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Inducement of Specific CTLs by Antigen-Peptides from Human Leukemia Cells and Their Cytotoxicity to Leukemia Cells

Journal of Huazhong University of Science and Technology(Medical Sciences) 2002年第4期22卷 265-269页

作者：冯作化张桂梅黄波李东王洪涛 departmentofbiochemistryandmolecularbiology

To investigate the inducement of cytotoxic T lym phocytes(CTL s) by antigen peptides m ixture from different leukemia cells and the cross- reaction of the m ixtures from different cell lines,antigen peptides m ixtures were prepared from different leukemia cell lines respectively and then bound with Hsp70 in *** and proliferation of PBMC were observed after stim u- lation with different Hsp70 - peptide *** ratio of CD8+ in proliferative cells was ana- lyzed by flow *** cytotoxicity of the activated PBMC to different target cells was as- *** results showed that the antigen peptides from different leukemia cell lines,bound with Hsp70 ,could activate PBMC effectively,and stimulate the activated PBMC to *** proliferative PBMC had specific cytotoxicity to corresponding leukem ia ***8+ cells,account- ing for a high proportion in proliferative cells,had a specific cytotoxicity to leukemia cells from which antigen peptides were prepared,suggesting that these CD8+ cells were CTL s specific to leukemia *** s activated by Hut78- peptides or Molt4 - peptides had a significantly stronger cytotoxicity to Hut78cells,Molt4 cells and Jurkat cells than that of CTL s activated by HL - 6 0 - peptides(P<0 .0 5 ) .And the cytotoxicity of CTL s activated by Hut78/ Molt4 - peptides to Jurkat cells was significantly stronger than that of CTL s activated by either Hut78- peptides or Molt4 - peptides alone(P<0 .0 5 ) .It is concluded that antigen peptides m ixtures from leukem ia cells can induce specific antitumor CTL *** exists cross- reactivity among antigen peptides m ixtures from different cell lines of the sam e type leukemia and more cross- reactive antigen peptides could be obtained from m ore cell lines,suggesting that antigen peptides m ixture with broad antigenic spectrum could be prepared by using multiple leukemia cell lines.

关键词： leukem ia antigen peptides mixture cross reaction

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Expression of Brassica napus L. γ-Glutamylcysteine Synthetase and Low-and High-Affinity Sulfate Transporters in Response to Excess Cadmium

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Journal of Integrative Plant Biology 2005年第2期47卷 243-250页

作者： XinSUN Xue-MeiSUN Zhi-MinYANG Shao-QiongLI JinWANG Song-HuaWANG departmentofbiochemistryandmolecularbiology CollegeofLifeSciencesNanjingAgriculturalUniversityNanjing210095China

In both the roots and leaves ofBrassica napus L. cv. Youyan No. 8 under treatment with 30 μmol/L Cd, massive production of non-protein thiols (NPT; mainly containing glutathione (GSH) and phytochelatins (PCs)) was induced, together with an increase in γ-glutamylcysteine synthetase (γ-ECS)mRNA transcripts. Because γ-ECS is the key enzyme catalyzing the first step in GSH biosynthesis, which, in turn, is converted to PCs, the Cd-induced increase in γ-ECS expression may be responsible for the observed increase in the production of NPT. Using a quantitative reverse transcription polymerase chain reaction (RT-PCR) approach, the expression of genes encoding a putative low-affinity sulfate transporter (LAST) and a putative high-affinity sulfate transporter (HAST) was determined at the transcriptional level. The RT-PCR analysis of relative transcript amounts indicates that the LAST gene in B. napus leaves showed a constitutive expression, which was hardly affected by Cd treatment. However, treatment with 30 μmol/L Cd for 2 or 3 d induced a marked increase in the expression of LAST in roots. Transcriptional expression of the HAST gene occurred in roots, but not in leaves. The expression of HAST only in the roots suggests that it has a specific function in sulfate uptake from soil and that the putative LAST may be responsible for the transport of sulfate from the roots to the shoots, as well as for the uptake of sulfate from soil. These results indicate that changes in transcriptional expression for sulfate transporters were required for the increased demand for sulfate during Cd stress.

关键词： Brassica napus L. cadmium γ-glutamylcysteine synthetase non-protein thiol sulfate transporters

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Cloning and activity analysis of in vitro expression of plant NAD-IDH genes

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Chinese Science Bulletin 2004年第4期49卷 328-336页

作者： CHENDefu CHENXiwen departmentofbiochemistryandmolecularbiology NankaiUniversityTianjin300071China

The plant NAD-dependent isocitrate dehydro-genase (NAD-IDH) is an important multifunctional enzyme. The cDNAs encoding three different subunits of the NAD-IDH were cloned successfully from leaves of Arabidop-sis thaliana ecotype Columbia gl1 and three lines (Shaan 2A, Shaan 2B and Ken C1) of Brassica napus by RT-PCR method. By searching the sequences in GenBank Database, it is shown that these sequences from B. napus were novel. Their encoding regions of a functional protein were then inserted into the multiple cloning sites of pMID1 vector for the expression of recombinant protein. All the recombinants were successfully expressed in the in vitro expression system. When the transcripts of subunits 0, 1 and 2 were added to-gether to the in vitro system with 36 靏/mL of protein disul-fide isomerase, the expressed products had NAD-IDH enzy-matic activity. Comparison of different kinds of gene and molecular ratio of the transcripts showed that the NAD-IDH enzyme is composed of three subunits designated subunit 0, subunit 1 and subunit 2. All the three subunits are essential to catalytic activity. Missing subunit 0 could abolish activity. Missing either subunit 1 or subunit 2 would cause severe impact on activity. Deletions, which would cause frameshift mutation or nonsense mutation, were also found in some transcripts of subunit 1 gene from Shaan 2A and Shaan 2B of B. napus. The mutated subunit 1 lost its proper function. This may explain why there is difference of the NAD-IDH activity among three lines of B. napus.

关键词：基因表达克隆技术 NAD-IDH基因异柠檬酸脱氢酶

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Specific targeted killing of ErbB2 positive breast cancer by retrovirus-mediated Immunocaspase-3 secreting T cells

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Chinese Science Bulletin 2004年第13期49卷 1380-1385页

作者： ZHANGLihong JIALintao ZHAOJing XUYanming WENWeihong BAOWei CAOYunxin SUChenazhi WANGChengji YANGAn-gang DepartmentofImmunology FourthMilitaryMedicalUniversityXi'an710032China departmentofbiochemistryandmolecularbiology FourthMilitaryMedicalUniversityXi'an710032China DepartmentofImmunology FourthMilitaryMedicalUniversityXi'an710032China//DepartmentofBiochemistryandMolecularBiologyFourthMilitaryMedicalUniversityXi'an710032China

In this study, lmmunocaspase-3 gene was transfected into Jurkat T lymphocytes and the targeted proapoptotic protein Immunocaspase-3 was stably *** entry to ErbB2 positive SKBr3 breast carcinoma cell line was observed by indirect immunofluorescence *** of SKBr3 cells was significantly inhibited when they were cultured with medium containing ***, lmmunocaspase-3 gene was cloned into retrovirus vector pLNCX, which was then transfected into PA317 cells to package. Packaged cells producing high titer pseudoviruses were acquired and the pseudoviruses were harvested to infect PBMCs, which had been stimulated to division. The latter were selected and administered to nude mice bearing SKBr3 tumors through tail vein. The results showed that the treatment contributed to an inhibition of tumor growth and prolonged the lifetime of nude mice bearing SKBr3 *** efficiency of inhibition of tumor reached 73.25%, and the average lifetime of treated nude mice was 80.95% longerthan that of control group. Immunohistochemical examination revealed the exclusive distribution of Immunocaspase-3 proteins only in the tumor tissue samples; and TUNEL assay confirmed the occurrence of apoptosis in tumor calls. Thepresent study suggests that Immunocaspase-3 secreted by T lymphocytes can selectively bind and enter into ErbB2 positive breast cancer cells, where it exhibits a proapoptotic activity and causes tumor suppression in an in vivo tumor model.

关键词： ErbB2 乳癌逆转录酶病毒免疫酶 T细胞 caspase-3 细胞凋亡基因治疗

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Immune response in Balb/c mice induced by recombinant spike protein of severe acute respiratory syndrome coronavirus

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Chinese Medical Journal 2005年第10期118卷 857-860页

作者： QINLi WUShao-ting WANGXi-ming YUANShi-shan HUANGDa-na LEIMing-jun PANHui-rong LINQi-ping GAOShi-tong ZHANGRen-li QUShen departmentofbiochemistryandmolecularbiology TongjiMedicalCollegeHuazhongUniversityofScienceandTechnologyWuhan430030China ShenzhenCenterforDiseaseControlandPrevention Shenzhen518020China

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly aggressive pathogen that caused SARS in 2003 and 2004.^(1-4) Spike protein (S) on the surface of virus particles mediates the attachment of the virus to cell surface receptors and induces the fusion of viral and cellular membranes. According to the epitope analysis and structure of S protein, we used two fragments of S protein S1 (108-488aa) and S2 (723-938aa) expressed in Escherichia coli and immunized Balb/c mice to investigate the immune response to the recombinant proteins in mice.~5

关键词： severe acute respiratory syndrome coronavirus spike protein IgG isotype T lymphocyte subset cytokine

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Activation of Proteinkinase ERK Mediates Induction of Macrophage MMP-12 by OxLDL

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Wuhan University Journal of Natural Sciences 2004年第1期9卷 120-124页

作者： HeChun-yan ZhouXin LiXiao-ming YuHong HongJia-ling ZhongnanHospital SchoolofMedicineWuhanUniversityWuhan430071HubeiChina departmentofbiochemistryandmolecularbiology SchoolofMedicineWuhanUniversityWuhan430071HubeiChina departmentofbiochemistryandmolecularbiology SchoolofMedicineWuhanUniversityWuhan430071HubeiChina

The present study was undertaken to investigate the effect of oxidized low density lipoprotein (oxLDL) on the expression of macrophage matrix metalloproteinase-12 (MMP-12), and the possible mechanisms. Activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was detected by Western blot Analysis. Enzymatic activity of MMP-12 was determined by β-casein zymography. RT-PCR analysis was used to measure the mRNA expression level of MMP-12. OxLDL-stimulated macrophages produced increased casein-degrading activities and oxLDL also significantly increased the mRNA level of MMP-12 in a dose-dependent manner. OxLDL stimulated the phosphorylation of ERK1/2 in macrophages. The use of the specific inhibitor indicated that the ERK1/2 signaling pathway was required for the induction of MMP-12. These data demonstrated that oxLDL induced MMP-12 expression in macrophages through an ERK1/2-dependent pathway. Key words oxidized low density lipoproteins - macrophage - matrix metalloproteinases CLC number Q 556+.9 Foundation item: Supported by the Natural Science Foundation of Hubei Province (99J138)Biography: He Chun-yan (1970-), female, Ph. D candidate, research direction: biochemistry diagnosis of atherosclerosis.

关键词： oxidized low density lipoproteins macrophage matrix metalloproteinases

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Expression and localization of Smadl, Smad2 and Smad4 proteins in rat testis during postnatal development

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Asian Journal of Andrology 2003年第1期5卷 51-55页

作者： JingHU Yuan-QiangZHANG Xin-PingLIU Rui-AnWANG YanJIN Ruo-JunXU DepartmentofHistologyandEmbryology departmentofbiochemistryandmolecularbiology DepartmentofOralHistologyandPathology SchoolofDentistryFourthMilitaryMedicalUniversityXi‘an710032China DepartmentofZoology UniversityofHongKongHongKongHKSARChina

Aim: To study the expression and regulation of Smadl, Smad2 and Smad4 proteins (intracellular signaling molecules of transforming growth factor-β family) in rat testis during postnatal development. Methods: The whole testes were collected from SD rats aged 3, 7, 14, 28 and 90 (adult) days. The cellular localization and developmental changes were examined by immunohistochemistry ABC method with the glucose oxidase-DAB-nickel enhancement technique. Quantitative analysis of the immunostaining was made by the image analysis system. The Smads proteins coexistence in the adult rat testis was tested by the double immune staining for CD14-Smad4 and Smad2-Smad4. The protein expression of Smad during rat testicular development was examined by means of Western blots. Results: Smadl, Smad2 and Smad4 were present throughout testicular development. The immunostaining of Smadl and Smad2 were present in spermatogenic cells. A positive immunoreactivity was located at the cytoplasm, but the nucleus was negative. Smadl was immunolocalized at the d14, d28 and adult testes, while Smad2, at the d7, d14, d28 and adult testis. There was positive irnmunoreaction in the Sertoli cells and Leydig cells as well. The immunolocalization of Smad4 was exclusively at the cytoplasm of Leydig cells and the nuclei were negative throughout the testicular development. No expression was detected in the germ cells. The results of image and statistical analysis showed that generally the expression of Smadl, Smad2 and Smad4 in the testis tended to increase gradually with the growth of the rat. Conclusion: The present data provide direct evidences for the molecular mechanism of TGF-βaction in rat testes during postnatal development and spermatogenesis.

关键词： TGF-β Smadl Smad2 Smad4 testis

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The Effect of cdk-5 Overexpression and Overactivation on Tau Hyperphosphorylation in Cultured N2a Cells

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Wuhan University Journal of Natural Sciences 2005年第2期10卷 472-476页

作者： CHENJuan LIHong-lian FENGYou-mei WANGjian-zhi departmentofbiochemistryandmolecularbiology TongjiMedicalCollegeHuazhongUniversityofScienceandTechnologyWuhan430030China DepartmentofPathophysiology TongjiMedicalCollegeHuazhongUniversityofScienceandTechnologyWuhan430030China

ISBN: (纸本)None

Neurofibrillary tangles (NFTs) are one of the neuro-pathological hallmarks ofAlzheimer's disease (AD) and abnormally hyperphosphorylated tau is the major protein of NFTs It wasreported that cyclin-dependent kinase5 (Cdk-5) could phosphorylate tau atmost AD-related epitopesin vivo. In this study, we investigated the effect of cdk-5 overexpression on tauhyperphosphbrylation in neuroblastoma N2a cells. We demonstrated that Overexpression of cdk-5 whichresulted in a 3.5-fold Cdk-5 activation in thetransfected cells induced a dramatic increase inphosphorylation of tau at severalphosphorylatiosites. Overexpression of cdk-5 led to a reducedstaining with antibody Tau-1 and an enhanced staining with antibody PHF-1, suggestinghy-perphosphorylation of tau at Serl99/202 and Ser396/404 sites. It implies that in vitroOverexpression of cdk-5 leads to Cdk-5 overactivation and tau hyperphosphorylation may be theunderline mechanism.

关键词： cyclin-dependent kinase5 (Cdk-5) tau Alzheimer's diseas

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A Paleogenomic Algorithm for Reconstruction of Ancient Operons from Complete Microbial Genome Sequences

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Chemical Research in Chinese Universities 2004年第5期20卷 665-667页

作者： WANGYu-hong LIWei FANGXue-xun JohnP.Rose WANGBi-Cheng LINDa-wei DepartmentofMolecularBiology JilinUniversityChangchun130023P.R.China SoutheastCollaboratoryforStructuralGenomics DepartmentofBiochemistryandMolecularBiologyUniversityofGeorgiaAthensGA30602Greece

Operons, or co-transcribed and co-regulated contiguous sets of genes, in microbial genomes are poorly conserved across different genomes due to gene fusion, deletion, duplication and other genome shuffling processes. The currently available genomes are the results of numerous reshuffling and acceptance iterations. We hypothesized that in ancient times, when life was more primitive, functionally related genes existed in close proximity and operated together as an operon to simplify regulation. As more sophisticated regulation mechanisms became available during evolution the genes forming an operon could be separated by the above mentioned processes. If gene shuffling is a random event, neighbor gene pairs are more likely to be preserved than distant gene pairs. Thus, if enough gene pairs can be identified, the original operon could be reconstructed by assembling the pairs. Here we propose a novel paleogenomic method to reconstruct present neighbor gene pairs into "ancient" operons that possibly existed at some point during evolution.

关键词： Operons Microbial genomes Random cost Global optimization

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MEF2C involved in the regulation of nicotinamide phosphoribosyl transferase expression through distinct mechanisms

MEF2C involved in the regulation of nicotinamide phosphoribo...

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中国生物化学与分子生物学会第十届会员代表大会暨全国学术会议

作者： Shaofei Yan Hongjie You Wei Ding departmentofbiochemistryandmolecularbiology CapitalMedicalUniversity

Nicotinamide phosphoribosyl transferase(Nampt,also known as PBEF1 or visfatin) is the rate-limiting enzyme along the salvage pathway of nicotinamide adenine dinucleotide(NAD) *** regul

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