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     

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.     

Spred2 Modulates the Erythroid Differentiation Induced by Imatinib in Chronic Myeloid Leukemia Cells

Differentiation induction is currently considered as an alternative strategy for treating chronic myelogenous leukemia (CML). Our previous work has demonstrated that Sprouty-related EVH1 domainprotein2 (Spred2) was involved in imatinib mediated cytotoxicity in CML cells. However, its roles in growth and lineage differentiation of CML cells remain unknown. In this study, we found that CML CD34⁺ cells expressed lower level of Spred2 compared with normal hematopoietic progenitor cells, and adenovirus mediated restoration of Spred2 promoted the erythroid differentiation of CML cells. Imatinib could induce Spred2 expression and enhance erythroid differentiation in K562 cells. However, the imatinib induced erythroid differentiation could be blocked by Spred2 silence using lentiviral vector PLKO.1-shSpred2. Spred2 interference activated phosphorylated-ERK (p-ERK) and inhibited erythroid differentiation, while ERK inhibitor, PD98059, could restore the erythroid differentiation, suggesting Spred2 regulated the erythroid differentiation partly through ERK signaling. Furthermore, Spred2 interference partly restored p-ERK level leading to inhibition of erythroid differentiation in imatinib treated K562 cells. In conclusion, Spred2 was involved in erythroid differentiation of CML cells and participated in imatinib induced erythroid differentiation partly through ERK signaling.     

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.     

Integrative investigation of lipidome and signal pathways in human endothelial cells under oxidative stress

Phospholipids in human endothelial cells (ECs), cell line EA.hy926, were profiled by a novel lipidomics approach, combining liquid chromatography (LC)-ion trap mass spectrometry (MS) and LC-tandem quadrupole MS. More than 200 species of phospholipids were quantified. Twenty-eight were identified as the most discriminant species in response to different levels of oxidative stress induced by hydrogen peroxide (H(2)O(2)). H(2)O(2) treatment induced phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) via the activation of extracellular-regulated kinase 1/2 (ERK1/2), increasing the production of lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC). The release of arachidonic acid (AA, 20?:?4) increased from no H(2)O(2) exposure to 1 h exposure, decreased from 1 h to 2 h, and increased again from 2 h to 4 h exposure time. The particular increase seen of phosphatidylcholine (PC) species that include AA chains from 1 h to 2 h indicates that the released AA is reincorporating into PC molecules to reduce the extension of the AA cascade. The change in free AA levels seen suggests possible defense mechanisms to oxidative injury in ECs. We further verified nine species as potential biomarkers by adding inhibitor and demonstrated direct correlation to the activity of the cPLA(2)-AA pathway. The oxidative injury to cell line EA.hy926 provided a novel application for a combined lipidomics and signal transduction approach. This combined approach has enabled future investigations for possible therapeutic interventions in phospholipids and cPLA(2) activity for defense against oxidative cellular stress.     

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.     

壳聚糖对玉米生长的调节作用

应用壳聚糖衍生物羧甲基壳聚糖(NCMC)水溶液处理玉米种子,能够促进玉米种子的萌发,提高发芽势和发芽率.处理玉米果穗能够提高玉米未成熟籽粒的可溶性蛋白以及成熟种子的贮藏蛋白含量.以上结果表明,羧甲基壳聚糖对作物生长有调节功能.本文还初步研究了羧甲基壳聚糖对作物生长调控的机理.     

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)。     

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.