Pseudomonas Type III effector AvrPto suppresses the programmed cell death induced by two nonhost pathogens in Nicotiana benthamiana and tomato

Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell. Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive. Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions. Pseudomonas syringae pv. tomato T1 causes an HR-like cell death on Nicotiana benthamiana. This rapid cell death was delayed significantly in plants inoculated with P. syringae pv. tomato expressing avrPto. In addition, P. syringae pv. tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines. Transient expression of avrPto in both N. benthamiana and tomato prf3 plants also was able to suppress nonhost HR. Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens. AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions. Experiments with avrPto mutants revealed several residues important for the suppression effects. AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and 196T enhanced suppression of cell death in N. benthamiana. These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion.     

在基于BAC的EB病毒基因组中引入突变

为了在Epstein-Barr病毒(EBV)172kb的基因组中引入突变以研究基因功能,建立了一种简单有效的基因操作方法.在载体pcDNA3.1( )上操作,将两端含有重组蛋白FLP识别位点(FRT)的卡那霉素筛选标记基因(kan)与鼻咽癌(NPC)来源的、包含LMP1基因全长ORF的gDNA"无缝"连接(无外源序列插入).连接后的kan-LMP1线性DNA片段经转化、由λ噬菌体中redαβγ系统介导在E.coli中发生同源重组(ET克隆),用kan-LMP1替代了BAC-EBV(p2089)中相应的LMP1基因区域,然后经过重组蛋白FLP对FRT-kan-FRT特异性的识别,切除了引入的kan基因,留下一个69bp的FRT"疤痕".通过抗性筛选和对菌液进行PCR扩增可以鉴定突变子.这种经改进并程序化的方法.也适应于引入其它突变或在其它BAC-疱疹病毒基因组中引入突变.     

浙江海岛盐生植被研究Ⅱ──天然植被类型及开发利用

天然盐生植被是海岛盐生植被的主体,类型丰富,分布广泛,在海岛植被的研究及开发利用中占据重要地位。目前,天然盐生植被的分类尚无统一标准与系统。本文根据作者1990～1993年在浙江海岛用样方法(Ｈ.ＶｏｎＰｏｓｔ,1851;陈彦卓,1965)测定的样地数据,参照《中国植被》的分类方法,根据群落的外貌与结构、生态地理、动态和种类组成等特征进行分类,其中对群系级单位侧重于种类组成,并采用建群植物的重要值(Ｊ.Ｔ.ＣｕｒｔｉｓａｎｄＲ.Ｐ.ＭｃＩｎｔｏｓｈ,1951)作为分类的定量指标。据此,浙江海岛天然盐生植被可划分为3个植被型、8个群系组、18…     

重组人白细胞介素－2在离子色谱分离时的复性

采用ＨＰＩＥＣ应用ＣＭ交换柱分离纯化Ｅ．ｃｏｌｉ工程菌表达的ｒＩＬ－２,与空气氧化复性相比较,又有１倍以上的变性ｒＩＬ－２在色谱过程中得到复性,且溶液ｐＨ影响ｒＩＬ－２的复性和纯化效果：ｐＨ７．０时ｒＩＬ－２复性率高于ｐＨ８．０时,但纯度明显低于ｐＨ８．０时分离的ｒＩＬ－２．     

生物种群动态微分方程模型参数估计方法

本文以数值分析和最优化技术的有机结合为基础,提出了一种新的对动态微分方程模型直接进行数据拟合和参数估计方法,并以Logistic微分方程、生物种间竞争关系微分方程以及一种复合形态的Logistic微分方程为例进行了数据拟合试验．结果表明,该方法对各种动态微分方程模型均能进行最优拟合分析并求解其参数．同时发现,以前有的作者〔1,2,3,4,5〕提出的方法所得到的参数估计值存在系统误差且误差较大．     

Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

Valproate-Induced Epigenetic Upregulation of Hypothalamic Fto Expression Potentially Linked with Weight Gain

Cellular and Molecular Neurobiology - Valproate (VPA), a widely-used antiepileptic drug, is a selective inhibitor of histone deacetylase (HDAC) that play important roles in epigenetic regulation....     

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 10⁷ independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near‐infrared fluorescent proteins with enhanced intracellular brightness. The developed near‐infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near‐infrared fluorescent proteins enabled crosstalk‐free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual‐color near‐infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.