丙型肝炎病毒核心蛋白上调层粘蛋白B1链基因启动子表达活性的研究

目的:探讨丙型肝炎病毒(HCV)核心蛋白对层粘蛋白B1链(LAMB)启动子转录的激活作用.方法:以我室构建的HCV核心蛋白反式调节基因的cDNA文库抑制性消减杂交(SSH)筛选结果为基础,利用生物信息学技术确定LAMB的启动子区域(LAMB-p),聚合酶链反应(PCR)扩增LAMB-p,克隆至真核表达载体pCAT3中,构建pCAT3-LAMB-p表达载体;以该质粒转染COS-7、NIH 3T3细胞系,用酶联免疫黏附方法(ELISA)法检测氯霉素乙酰转移酶(CAT)的表达活性;与pcDNA3.1(-)-core共转染NIH 3T3细胞系,用ELISA法检测CAT的表达活性.结果:质粒pCAT3-LAMB-p在NIH 3T3、COS-7细胞中能够激活CAT的表达;共转染实验中pCAT3-LAMB-p+pcDNA3.1(-)-core组CAT的表达活性是pCAT3-LAMB-p的3.3倍.结论:构建的pCAT3-LAMB-p具有启动子活性;HCV核心蛋白对LAMB-p有激活作用.本研究为利用SSH技术研究HCV核心蛋白致肝细胞癌机制提供新的实验及理论基础.

人CNTF突变体(CNTFM)基因的克隆、表达和产物纯化及活性鉴定

采用PCR的方法对睫状神经营养因子(CNTF)基因进行改造,获得CNTF突变体基因(CNTFM) ,将CNTFM基因克隆入表达载体pBV2 2 0 ,在大肠杆菌BL 2 1(Gold)中进行了表达.目的蛋白占细胞总蛋白5 5 %左右,以包涵体形式存在,经Superdex 75凝胶过滤柱一步纯化和复性,获得纯度达90 %目的蛋白.纯化的重组CNTFM蛋白能促进培养的鸡胚背根神经节长出神经突起,能明显减轻实验小鼠的体重,表明CNTFM具有良好的体内、体外生物学活性,为开发新型高效的减肥药奠定了基础.

羧肽酶N调节乙型肝炎病毒核心启动子表达活性的研究

目的:乙型肝炎病毒(HBV)的感染,不仅引起急、慢性病毒性肝炎,而且与肝纤维化、肝细胞癌的发生发展密切相关。为深入研究HBV调节表达的机制,我们应用噬菌体展示技术,以HBV核心启动子DNA片段为固相支持物,筛选肝细胞cDNA文库,获得HBV核心启动子的肝细胞结合蛋白-羧肽酶N(CPN)。CPN是一种从多肽和蛋白质C端氨基酸残基分离的血浆金属蛋白酶,他在调节激肽和过敏毒素的生物活性上起关键作用。CPN是分子量280kD的四聚体,包含两个50KD酶性亚单位和两个83kD调节亚单位。核心启动子产生两个3.5kb RNA:前-核心和前基因组RNA。前-核心RNA编码前-核心蛋白和e抗原,前基因组RNA不仅作为mRNA编码核心蛋白和聚合酶蛋白,而且与病毒蛋白一起包埋入核衣壳,作为模板逆转录。前基因组RNA的表达调控在病毒复制周期中起着关键作用。核心启动子分为两部分:基本核心启动子和核心上游调节元件(CURS),其上游为负性调节元件(NER,1616-1621nt)。CPD在体内与HBV核心启动子结合的作用还不清楚,我们分别构建HBV核心启动子及羧肽酶N的重组载体,通过脂质体转染NIH 3T3细胞,研究CPN对核心启动子的调节表达。方法:根据HBV核心启动子及羧肽酶N的序列设计引物,在核心启动子的引物两端引入MluⅠ和NheⅠ的酶切位点,在羧肽酶N的引物两端引入EcoRⅠ和BamHⅠ的酶切位点。用聚合酶链反应(PCR)的方法分别扩增HBV核心启动子和羧肽酶N基因,克隆到pGEM-Teasy载体上。核心启动子经MluⅠ和NheⅠ双酶切回收连接到同样酶切的pCAT载体上,羧肽酶N经EcoRⅠ和BamHⅠ双酶切回收连接到同样酶切的pcDNA3.1(-)质粒上,构建HBV核心启动子的报告载体及羧肽酶N的真核表达载体,脂质体法瞬时转染NIH 3T3细胞。结果:HBV核心启动子和羧肽酶N(CPN)的PCR产物经1％琼脂糖电泳鉴定与预期大小符合,分别为206 bp和580 bp。重组载体经双酶切鉴定后,证明HBV核心启动子的报告载体及羧肽酶N(CPN)的真核表达载体构建成功。脂质体法瞬时转染NIH 3T3细胞48h后,用ELISA法检测β-gal的表达,显示核心启动子在羧肽酶N(CPN)的影响下,其活性有大约5-6倍的增加。通过体内实验证明羧肽酶N(CPN)可以上调HBV核心启动子的表达。结论:HBV核心启动子结合蛋白羧肽酶N(CPN)与HBV核心启动子共转染细胞,明显调节HBV核心启动子的表达,为进一步研究HBV复制的分子生物学机制提供了新的理论基础。

烟草合子时期表达基因NtZE1的克隆及结构分析

通过分析烟草Nicotiana tabacum长形合子和卵细胞的cDNA差减文库,获得一条合子时期表达的基因NtZE1的EST序列。分析显示,EST序列中含有315bp编码序列,通过RT-PCR(reverse transcription polymerase chain reaction)成功克隆到该序列。生物信息学分析显示,NtZE1基因编码105个氨基酸残基;NtZE1蛋白为不稳定的亲水性蛋白,具有信号肽;蛋白二级结构多为α-螺旋,软件预测该蛋白可能定位于细胞外。利用Genome walking技术获得ATG起始密码子前共2578bp的5’侧翼序列(启动子和5’UTR),将该序列连入EGFP核定位载体中并通过瞬时表达技术使其在小叶烟草N.benthamiana叶表皮细胞中表达,有明显的绿色荧光,证实所获得的5’侧翼序列具有启动基因表达的活性。

人Tumstatin在毕赤酵母中的表达和活性分析

利用PCR技术从重组质粒pET-3c-tum中扩增人tumstatin的cDNA片段,连入pPICZαA酵母表达载体,获得的重组质粒pPICZα-tum电激法转化毕赤酵母GS115。经表型鉴定、诱导表达筛选,得到可分泌表达人tumstatin的重组酵母转化子,表达蛋白质的相对分子量约30kD,表达量约25mg/L。表达上清经超滤浓缩和离子交换法初步纯化,所得产物具有免疫活性,能够抑制内皮细胞增殖,诱导其发生细胞凋亡,并能抑制鸡胚尿囊膜血管生成。

梭鱼脂肪酸合成酶基因部分片段的克隆和表达分析

分离、克隆梭鱼脂肪酸合成酶(fatty acid synthase,简称FAS)基因的部分c DNA片段(Gen Bank登录号为KJ848474),共920 bp,编码303个氨基酸,序列分析表明梭鱼FAS基因与其他物种的同源性为74%~95%,其中与军曹鱼、斑马拟丽鱼相似性达95%。FAS基因mRNA在梭鱼鱼皮、肌肉、肝脏、心脏、脾脏、肾脏、胃、肠、腹腔脂肪、脑和鳃组织中表达丰度差异显著(P<0.05),脑中含量最高,其次是肝脏和腹腔脂肪组织,肌肉中表达量最低,其余7种组织中的表达量均显著低于脑和肝脏(P<0.05)。此外,还研究了饲料脂肪水平对梭鱼FAS活性和mRNA表达的影响,平均质量为(9.5±0.3)g的梭鱼幼鱼投喂6种不同脂肪含量的等氮等能饲料(脂肪水平分别为2.04%、4.83%、7.47%、9.79%、12.01%、14.59%),饲养60 d,试验结束后测定各组梭鱼肝脏中FAS的生物活性及肝脏、腹腔脂肪、肌肉中FAS mRNA的表达丰度。结果表明,随着饲料脂肪水平的升高,肝脏中FAS活性呈降低趋势,14.59%组的活性显著低于2.04%、4.85%组(P<0.05);肝脏中FAS的mRNA表达量显著下降(P<0.05);肌肉和腹腔脂肪中FAS的mRNA表达量呈下降趋势,但各组之间差异不显著(P>0.05)。

鱼类糖代谢几种关键酶的研究进展

鱼类饲料中的碳水化合物主要用作代谢能源,然而与陆生动物相比,鱼类对饲料中碳水化合物的利用能力较低。在糖代谢过程中,鱼体内糖代谢酶起了重要的作用。本文主要从糖代谢酶的活性和基因表达等方面,较系统地阐述了糖酵解和糖异生过程中的一些关键酶的研究现状。分析认为,饲料中碳水化合物的含量对部分糖代谢酶的活性和基因表达存在影响,但是一些研究还不明确,有待于更深入地研究。

氯原酸对成骨细胞Ⅰ型胶原及骨钙蛋白基因表达的影响

为了探讨陆英作为骨折常用中草药对成骨细胞的影响和相关机理。以陆英提取物氯原酸(chlorogenic acid,简称CHA)对成骨细胞的作用为研究模型,通过提取纯化获得原代成骨细胞,并经细胞活性观察、组织化学染色及荧光定量PCR检测技术,分别对CHA作用于成骨细胞后,检测成骨细胞活性及CHA对成骨细胞生长及Ⅰ型胶原及骨钙蛋白基因表达的影响。结果表明:10μmol/L,100μmol/L CHA能促进成骨细胞钙化结节形成,且能促进I型胶原基因持续增长;在培养第3d时,CHA对骨钙蛋白的表达具有促进作用。陆英有效成分氯原酸能明显增强成骨细胞I型胶原蛋白和骨钙蛋白基因的表达,合成和分泌骨基质,具有一定的成骨活性。

Cloning and Differential Gene Expression of Two Catalases in Suaeda salsa in Response to Salt Stress

Two different cDNA clones (Sscat1 and Sscat2) encoding catalase, the primary important H2O2-scavenging enzyme, were isolated from a AZap-cDNA library constructed from a 400 mmol/L NaCl-treated library of Suaeda salsa ( L.) Pall aerial tissue. Sscat1 (1.7 kb) contains a full open reading frame of 492 amino acids and Sscat2 (1.1 kb) is a partial clone. BLAST analysis indicates that the two clones share 71.9% identity in nucleotide sequence and 75% identity in deduced amino acid sequence within the last 287 amino acid residues of Sscat1. Southern blotting analysis showed that Sscat1 is multicopy in S. salsa genome, while Sscat2 is a single copy gene. Northern blotting analysis showed a rapid increase in the steady-level of both genes in roots after 48 It salt treatment, but only Sscat1 was induced in salinity treated leaves. Time-course analysis carried out in leaves confirmed that Sscat1 was induced by salt stress, in contrast to Sscat2. These implied that the expression of Sscat1 and Sscat2 genes are differentially regulated in S. salsa. The activity of total catalase is dramatically increased in response to salt stress.

茶树凋落叶浸提液对菘蓝生理生化的化感效应

为研究茶树凋落叶浸提液对菘蓝生长与生理生化的化感效应,以模拟自然条件下雨雾淋溶方式,采用不同浓度的茶树凋落叶浸提液(CK:0 mg·mL^-1、T1:6.25 mg·mL^-1、T2:12.5 mg·mL^-1、T3:25 mg·mL^-1和T4:50 mg·mL^-1)处理菘蓝,测定其生长、抗氧化酶活性及其基因表达量、细胞膜损伤率以及丙二醛(MDA)、过氧化氢(H2O2)、渗透调节物质和次生代谢物含量的变化。结果表明,茶树凋落叶浸提液对菘蓝生长表现出"低促高抑"的浓度效应,与CK相比,T1对菘蓝生长具有一定的促进作用,而浸提液浓度超过菘蓝的耐受阈值时,会对其生长产生不利的影响。随着浸提液浓度的升高,可溶性糖和可溶性蛋白含量呈先上升后下降的趋势,而脯氨酸含量则持续增加。与CK相比,T1的MDA含量、细胞膜损伤率和H2O含量差异不显著,而T3、T4则显著升高。茶树凋落叶浸提液对抗氧化酶活性及其基因表达有不同的影响,T1的过氧化物酶(POD)与抗坏血酸氧化物酶(APX)活性最高,T2的过氧化氢酶(CAT)活性最高,而T3、T4的4种抗氧化酶活性均显著低于CK。T1的Pod、Cat、Apx基因表达量最高,而T4则抑制了抗氧化酶基因的表达。此外,POD活性与其基因表达量呈显著正相关,而超氧化物歧化酶(SOD)、CAT和APX活性与其基因表达量的相关性不显著。不同浓度的茶树凋落叶浸提液对于菘蓝次生代谢物质积累的影响存在显著差异,低浓度茶树凋落叶浸提液对菘蓝生长、靛蓝和靛玉红含量积累有促进作用,而高浓度茶树凋落叶浸提液对总黄酮含量积累有一定的促进作用。本研究结果可为幼龄茶园中茶树-菘蓝复合种植提供理论参考。

Expression, purification and bioactivity of human augmenter of liver regeneration

AIM： To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration （hALR） and to study their biological activity. METHODS： hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase （GST） sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. RESULTS： The product of PCR from plasmid pGEM-T- hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups （P 〈 0.01）.CONCLUSION： Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.

Science Letters:Transient expression of chicken alpha interferon gene in lettuce

We investigated the possibility of producing chicken alpha interferon （ChlFN-α） in transgenic plants. The cDNA encoding ChlFN-α was introduced into lettuce （Lactuca sativa L.） plants by using an agro-infiltration transient expression system. The ChlFN-a gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus （VSV） replication on chicken embryonic fibroblast （CEF）. The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.

Effect of NaCI and Helicobacter pylori vacuolating cytotoxin on cytokine expression and viability

AIM; To determine whether Helicobacter pylori （H pylon） vacuolating cytotoxin （VacA） regulates release of proinflammatory cytokines （IL-1β, IL-8, TNF-α, and IL-6） or alters gastric epithelial cell viability and to determine whether NaCl affects these VacA-induced changes. METHODS： Vacuolating activity was determined by measuring the uptake of neutral red into vacuoles of VacA-treated human gastric epithelial （AGS） cells. AGS cell viability was assessed by direct cell counting. Specific enzyme-linked immunosorbent assays （ELISA） and reverse transcdptase-polymerase chain reaction（RT-PCR） were performed to examine the effects of H pylori VacA and NaCl on cell pro-inflammatory cytokine production in AGS cells. Immunohistochemical staining of gastric tissue from Mongolian gerbils was used to confirm VacAinduced pro-inflammatory cytokine production and the effects of NaCl on this VacA-induced response. RESULTS： Addition of VacA alone reduced AGS cell viability （P〈 0.05）, and this reduction was enhanced by high doses of NaCl （P〈0.05）. VacA alone induced expression of TNF-α, IL-8 and IL-1β, while NaCl alone induced expression of TNF-α and IL-1β. Changes in mRNA levels in the presence of both VacA and NaCl were more complicated. For the case of TNF-α, expression was dosedependent on NaCl. IL-6 mRNA was not detected. However, low levels of IL-6 were detected by EUSA. Positive immunohistochemical staining of IL-1, IL-6, and TNF-α was found in gastric tissue of H pylori-infected gerbils fed with either a normal diet or a high salt diet. However, the staining of these three cytoldnes was sb＇onger in H pylori-infected animals fed with a 5g/kg NaCl diet. CONCLUSION： VacA decreases the viability of AGS cells, and this effect can be enhanced by NaCl. NaCl also affects the production of pro-inflammatory cytokines in- duced by VacA, suggesting that NaCl plays an important role in Hpylori-induced gastric epithelial cell cytotoxicity.

Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer

AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters. METHODS: Western blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3, p38 and mitogen or ERK activated protein kinaseMEK-1 proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients. Immunohistochemistry was employed for their localization. RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526?5 760 vs 122 807±65 515, P= 0.001), ERK-2 (168 471±95 051 vs 120 469±72 874, P<0.001), ERK-3 (118 651±71 513 vs 70 934±68 058,P<0.001), P38 (104 776±51 650 vs 82 930±40 392, P= 0.048) and MEK-1 (116 486±45 725 vs 101 434±49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging [average ratio of integral optic density (IOD)tumor: IODnormal in TNM I, II, III, IV tumors was 1.43±0.34, 5.08±3.74, 4.99±1.08, 1.44±1.02, n = 42, P= 0.023] and serosa invasion (4.31±4.34 vs 2.00±2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage III and IV tumors were higher than those in stage I and II tumors (2.64+3.01 vs 1.01±0.33, P= 0.022; 2.05±1.54 vs1.24±0.40, P= 0.030). Gastric cancer tissues with either lymph node involvement (2.49±2.91 vs1.03±0.36, P= 0.023; 1.98±1.49vs1.24±0.44, P= 0.036) or serosa invasion (2.39±2.82 vs 1.01±0.35, P= 0.022; 1.95±1.44 vs1.14±0.36, P=0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann II tumors, expression of ERK-2 and ERK-3 was increased compared with Borrmann III tumors (2.57±1.86 vs1.23±0.60, P= 0.022; 5.50±5.05 vs1.83±1.21, P= 0.014). Borrmann IV tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P>0.05). Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site. CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers. Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MARK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.

Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier. METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR. RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preSl (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preSl (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro. CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

Effects of psychological stress on small intestinal motility and expression of cholecystokinin and vasoactive intestinal polypeptide in plasma and small intestine in mice

AIM: To investigate the effects of psychological stress on small intestinal motility and expression of cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP) in plasma and small intestine, and to explore the relationship between small intestinal motor disorders and gastrointestinal hormones under psychological stress. METHODS: Thirty-six mice were randomly divided into psychological stress group and control group. A mouse model with psychological stress was established by housing the mice with a hungry cat in separate layers of a two-layer cage. A semi-solid colored marker (carbon-ink) was used for monitoring small intestinal transit. CCK and VIP levels in plasma and small intestine in mice were measured by radioimmunoassay (RIA). RESULTS: Small intestinal transit was inhibited (52.18±19.15% vs70.19±17.79%, P<0.01) in mice after psychological stress, compared to the controls. Small intestinal CCK levels in psychological stress mice were significantly lower than those in the control group (0.75±0.53 μg/g vs1.98±1.17 μg/g, P<0.01), whereas plasma CCK concentrations were not different between the groups. VIP levels in small intestine were significantly higher in psychological stress mice than those in the control group (8.45±1.09 μg/g vs7.03±2.36 μg/g, P<0.01), while there was no significant difference in plasma VIP levels between the two groups. CONCLUSION: Psychological stress inhibits the small intestinal transit, probably by down-regulating CCK and up-regulating VIP expression in small intestine.

Expression profiling of gastric cancer samples by oligonucleotide microarray analysis reveals low degree of intra-tumor variability

AIM： Gene expression profiling provides an unique opportunity to gain insight into the development of different types of gastric cancer. Tumor sample heterogeneity is thought to decrease the sensitivity and tumor specificity of microarray analysis. Thus, microdissection and preamplification of RNA is frequently performed. However, this technique may also induce considerable changes to the expression profile. To assess the effect of gastric tumor heterogeneity on expression profiling results, we measured the variation in gene expression within the same gasbic cancer sample by performing a gene chip analysis with two RNA preparations extracted from the same tumor specimen. METHODS： Tumor samples from six intestinal T2 gastric tumors were dissected under liquid nitrogen and RNA was prepared from two separate tumor fragments. Each extraction was individually processed and hybridized to an Affymetrix U133A gene chip covering approximately 18 000 human gene transcripts. Expression profiles were analyzed using Microarray Suite 5.0 （Affymetrix） and GeneSpring 6.0 （Silicon Genetics）. RESULTS： All gastric cancers showed little variance in expression profiles between different regions of the same tumor sample. In this case, gene chips displayed mean pair wise correlation coefficients of 0.94±0.02 （mean±SD）, compared to values of 0.61±0.1 for different tumor samples. Expression of the variance between the two expression profiles as a percentage of “total change” （Affymetrix） revealed a remarkably low average value of 1.18±0.78 for comparing fragments of the same tumor sample. In contrast, comparison of fragments from different tumors revealed a percentage of 24.4±4.5. CONCLUSION： Our study indicates a low degree of expression profile variability within gastric tumor samples isolated from one patient. These data suggest that tumor tissue heterogeneity is not a dominant source of error for microarray analysis of larger tumor samples, making total RNA extraction an appropriate strategy for performing gene chip expression profiling of gastric cancer.

Tumor Antigen Specific Activation of Primary Human T-Cells Expressing a Virally Encoded Chimeric T-Cell Receptor Specific for p185HER2

We have developed and tested chimeric T-cell receptors (TCR) specific for p185HER2. In these experiments, retroviral vectors expressing the N29γ or N29ζ receptors were constructed in pRET6. Amphotropic viral producer cells were established in the GALV-based PG13 packaging cell line. Ficoll purified human peripheral blood lymphocytes (PBL) were virally transduced using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28 monoclonal anti- bodies, followed by viral infection in the presence of fibronectin fragment CH296. Transduced cells were co-cultured with human tumor cell lines that overexpress (SK-OV-3) or underexpress (MCF7) p185HER2 to assay for antigen specific im- mune responses. Both CM+ and CD8+ T-cells transduced with the N29γ or N29ζ chTCR demonstrated HER2-specific anti- gen responses, as determined by release of Th1 like cytokines, and cellular cytotoxicity assays. Our results support the fea- sibility of adoptive immunotherapy with genetically modified T-cells expressing a chTCR specific for p185HER2.

Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-Receptor and identification of its activity

Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF R, permitting the recruitment of gp130 and LIF R, forming a tripartite receptor complex. Cells that only express gp130 and LIF R, but not CNTF R are refractory to stimulation by CNTF. On many target cells CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH terminus of sCNTF R to the NH 2 terminus of CNTF. Recombinant CNTF/sCNTF R fusion protein (Hyper CNTF) was successfully expressed in COS 7 cells. The apparent molecular mass of the Hyper CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of transfected BAF/3 cells in response to CNTF and Hyper CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gp130, CNTF R and LIF R receptor subunit whereas Hyper CNTF required heterodimerization of the gp130 and LIF R receptor subunit. We concluded that the fusion protein Hyper CNTF had superagonistic activity on target cells expressing gp130 and LIF R, but lacking membrane bound CNTF R.

Dynamic change of xylanase activity and gene expression during wheat germination on As（III） stress

Through water cultivating method, the dynamic changes of xylanase activity in seed, root and plumule of wheat with different As （III） concentration treatment were studied. The results indicated that the order of average xylanase activity was seed〉plumule〉root. With the increasing concentration of As （III）, the xylanase activity elevated first then dropped in seed, but it descended first then ascended in root and plumule. As the sampling time prolonged, the xylanase activity of seeds climbed first then dropped on the four as （III） concentration, the same trend also appeared in pulume, as the as （Ill） concentration went up, the xylanase activity moved up simultaneity. Semi-quantity Reverse Transcription Polymerase Chain Reaction was used in the study, the results indicated that, the xylanase gene began to express at 132 h on 0 mg/L As （III） concentration and at 120h on other concentration in the leaves of wheat.