狂犬病病毒M蛋白胞内合成和分布初步研究

【目的】狂犬病病毒(Rabies virus,RABV)是一种主要由核蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)、依赖RNA的RNA聚合酶蛋白(L)5种结构蛋白组成的高度嗜神经性病毒。RABV基因组RNA与N蛋白、P蛋白和L蛋白组成核糖核蛋白复合体(RNP),M蛋白在调控RABV病毒结构蛋白的合成和装配过程中起重要作用。但RABV不同毒株M蛋白胞内合成、分布场所及其是否存在差异尚不清楚。研究不同RABV毒株M蛋白与内质网、高尔基体以及M蛋白与G蛋白、M蛋白与RNP在N_2A和BHK细胞内的共定位,以期确定RABV M蛋白的胞内合成途径和分布场所。【方法】测定RABV不同毒株(CVS-11、SRV-9、PB4)在N_2A或BHK细胞的TCID_(50),以相同的MOI接毒,通过免疫荧光研究M蛋白与内质网、高尔基体的共定位,以及M蛋白与G蛋白、M蛋白与RNP(为N蛋白代表)的共定位。【结果】RABV不同毒株效价存在一定差异。在N_2A和BHK细胞中,不同RABV毒株的M蛋白与内质网、高尔基体均存在共定位;M蛋白与G蛋白、M蛋白与N蛋白也存在共定位,但M蛋白与G蛋白主要共定位于内质网、高尔基体上,而M蛋白与N蛋白主要共定位在胞浆中。【结论】RABV M蛋白在胞内主要通过内质网-高尔基体途径合成加工。研究结果明确了RABV M蛋白胞内合成分布部位,为进一步研究RABV病毒粒子的装配和出芽过程丰富了数据。     

Effects of GOLPH3 gene silencing mediated by lentivirus on proliferation,invasion and metastasis of human gastric cancer cell line SGC-7901

AIM：To investigate the effect of Golgi phosphoprotein 3 (GOLPH3) gene silencing on the proliferation, invasion and metastasis of human gastric cancer SGC-7901 cell in vitro. METHODS：SGC-7901 cells were transfected with lentiviral vector carrying GOLPH3 shRNA to construct a stable GOLPH3-silencing cell line LV-GOLPH3-RNAi. The expression of GOLPH3 at mRNA and protein levelss were detected by real-time PCR and Western blotting, respectively. The cell proliferation was analyzed by MTT assay. Transwell migration and invasion experiments were performed to measure the migration and invasion abilities, respectively. RESULTS：The stable GOLPH3-silencing cell line was successfully established. The expression of GOLPH3 at mRNA and protein levels was reduced significantly (P<0.05), leading to the inhibition of cell proliferation in LV-GOLPH3-RNAi group compared with scrambled group and blank control group, as well as the capacities of migration (56.7±1.5 vs 186.0±3.4 and 183.3±4.2, P<0.05) and invasion (33.5±3.0 vs 85.0±3.9 and 83.1±4.4, P<0.05). CONCLUSION：GOLPH3 silencing suppresses the capacities of proliferation, migration and invasion of human gastric cancer SGC-7901 cells, suggesting that GOLPH3 may be a potential tumor marker and independent prognostic factor.     

大豆根瘤侵染细胞发育中高尔基体的变化

为了探讨高尔基体与细菌增殖和周膜扩展的关系,用电子显微镜研究了大豆根瘤侵染细胞发育中高尔基体的变化.结果表明:幼龄侵染细胞中有高尔基体,但在成熟和衰老的侵染细胞中却几乎没有这种结构.高尔基体随幼龄侵染细胞中细菌的增多而增多,它们主要位于细菌附近.这些高尔基体的生理活性较高,不断向周围的细胞质分泌含有纤维状物质的小泡,有的小泡还在向细菌运动.一些小泡位于细菌的周膜上,其附近细菌的周膜常向外形成隆起,并有纤维状物质出现在其中.由此说明,大豆根瘤侵染细胞中的高尔基体参与了细菌的增殖和周膜的扩展.     

An evaluation of two cypermethrin-based pour-on formulations on sheep infested with the biting louse,Bovicola ovis

Two synthetic pyrethroid (cypermethrin) based “pour-on” insecticide formulations, with high cis/trans isomer ratios (80:20) but differing in their respective active ingredient concentrations and solvent component(s), were applied to sheep infested with the biting-louse, Bovicola ovis. All treated sheep were penned with louse-infested sheep 9, 12 and 15 weeks after the insecticide was applied. The 2% cypermethrin formulation achieved a higher level of control than the 1.25% cypermethrin formulation at each challenge interval when applied 12 weeks after shearing. The 2% cypermethrin formulation provided 97-100% control of lice from 4 to 16 weeks after application on sheep shorn 6 or 12 weeks prior to treatment. The 1.25% cypermethrin formulation provided 85% control of lice 4 weeks after application on sheep shorn 12 weeks prior to treatment, the level of control increasing to a maximum of 100% by week 9, and declining thereafter. The 2% cypermethrin formulation may provide a better level of control in long-woolled sheep than 1.25% cypermethrin, by compensating for the diluent effect of lipid.     

伪狂犬病病毒UL56蛋白与宿主蛋白Nedd4相互作用的研究

伪狂犬病病毒(PRV)UL56蛋白(pUL56)属于Ⅱ型膜蛋白。近期研究结果表明PRVpUL56与PRV对啮齿动物的致病性相关。为揭示PRVpUL56与宿主相互作用的分子基础,本研究通过免疫共沉淀(Co-IP)试验确定了pUL56与神经前体细胞表达的发育下调蛋白4(Nedd4)存在相互作用。进一步,利用激光共聚焦试验确定过表达pUL56与Nedd4在转染HEK293T细胞的高尔基体存在共定位。通过互作,pUL56可以将细胞质分布的Nedd4重新定位于高尔基体。前期研究表明,含有WW结构域的宿主蛋白可以与含有PPxY(PY)基序的病毒蛋白互作。因此,本研究构建了pUL56PY基序丙氨酸突变体(PPxY/AAxY),利用Co-IP试验对pUL56突变体和Nedd4相互作用进行验证,结果表明随着pUL56PY基序突变的增加,蛋白互作能力逐渐减弱。此外,pUL56通过其PY基序显著下调Nedd4的表达。这些结果证实了PRVpUL56与Nedd4主要通过WW结构域/PY基序模式发挥相互作用,并下调Nedd4的表达。因此,本研究为阐明PRVpUL56与宿主相互作用的分子机制提供了参考依据。     

Differential expression of Golgi mannosidaseⅡ in different differentiated gastric carcinoma cell lines and tissues

AIM: To explore the role of Golgi mannosidase Ⅱ(GMⅡ) in the development of gastric carcinoma by analysis of the relationship between differential expression of GMⅡ and differentiation of gastric carcinoma cell lines and tissues. METHODS: Thirty cases of human normal gastric tissues and 38 cases of gastric adenocarcinoma tissues were selected. Three different differentiated gastric carcinoma cell lines (MKN-28, SGC-7901 and BGC-823) and a normal gastric epithelial cell line GES-1 were cultured in vitro. The mRNA levels of GMⅡ were detected by RT-PCR, and the protein expression was detected by immunohistochemistry and Western blotting. RESULTS: GMⅡ was mainly distributed in cytoplasm. The positive rates of GMⅡ in 30 cases of human normal gastric tissues, 8 cases of well-differentiated, 18 cases of moderately-differentiated and 12 cases of poorly-differentiated gastric cancer tissues were 53% (16/30), 63% (5/8), 83% (15/18) and 100% (12/12), respectively. The expression of GMⅡ was gradually increased in normal gastric epithelial cell line and in well, moderately and poorly-differentiated gastric cancer cell lines by cell-attached coverslip. Compared with normal gastric epithelial cell line, 3 gastric carcinoma cell lines showed the higher expression of GMⅡ at mRNA and protein levels (P<0.05). Furthermore, GMⅡ expression in poorly-differentiated gastric carcinoma cell line BGC823 was the highest, and the lowest expression of GMⅡ was the well-differentiated cell line MKN-28. Compared with normal gastric epithelial tissues, gastric carcinoma tissues showed the higher expression of GMⅡ at mRNA and protein levels (P<0.05), and the highest was the poorly-differentiated carcinoma tissues. The expression of GMⅡ at mRNA and protein levels in normal gastric tissues was the lowest. CONCLUSION: GMⅡ is involved in the development and progression of gastric cancer. The expression of GMⅡ is highly related to the poorly-differentiated gastric cancer.