盘基网柄菌柄细胞分化过程中gp150 分子的作用

饥饿的盘基网柄菌进入多细胞发育期后,在细胞疏松结合阶段,gp150分子分布在多细胞体外周;在蛞蝓体阶段,gp150分子把蛞蝓体分成前孢子细胞区和前柄细胞区两个部分,在分化成柄细胞的区域内gp150分子的量逐渐增多.实验结果表明gp150分子与柄细胞分化有密切关系.     

Altruism and social cheating in the social amoeba Dictyostelium discoideum

The social amoeba, Dictyostelium discoideum, is widely used as a simple model organism for multicellular development, but its multicellular fruiting stage is really a society. Most of the time, D. discoideum lives as haploid, free-living, amoeboid cells that divide asexually. When starved, 10(4)-10(5) of these cells aggregate into a slug. The anterior 20% of the slug altruistically differentiates into a non-viable stalk, supporting the remaining cells, most of which become viable spores. If aggregating cells come from multiple clones, there should be selection for clones to exploit other clones by contributing less than their proportional share to the sterile stalk. Here we use microsatellite markers to show that different clones collected from a field population readily mix to form chimaeras. Half of the chimaeric mixtures show a clear cheater and victim. Thus, unlike the clonal and highly cooperative development of most multicellular organisms, the development of D. discoideum is partly competitive, with conflicts of interests among cells. These conflicts complicate the use of D. discoideum as a model for some aspects of development, but they make it highly attractive as a model system for social evolution.     

盘基网柄菌发育期间蛞蝓体的形态学研究

用吖啶橙和Hoechst 33342两种活体荧光染料,通过荧光显微镜观察,来了解盘基网柄菌多细胞发育期间蛞蝓体阶段中出现的细胞分化及凋亡特点.结果发现:随着发育进程,将发育成柄细胞的前柄细胞先被染成蓝绿色,然后被染成绿色,再成橙色,最后为无色,这些分别表示了前柄细胞不同的凋亡阶段.在蛞蝓体后期其内部出现一条"通道".得出结论,蛞蝓体是盘基网柄菌细胞分化和衰退的起始阶段,其形态发生了巨大的变化.前柄细胞从蛞蝓体前期开始凋亡,但并不是马上死亡,而是一个渐进的凋亡过程.     

盘基网柄菌KAx-3中类免疫细胞的初步研究

以Ethidium bromide(EB)代替盘基网柄菌生长环境中所遇到的毒物,检测盘基网柄菌中是否存在能吞噬毒物的特定细胞.荧光显微镜下观察,细胞丘时期仅能看到EB加入后显现的暗红色轮廓;Hoechst33342定位细胞核,以确证EB被吞噬进细胞内;蛞蝓体时期少数细胞有明显的强荧光反应,随着蛞蝓体的爬行,含EB的细胞团被丢弃在遗弃的粘液鞘中.表明多细胞发育至蛞蝓体阶段存在一种能吞噬EB的细胞,最终将EB排出体外.流式细胞术检测发现,该类特殊的细胞主要来自于前柄细胞.结果显示,盘基网柄菌中存在简单的内在免疫细胞,为研究免疫系统在生物体的发生和发展提供了研究基础.     

Pleiotropy as a mechanism to stabilize cooperation

Most genes affect many traits. This phenomenon, known as pleiotropy, is a major constraint on evolution because adaptive change in one trait may be prevented because it would compromise other traits affected by the same genes. Here we show that pleiotropy can have an unexpected effect and benefit one of the most enigmatic of adaptations--cooperation. A spectacular act of cooperation occurs in the social amoeba Dictyostelium discoideum, in which some cells die to form a stalk that holds the other cells aloft as reproductive spores. We have identified a gene, dimA, in D. discoideum that has two contrasting effects. It is required to receive the signalling molecule DIF-1 that causes differentiation into prestalk cells. Ignoring DIF-1 and not becoming prestalk should allow cells to cheat by avoiding the stalk. However, we find that in aggregations containing the wild-type cells, lack of the dimA gene results in exclusion from spores. This pleiotropic linkage of stalk and spore formation limits the potential for cheating in D. discoideum because defecting on prestalk cell production results in an even greater reduction in spores. We propose that the evolution of pleiotropic links between cheating and personal costs can stabilize cooperative adaptations.     

细菌环二鸟苷酸介导的信号转导途径研究进展

环二鸟苷酸(c-di-GMP)是在细菌中广泛存在的第二信使分子,它介导的信号转导途径涉及调控细菌的运动、致病性、生物膜形成、细胞周期进程和有机溶剂耐受性等多种重要功能.该文主要从c-di-GMP的合成与降解、c-di-GMP的效应子及c-di-GMP的生理作用3方面对c-di-GMP介导的信号转导途径进行总结,并对该信号转导途径的研究前景进行了展望.     

WspR在大肠杆菌中的表达及其对c-di-GMP合成的影响

可得胶是土壤农杆菌ATCC31749分泌的一种胞外多糖,大多数细菌胞外多糖的合成受控于新近发现的第二信使环二鸟苷酸(c-di-GMP).细胞内c-di-GMP的产生受二鸟苷酸环化酶(diguanylate cyclase,DGC)合成和磷酸二酯酶(phosphodiesterase,PDE)降解两条途径调控.在结构上,通常DGC含有GGDEF结构域,PDE含有EAL结构域.研究表明从绿脓杆菌(P.aeruginosa)中得到的类趋化系统蛋白WspR具有GGDEF结构域,具有DGC活性.本实验将WspR转入大肠杆菌中得到了表达,并用颜色反应得到证实c-di-GMP在E.coli大量合成.为进一步研究ATCC31749中WspR的作用机制,促进可得胶的合成提供切实可行的依据.     

环二鸟苷酸对细菌运动调控的研究进展

细菌中的第二信使环二鸟苷酸(c-di-GMP)对细菌的运动性有调节作用.c-di-GMP调控鞭毛的生物合成、菌毛形成和菌毛蛋白的组成,以及其他一些与运动相关的蛋白的合成.细菌的运动性与其毒力、致病性、粘附性、趋化性、生物膜组成等密切相关.在革兰氏阴性细菌中,关于c-di-GMP的信号通路的研究较为清晰,而在革兰氏阳性细菌中,关于该信号转导通路的研究较少.此外,有关c-di-GMP的信号通路的研究主要集中在病原菌.该文主要综述了一些常见病原菌中c-di-GMP对其运动性的调控机制,为研究其他细菌c-di-GMP信号通路提供思路.     

Aminoglycoside antibiotics induce bacterial biofilm formation

Biofilms are adherent aggregates of bacterial cells that form on biotic and abiotic surfaces, including human tissues. Biofilms resist antibiotic treatment and contribute to bacterial persistence in chronic infections. Hence, the elucidation of the mechanisms by which biofilms are formed may assist in the treatment of chronic infections, such as Pseudomonas aeruginosa in the airways of patients with cystic fibrosis. Here we show that subinhibitory concentrations of aminoglycoside antibiotics induce biofilm formation in P. aeruginosa and Escherichia coli. In P. aeruginosa, a gene, which we designated aminoglycoside response regulator (arr), was essential for this induction and contributed to biofilm-specific aminoglycoside resistance. The arr gene is predicted to encode an inner-membrane phosphodiesterase whose substrate is cyclic di-guanosine monophosphate (c-di-GMP)-a bacterial second messenger that regulates cell surface adhesiveness. We found that membranes from arr mutants had diminished c-di-GMP phosphodiesterase activity, and P. aeruginosa cells with a mutation changing a predicted catalytic residue of Arr were defective in their biofilm response to tobramycin. Furthermore, tobramycin-inducible biofilm formation was inhibited by exogenous GTP, which is known to inhibit c-di-GMP phosphodiesterase activity. Our results demonstrate that biofilm formation can be a specific, defensive reaction to the presence of antibiotics, and indicate that the molecular basis of this response includes alterations in the level of c-di-GMP.     

The 2.8 Å crystal structure of the dynein motor domain

Dyneins are microtubule-based AAA(+) motor complexes that power ciliary beating, cell division, cell migration and intracellular transport. Here we report the most complete structure obtained so far, to our knowledge, of the 380-kDa motor domain of Dictyostelium discoideum cytoplasmic dynein at 2.8?? resolution; the data are reliable enough to discuss the structure and mechanism at the level of individual amino acid residues. Features that can be clearly visualized at this resolution include the coordination of ADP in each of four distinct nucleotide-binding sites in the ring-shaped AAA(+) ATPase unit, a newly identified interaction interface between the ring and mechanical linker, and junctional structures between the ring and microtubule-binding stalk, all of which should be critical for the mechanism of dynein motility. We also identify a long-range allosteric communication pathway between the primary ATPase and the microtubule-binding sites. Our work provides a framework for understanding the mechanism of dynein-based motility.     

KAx-3细胞和AK127细胞发育期间细胞骨架蛋白组分比较研究

采用生化抽提骨架蛋白和SDS-聚丙烯酰胺凝胶电泳等方法比较盘基网柄菌AK127细胞和KAx-3细胞骨架蛋白的结果显示，发育各时期的KAX-3和AK127细胞骨架蛋白在含量和组分方面存在一定的差异.在整个发育阶段两种类型细胞共有的蛋白组分是103.4kD、49.6kD、44.2kD、30.8kD、19.8kD和13.5kD条带，其中44.2kD和30.8kD是最稳定的细胞骨架成分，并不因AK127细胞缺失gp150分子而有所改变.它们与其他骨架蛋白组分一起为细胞提供有力的支持，保证发育的顺利进行.差别最为明显的蛋白条带是87.2kD、 68.2kD和40.7kD蛋白，由于同时期的突变型细胞不能显示这些蛋白条带；说明这些条带是发育所需的蛋白.值得注意的是突变型细胞也出现一条特征性的21.1kD的蛋白条带.分析认为这些变化一方面与盘基网柄菌发育过程有关，另一方面与突变细胞缺乏gp150分子有密切关系.     

注CO2前置段塞+N2顶替提高采收率机理

SJ油田为一低渗透油藏,天然能量低。受CO2气源不足以及油井管柱抗腐蚀能力差的限制,持续的CO2-EOR不适合SJ油田的实际情况。鉴于此,一个改进的对策是用N2推动的CO2前置段塞驱代替持续的CO2驱油。本文根据SJ油田先导试验区的流体特征,对比了连续注入CO2驱油和N2推动的CO2前置段塞混相驱油的效果和机理。在注CO2、N2细管驱替效率及最小混相压力实验测试基础上,通过注CO2、N2与地层原油多级接触混相驱机理相态模拟,长细管前置CO2混相驱替+后续N2段塞顶替驱替机理一维数值模拟研究,分析了前置CO2段塞+后续N2顶替驱油时原油与注入气的互溶情况、气驱界面张力变化规律、气驱过程中C2—C6的中间烃组分在油气两相中的分布、气驱过程中油气两相的黏度以及密度变化。结果表明SJ油田实施前置CO2段塞+后续N2顶替驱油时,后续的N2与前置的CO2段塞不会出现严重的扩散弥散,注气前缘仍能保持CO2的富集并实现稳定的混相驱油即在注气总量相同的情况下,N2推动的CO2前置段塞驱可以获得与持续的CO2驱相同的驱油效果;同时减少了CO2的注入量,从而可减缓CO2长期注入对油井管柱产生的腐蚀。所得认识对CO2驱提高采收率技术的改进和发展具有一定的启发作用。     

盘基网柄菌细胞发育中gp150分子与凋亡蛋白作用分析

用Percoll密度梯度技术分离和收集盘基网柄菌前柄和前孢子细胞,Western blot分析gp150分子和胱天蛋白酶在前孢子细胞和前柄细胞两种类型细胞中的表达情况.结果显示：只能在前柄细胞中检测到gp150蛋白条带,并随细胞发育蛋白的量逐渐增加,提示gp150蛋白的表达量与发育时间,前柄细胞分化有密切关系;在前柄细胞中能检测到31.5 kD和37.5 kD分子量大小的凋亡蛋白,且蛋白量也是随发育时间有所增加,在两种类型细胞中都可检测38.2 kD的凋亡蛋白.这些数据表明盘基网柄菌细胞凋亡过程中有类似Caspase-3的蛋白表达,它们的存在与细胞凋亡存在密切关系; gp150分子的表达与胱天蛋白酶的激活可能存在一定关系.     

反相高效液相色谱法分析盘基网柄菌对氨基酸的利用

为观察盘基网柄菌(Dictyostelium discoideum)在SIH培养基上对氧基酸的利用情况,以2.4-二硝基氯苯为衍生化试剂,研究了用反相高效液相色谱(RP-HPLC)测定氨基酸的方法.在60 min内16种氨基酸达到基线分离,峰面积与氨基酸浓度的线性相关系数为0.992～0.999.对发酵液样品的分析结果表明,盘基网柄菌对赖氨酸的利用最为彻底,发酵后期赖氨酸已被消耗完全.蛋氯酸、色氨酸,精氨酸、组氧酸也较易被盘基网柄菌利用,而对天冬氨酸、谷氨酸、甘氨酸、苏氨酸、脯氨酸、缬氨酸的需求不大.这一代谢特点为改进盘基网柄菌培养基组成提供依据.     

段塞流诱导柔性立管振动响应实验分析

在气液两相循环实验系统中开展了水动力段塞流诱导的悬链线型柔性立管振动响应测试,利用高速摄像非介入测试方法同步捕捉了柔性立管的振动位移与管内的段塞流动细节,预测了气体表观流速对水动力段塞流诱导柔性立管振动响应的影响规律,分析了振幅响应、模态权重、频谱变化以及管内流动特性.结果 表明,随着气体表观流速的增大,振动幅度逐渐增...     

C-di-GMP signaling and implications for pathogenesis of Mycobacterium tuberculosis

C-di-GMP is a ubiquitous bacterial second messenger that regulates a wide range of bacterial physiological processes including biofilm formation, virulence, motility and cell differentiation. Here, we have summarized our current knowledge on the upstream signaling factors and downstream effectors of c-di-GMP in addition to the interaction between c-di-GMP and eukaryotic organisms. New discoveries in these areas have enriched our understanding of the diversity of c-di-GMP signaling pathways and provide important clues for us to explore the roles of c-di-GMP signaling in human pathogens such as Mycobacterium tuberculosis.     

Adherens junctions and beta-catenin-mediated cell signalling in a non-metazoan organism

Mechanical forces between cells have a principal role in the organization of animal tissues. Adherens junctions are an important component of these tissues, connecting cells through their actin cytoskeleton and allowing the assembly of tensile structures. At least one adherens junction protein, beta-catenin, also acts as a signalling molecule, directly regulating gene expression. To date, adherens junctions have only been detected in metazoa, and therefore we looked for them outside the animal kingdom to examine their evolutionary origins. The non-metazoan Dictyostelium discoideum forms a multicellular, differentiated structure. Here we describe the discovery of actin-associated intercellular junctions in Dictyostelium. We have isolated a gene encoding a beta-catenin homologue, aardvark, which is a component of the junctional complex, and, independently, is required for cell signalling. Our discovery of adherens junctions outside the animal kingdom shows that the dual role of beta-catenin in cell-cell adhesion and cell signalling evolved before the origins of metazoa.     

Myosin I is located at the leading edges of locomoting Dictyostelium amoebae

Movement of a eukaryotic cell along a substrate occurs by extension of lamellipodia and pseudopodia at the anterior and retraction at the posterior of the cell. The molecular and structural mechanisms of these movements are uncertain. Dictyostelium discoideum contains two forms of myosin. Here we show by immunofluorescence microscopy that non-filamentous myosin I occurs at the leading edges of the lamellipodial projections of migrating Dictyostelium amoebae, which are devoid of myosin II, whereas filamentous myosin II is concentrated in the posterior of the cells. On the basis of these locations of the two forms of myosin and their known biochemical and biophysical properties, we suggest that actomyosin I may contribute to the forces that cause extension at the leading edge of a motile cell, while the contraction of actomyosin II at the rear squeezes the cell mass forward. Myosin I isozymes might have similar roles in metazoan cells, for example at the leading edges of neuronal growth cones, and in the extension of lamellipodia and pseudopodia of leukocytes, macrophages and fibroblasts.     

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.