CD40 signal expression in gastric cancer tissue and its correlation with prognosis of gastric cancer patients

CD40 signaling plays a critical role in the survival rate of gastric cancer patients. Tumour samples were collected from 73 patients with who were diagnosed as gastric cancer in general surgery department in the 1st affiliated hospital of Suzhou University between September 2002 and July 2003. All patients had not received radiotherapy and chemotherapy before operation. These patients include 46 male and 27 female. Here we show that CD40 is constitutively expressed in the human gastric carcinoma tissues, and CD40 protein and mRNA positive expression in gastric cancer tissues closely correlated with lymph node metastasis and tumour TNM stage. CD40 positive expression in gastric cancer patients with lymph node metastasis was markedly higher than that in gastric cancer patients without lymph node metastasis. CD40 positive expression in stage III-IV gastric cancer patients was markedly higher than that in stage I-II gastric cancer patients. Moreover, CD40 expression closely correlated with prognosis of gastric cancer patients. Therefore, CD40 was taken as grouping variable, and lymph node metastasis and clinical staging were taken as stratification variables, respectively, further analysis showed that prognosis in gastric cancer patients with lymph node metastasis and CD40 positive expression was markedly worse than that in gastric cancer patients without lymph node metastasis and CD40 negative expression (P = 0.0076). These results suggest that CD40 signaling plays a critical role in the survival of gastric cancer patients.     

A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting

To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K₂, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl₂ before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.     

丙型肝炎病毒NS4区基因的克隆,序列分析及其表达

用RT－PCR法从一名抗-HCV阳性献血员血清中扩增到约900bp的DNA片段,经EcoRI酶切后插入pET17Xb表达质粒。SDS－PAGE表明重组质粒在约65kD处有融合蛋白表达,表达蛋白含量约为24％。对其插入片段进行序列分析：核着酸长度为945bp,A：T：G：C比例为18．6：21.9:30.5：29.0,G/C含量占59％,与其它一些HCV殊比较,核着酸同源性Ⅰ型为77．1％．Ⅲ、Ⅳ、Ⅴ、Ⅵ型均＜70％．Ⅱ型各株均＞90％;氨基酸序列比较的同源性Ⅰ型为857％,Ⅲ、Ⅳ、ⅤⅥ型各株均＞70％,Ⅱ型各株均＞93％。NS4基因的克隆和表达为其基因和产物作用的进一步研究奠定了基础。     

腺相关病毒与人多药耐药基因重组载体的构建及在NIH3T3细胞中的表达

腺相关病毒 (adeno- associated virus,AAV)属细小病毒科 ,是一种最小的动物病毒 .具有其他病毒载体所没有的优点 ,在基因治疗中日益受到瞩目 .以 AAV的一种多克隆载体为基础 ,构建了携带 MDR1基因的重组腺相关病毒载体 (r AAV- MDR1 ) ,经 2 93细胞包装成重组病毒 .将重组质粒、重组病毒分别转染和感染 NIH3T3细胞 ,用 PCR和 MTT法检测了人 MDR1基因的转导及表达 .为 MDR1基因用于临床和腺相关病毒载体在基因治疗中的应用提供了依据     

SARS冠状病毒M蛋白的生物信息学研究

针对GenBank上发布的来自不同国家地区的39条SARSCoV推测M蛋白,采用生物信息学软件分析其核酸和氨基酸序列,获得其分子生物学特征,确定突变位点,预测功能结构区、Motif及抗原决定簇,比较基因突变对这些功能结构的影响.结果表明:在39个病毒株M蛋白的666 bp中,共有18个病毒株在7个位点上发生了25次变异.在M蛋白序列上预测获得3个跨膜螺旋序列和一个可能的信号肽序列.氨基酸序列的变异主要发生在其跨膜和胞外区域,胞内区域相对较少.预测发现12个Motif和7个抗原决定簇.提示突变对M蛋白的结构功能区的影响不大,也未造成M蛋白的Motif的数量和构成发生改变.对抗原决定簇的影响也主要体现在序列成分构成的改变上,在设计疫苗时,应考虑由其导致的抗原特性改变.     

CTAB-PEG DNA Extraction from Fungi with High Contents of Polysaccharides

Polysaccharides influence concentration and purity of extracted DNA. Here we present rapid and efficient protocol for DNA extraction from samples rich in polysaccharides. The technique has been developed using cultures of Schizophyllum commune and involves a modification of known Cetyltrimethyl Ammonium Bromide (CTAB) protocol. To remove polysaccharides, Polyethylene Glycol (PEG) 8000 was added during DNA precipitation. Genomic DNA obtained with the CTAB-PEG method had high integrity, with average fragment size >30 kb, the concentration higher than 100 ng/μL, and the yield more than 30 μg/g. Presented technique is suitable for DNA extraction from fungi, bacteria, archaea or even mollusks with high polysaccharide content.     

Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5‘-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.     

Pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety as potential antiviral agents

A series of pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety were synthesized, characterised, and evaluated for their activity against tobacco mosaic virus (TMV). Biological assays indicated that several of the derivatives exhibited significant activity against TMV. In particularly, compounds 5y and 5aa displayed excellent inactivating activity against TMV, with half maximal effective concentration (EC₅₀) values of 70.3 and 53.65?μg/mL, respectively, which were much better than that of ribavirin (150.45?μg/mL), and 5aa was superior to ningnanmycin (EC₅₀?=?55.35?μg/mL). Interactions of compounds 5y and 5aa with TMV coat protein (TMV-CP) were investigated using microscale thermophoresis and molecular docking. Compounds 5y and 5aa displayed strong binding capability to TMV-CP with dissociation constant (K_d) values of 22.6 and 9.8?μM, respectively. These findings indicate that pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base may be potential antiviral agents.     

中水技术污水处理工艺去除病毒的效果

中水技术开发既可缓解水资源的紧缺,又能改善城市环境。本文报道用石英粉吸附/洗脱和浓缩棒脱水的二步浓集水病毒技术(回收率为25.4～37.3％),研究中水技术不同处理工艺去除病毒的效果。结果,二级处理可去除掺入病毒的98.9％,结合三级处理的混凝沉淀和过滤,可去除99.976％,再加活性炭吸附或加氯消毒,所得中水分别去除掺入病毒的99.986％和99.991％。经首都机场中水道试验厂水样检测表明,中水中未检测到病毒(<0.23PFU/L)。     

NaCl、Na_2SO_4和Na_2CO_3胁迫对小麦叶片自由基含量及质膜透性的比较研究

分别用浓度为２５ｍｍｏｌ／Ｌ、５０ｍｍｏｌ／Ｌ、１００ｍｍｏｌ／Ｌ和２００ｍｍｏｌ／Ｌ的ＮａＣｌ、Ｎａ２ＳＯ４和Ｎａ２ＣＯ３的营养液培养小麦４ｄ,较之不含盐的营养液,其自由基含量上升,产生速率增加,叶片质膜透性增加。不同盐的影响也不同,在低浓度时,ＮａＣｌ的影响大于Ｎａ２ＳＯ４,高浓度时,ＮａＣｌ影响小于Ｎａ２ＳＯ４,Ｎａ２ＣＯ３的影响最为显著。实验结果也表现出小麦叶片自由基含量和质膜透性呈现较好的相关性。因此可认为,盐胁迫促使自由基含量增加,自由基通过过氧化作用影响质膜透性,从而影响植物的生长。