocyst to 17.5 dpc embryo,and was only detected in testis and ovary tissues in ***,ESGP was only expressed in pluripotent cell lines,such as embryonic stem cells,embryonic carcinomacells and embryonic germ cells,but not in their differentiated *** its specific expression,forced expression of ESGP is not indispensable for the effect of Oct-4 on ES cell self-renewal,and does notaffect the differentiation to three germ layers.

关键词： embryonic stem cell and germ cell specific protein(ESGP)gene embryonic stem cell Oct-4 self-renewal

Toosendanin interferes with pore formation of botulinum toxin type A in PC12 cell membrane

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Acta Pharmacologica Sinica 2006年第1期27卷 66-70页

作者： Mu-feng LI Yu-liang SHI institute of Physiology,shanghai institutes for biological sciences,Chinese Academy of sciences,shanghai 200031,china institute of Physiology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

Aim:Botulinum neurotoxins (BoNT) abort the process of neurotransmitter re-lease at presynaptic motor nerve terminals,causing muscle *** abilityof botulinum toxin to produce its effect is dependent on the ability of the lightchain to cleave the SNARE proteins associated with transmitter ***-cation of the light chain protease through the heavy chain-formed channel is apivotal step in the intoxication *** (TSN),a triterpenoid de-rivative extracted from a Chinese traditional medicine,has been demonstrated tobe an effective cure for experimental *** study was designed to explorethe antibotulismic mechanisms of ***:The inside-out single-channel recording patch-clamp technique was used to record the BoNT/A-in-duced currents in the presence and absence of ***:Channel formationwas delayed and the sizes of the channels were reduced in the TSN-treated PC12cell ***:The antibotulismic effect of TSN might occur viainterference with toxin translocation.

关键词： toosendanin botulinum toxin type A ion channel antibotulismic mechanism single channel recording