猪大肠杆菌水肿毒素SLT-IIeA基因的克隆和序列分析

研究以本地猪水肿病大肠杆分离物ED1株为材料 ,利用 PCR克隆了含猪水肿病大肠杆菌 (VTEC) slt-IIe A基因 987bp的片段 ,并测定了含该克隆片段的 Bam HI、Hind 酶切片段的核苷酸序列。结果表明 ,slt-IIe A基因的编码区全长 960 bp,编码 3 1 9个氨基酸的蛋白质 ,序列与国外报导的 S1 1 79株进行比较发现其核苷酸同源性为 98.9%。经推导的氨基酸序列的同源性为 99.7%。这为进一步研究 slt-IIe A的生物学特性、水肿病的分子诊断及其防制打下基础     

利用紫茎泽兰栽培田头菇的研究

利用紫茎泽兰作为原料栽培田头菇 ,同时利用稻草和棉籽壳为原料栽培田头菇作对照。研究结果表明 ,代料熟料栽培田头菇的培养基配方中 ,以紫茎泽兰为主要原料的配方A效果最好 ,生物学效率比对照的稻草培养基平均增产 80 %以上。利用紫茎泽兰鲜草和干草作培养基的单产量差异不明显。     

三尖杉枝叶粉末防治花生根结线虫病

采用盆栽及大田小区试验,研究了三尖杉Cephalotaxus fortunei枝叶干粉末对花生根结线虫病的防治效果.盆栽试验结果表明,每盆(2kg土)施用三尖杉枝叶干粉末10、15及20 g三种处理,与对照组相比,初侵染相应推迟4、6、10天,并能减少侵染量,降低根结增长率,显著减轻花生根结线虫病病情.大田小区试验结果与盆栽试验结果相似,每小区(5m2)沟施三尖杉枝叶干粉末120、80、50 g,处理后34及53天的2次平均防治效果分别为89%、82%及50%,施药对照10%益舒宝颗粒剂25g处理(相当于45kg/hm2)为74%.综合使用剂量及花生生长情况,建议沟施150kg/hm2三尖杉枝叶干粉为宜.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

Cortical gene expression pattern in rat focal cerebral ischemia

AIM: To investigate the genes differential expression in cortex during rat focal cerebral ischemia.METHODS: cDNA microarray chips containing numerous cDNAs were used to investigate the gene expression pattern between samples of focal cerebral ischemia and sham-control operation rats. RESULTS: Two hundred and eleven genes differentially expressed were screened out, among these genes, up-and down-regulated genes were 199 and 12, respectively. CONCLUSIONS: The analysis of gene expression pattern of focal cerebral ischemia based on cDNA microarray can realize high-throughput screening of the genes associated with the focal cerebral ischemia. The differential expression of genes may be related to the pathogenesis of focal cerebral ischemic diseases.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.     

Regulation of MMP-2 and TIMP-3 expression by uPA signal transduction system in human bone giant cell tumor

AIM: To study effects of urokinase-type plasminogen activator (uPA) signal transduction on expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) in giant cell tumor of bone (GCT). METHODS: Expression of uPAR, MMP-2 and TIMP-3 in GCT tissue was detected by immunohistochemistry. Phosphorylation level of mitogen-activated protein kinase (p44) in uPA/uPAR signal pathway in cultured GCT cells was detected by immunoprecipitation. The expression of MMP-2 and TIMP-3 in cultured cells after treatment with uPA-ATF or anti-uPAR antibody was also detected by Western blotting. RESULTS: 1) Urokinase-type plasminogen activator receptor (uPAR) was positive on the cell membrane and in cytoplasm of some mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs); 2) MMP-2 was positive in the cytoplasm and on the cell membrane of almost all of MSCs and some of MGCs. The polar distribution of MMP-2 in the cytoplasm of MGCs was especially obvious; 3) The expression of TIMP-3 of some MSCs and MGCs in GCT was much lower than MMP-2. The positive signal also showed a prominent polarity; 4) After treatment with uPA-ATF, the phosphorylation level of p44 in GCT cultured cells was much higher than the control. Addition of anti-uPAR antibody in the cells remarkably down-regulated the phosphorylation level of p44 as compared with the control group, suggesting that uPA-ATF participates cell signal transduction and this reaction can be inhibited by anti-uPAR antibody; 5) uPA-ATF cell signal pathway up-regulated expression of MMP-2 and TIMP-3, while anti-uPAR antibody down-regulated the expression of MMP-2 and TIMP-3. CONCLUSION: These results demonstrate for the first time that uPA-ATF directly regulates the expression of MMP-2 and TIMP-3 by signal transduction pathway, and the over-expression of MMP-2 and TIMP-3 may play an important role in local osteolysis of GCT.     

Comparison of different conditions inducing embryonic stem cells

AIM: To evaluate the different conditions inducing mouse embryonic stem cells (ESC) in vitro to differentiate into cardiomyocytes. METHODS: BRL conditioned medium was used to promote the growth of ESC and maintain them in an undifferentiated state. During the inducing process, retinoic acid (RA), DMSO, activin-A and TGF-β₁ were used as inducing reagents, and made up six kinds of differentiating medium. Then a three-step method inducing ESC cultured in hanging drops, in suspension and in plating was used to induce the differentiation of ESC. RESULTS: ESC were induced in vitro to differentiate into cardiomyocytes. Of all groups, the highest differentiating rate was observed in the group induced by activin-A (20 μg/L) and TGF-β₁ (2 μg/L). CONCLUSION: The inducing conditions including activin-A (20 μg/L) and TGF-β₁ (2 μg/L) is very valuable in inducing ESC differentiation into cardiomyocytes.