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.     

The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium

Cyclic di-(3′:5′)-guanosine monophosphate (c-di-GMP) is a major prokaryote signalling intermediate that is synthesized by diguanylate cyclases and triggers sessility and biofilm formation. We detected the first eukaryote diguanylate cyclases in all major groups of Dictyostelia. On food depletion, Dictyostelium discoideum amoebas collect into aggregates, which first transform into migrating slugs and then into sessile fruiting structures. These structures consist of a spherical spore mass that is supported by a column of stalk cells and a basal disk. A polyketide, DIF-1, which induces stalk-like cells in vitro, was isolated earlier. However, its role in vivo proved recently to be restricted to basal disk formation. Here we show that the Dictyostelium diguanylate cyclase, DgcA, produces c-di-GMP as the morphogen responsible for stalk cell differentiation. Dictyostelium discoideum DgcA synthesized c-di-GMP in a GTP-dependent manner and was expressed at the slug tip, which is the site of stalk cell differentiation. Disruption of the DgcA gene blocked the transition from slug migration to fructification and the expression of stalk genes. Fructification and stalk formation were restored by exposing DgcA-null slugs to wild-type secretion products or to c-di-GMP. Moreover, c-di-GMP, but not cyclic di-(3′:5′)-adenosine monophosphate, induced stalk gene expression in dilute cell monolayers. Apart from identifying the long-elusive stalk-inducing morphogen, our work also identifies a role for c-di-GMP in eukaryotes.     

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.     

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.     

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.     

Social evolution: kin preference in a social microbe

Kin recognition helps cooperation to evolve in many animals, but it is uncertain whether microorganisms can also use it to focus altruistic behaviour on relatives. Here we show that the social amoeba Dictyostelium purpureum prefers to form groups with its own kin in situations where some individuals die to assist others. By directing altruism towards kin, D. purpureum should generally avoid the costs of chimaerism experienced by the related D. discoideum.     

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.     

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.     

Primitive agriculture in a social amoeba

Agriculture has been a large part of the ecological success of humans. A handful of animals, notably the fungus-growing ants, termites and ambrosia beetles, have advanced agriculture that involves dispersal and seeding of food propagules, cultivation of the crop and sustainable harvesting. More primitive examples, which could be called husbandry because they involve fewer adaptations, include marine snails farming intertidal fungi and damselfish farming algae. Recent work has shown that microorganisms are surprisingly like animals in having sophisticated behaviours such as cooperation, communication and recognition, as well as many kinds of symbiosis. Here we show that the social amoeba Dictyostelium discoideum has a primitive farming symbiosis that includes dispersal and prudent harvesting of the crop. About one-third of wild-collected clones engage in husbandry of bacteria. Instead of consuming all bacteria in their patch, they stop feeding early and incorporate bacteria into their fruiting bodies. They then carry bacteria during spore dispersal and can seed a new food crop, which is a major advantage if edible bacteria are lacking at the new site. However, if they arrive at sites already containing appropriate bacteria, the costs of early feeding cessation are not compensated for, which may account for the dichotomous nature of this farming symbiosis. The striking convergent evolution between bacterial husbandry in social amoebas and fungus farming in social insects makes sense because multigenerational benefits of farming go to already established kin groups.     

盘基网柄菌allC的克隆、表达和多克隆抗体的制备

运用RT-PCR技术从盘基网柄菌(Dictyostelium discoideum)总mRNA中克隆到了尿囊酸酶基因(allC),该基因编码区开放读框长1 100bp,编码的蛋白约42kD.由于是在野生型和突变型细胞中差异表达的片段,表明该基因在盘基网柄菌多细胞发育中起到重要作用,因此将allC克隆入融合表达载体pET-32a(+),在大肠杆菌E.coliBL21(DE3)中进行诱导表达带有6个组氨酸标签的尿囊酸酶(ALC)融合蛋白,经镍柱亲和层析,获得了电泳纯蛋白.用纯化的融合蛋白免疫新西兰大白兔,制备多克隆抗体.ELISA测得制备的抗ALC蛋白的多克隆抗体的效价可达1∶64 000,Western Blotting检测证明该抗体有较强的针对ALC蛋白的专一性.这些数据表明重组质粒表达的ALC融合蛋白具有良好的抗原性,制备ALC的多克隆抗体有良好特异性和效价,能够满足针对ALC免疫印迹和细胞内定位检测等实验要求,为深入研究ALC蛋白在盘基网柄菌多细胞发育的功能作用提供了有力工具.     

面向组织工程的多细胞结构三维自组装成形方法的研究进展

多细胞结构是由细胞群体构成的有机体,其体外构建对于组织工程和再生医学的发展具有重要的基础意义。利用细胞自身的自组织特性构建三维(3D)多细胞结构正成为生物制造和组织再生的一种重要途径,并受到越来越多的关注。对三维多细胞结构的自组装式构建与调控的相关基础研究及关键技术进行了综述及分析,主要涉及凝胶内3D细胞培养、多细胞结构可控形成,及其与图灵反应-扩散机制的联系等方面的研究工作。为进一步研究多细胞3D自组装机理,使得该自组装过程可控,且满足同时调控外部施加和细胞自身分泌的作用因子的浓度梯度分布的需求,提出对内部结构特征梯度化的3D凝胶体内细胞3D自组装模型进行研究,以推进三维多细胞结构及组织前体形成的理性调控技术。     

混输管路气液两相流模型的建立与验证

针对管路气液两相流,建立了一种捕获段塞流的形成和发展过程;并进行气液两相流水力计算的组合模型。以滑移速度和液相连续性方程为基础,通过建立动坐标系来求解液相连续性方程,得到以持液率的变化来反映段塞的形成和发展。加入地形起伏的影响和压降计算关系式,对气液混输管路进行水力计算。分别使用实验数据和工程现场数据来验证模型的准确性。实验数据分别来自于实验环道和大庆油田现场数据。结果显示:模型计算大部分压降误差在±17%以内,大部分积液量误差在±11%以内,具有较好的精度。     

Mining potential social relationship with active learning in LBSN

Rapid development of local-based social network ( LBSN ) makes it more convenient for re-searchers to carry out studies related to social network.Mining potential social relationship in LBSN is the most important one.Traditionally, researchers use topological relation of social network or tel-ecommunication network to mine potential social relationship.But the effect is unsatisfactory as the network can not provide complete information of topological relation.In this work, a new model called PSRMAL is proposed for mining potential social relationships with LBSN.With the model, better performance is obtained and guaranteed, and experiments verify the effectiveness.     

盘基网柄菌发育中尿囊酸酶表达的定量定位研究

用免疫荧光技术对KAx-3多细胞发育不同阶段的尿囊酸酶进行定位观察,用Western blot分析野生型细胞KAx-3 和突变型细胞AK127多细胞发育中尿囊酸酶的表达情况.结果显示：在细胞聚集阶段,尿囊酸酶在盘基网柄菌细胞膜附近存在较多;在细胞丘阶段,尿囊酸酶在细胞丘外层细胞中荧光强度较强;在蛞蝓体阶段,尿囊酸酶在前柄细胞中的表达量明显多于前孢子细胞;在子实体成熟的过程中,在前柄细胞区与前孢子细胞区交界处荧光强度最强,该区域内细胞将分化成前柄细胞B.据此推测尿囊酸酶的定位表达可能与盘基网柄菌细胞分化的类型相关.Western blot结果显示：在KAx-3 发育过程中尿囊酸酶的表达量呈现出逐渐上升的趋势,发育至18 h左右达到最大值;而AK127中尿囊酸酶的表达量始终在低水平徘徊.这表明gp150 的缺失影响了尿囊酸酶的表达.实验结果提示,尿囊酸酶的表达量与发育时间有关,并且这种表达量的变化与gp150存在着密切的关系.     

Mechanism of sequential induction of cell-type specific mRNAs in Dictyostelium differentiation

Upon starvation, the cellular slime mould Dictyostelium discoideum initiates a 24-h programme of differentiation. Within 6 h, cells move towards aggregation centres in response to pulsatile synthesis and secretion of cyclic AMP. At about 12 h, aggregates of 10(5) cells are formed, held together by newly made surface adhesion molecules. The cells then differentiate into the two principal types found in the terminal stage of development, spores and stalks. Here we show that the chemotaxis and aggregation stages of this developmental programme can be described as a series of sequential events in which these extracellular signals--starvation, cyclic AMP and cell-cell contact--induce specific, sequential changes in the pattern of gene expression.     

Developmental cheating in the social bacterium Myxococcus xanthus

Cheating is a potential problem in any social system that depends on cooperation and in which actions that benefit a group are costly to individuals that perform them. Genetic mutants that fail to perform a group-beneficial function but that reap the benefits of belonging to the group should have a within-group selective advantage, provided that the mutants are not too common. Here we show that social cheating exists even among prokaryotes. The bacterium Myxococcus xanthus exhibits several social behaviours, including aggregation of cells into spore-producing fruiting bodies during starvation. We examined a number of M. xanthus genotypes that were defective for fruiting-body development, including several lines that evolved for 1,000 generations under asocial conditions and others carrying defined mutations in developmental pathways, to determine whether they behaved as cheaters when mixed with their developmentally proficient progenitor. Clones from several evolved lines and two defined mutants exhibited cheating during development, being overrepresented among resulting spores relative to their initial frequency in the mixture. The ease of finding anti-social behaviours suggests that cheaters may be common in natural populations of M. xanthus.     

可扩展的社交多媒体安全分享方法研究

社交多媒体在社交网络中的分享面临安全与隐私威胁,多媒体加密可以解决保密性问题,数字指纹技术可以实现叛逆者追踪,但现有数字指纹技术还不能应用于大规模社交网络。现有指纹码不能容纳社交网络中数以亿计的海量用户,为每位用户单独生成指纹拷贝,对数亿计的用户而言,会造成巨额的空间和时间开销,此类问题的产生源于现有的安全分享算法缺乏可扩展性。面向社交多媒体安全分享在确保保密性、可追踪性的同时,更需要保证可扩展性,以适应社交网络的动态变化。基于混沌加密和社交网络指纹技术,提出一种面向社交多媒体安全分享的树结构 Haar（tree structure Haar,TSH）变换域的联合多媒体指纹与加密技术,实验结果与理论分析证明了该方法不仅可以保证社交多媒体的安全分享,而且可以实现可扩展性。     

Autonomous chaotic behaviour of the slime mould Dictyostelium discoideum predicted by a model for cyclic AMP signalling

How sustained oscillations lose their periodicity and thus give rise to chaos was first analysed in mathematical models, then observed in chemical systems such as the Belousov-Zhabotinsky reaction where chaos is autonomous because it originates from endogenous kinetic mechanisms. In contrast, chaos can also be obtained by periodically forcing an oscillatory system, as shown, for example, in cardiac cells and yeast glycolysis. Biochemical evidence for autonomous chaos has been obtained both in vitro for the peroxidase reaction and in enzymatic models not based directly on experimental systems. We report here the occurrence of autonomous chaos in a realistic model for the cyclic AMP signalling system of the slime mould Dictyostelium discoideum, based on receptor modification. This model is also capable of bursting, a phenomenon characteristic of some pacemaker neurones such as R15 in Aplysia. Whereas bursting has not been observed in D. discoideum, our model suggests that 'aperiodic signalling' in the mutant Fr17 provides the first example of autonomous chaos occurring spontaneously at the cellular level.     

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.     

Pinyon jays use transitive inference to predict social dominance

Living in large, stable social groups is often considered to favour the evolution of enhanced cognitive abilities, such as recognizing group members, tracking their social status and inferring relationships among them. An individual's place in the social order can be learned through direct interactions with others, but conflicts can be time-consuming and even injurious. Because the number of possible pairwise interactions increases rapidly with group size, members of large social groups will benefit if they can make judgments about relationships on the basis of indirect evidence. Transitive reasoning should therefore be particularly important for social individuals, allowing assessment of relationships from observations of interactions among others. Although a variety of studies have suggested that transitive inference may be used in social settings, the phenomenon has not been demonstrated under controlled conditions in animals. Here we show that highly social pinyon jays (Gymnorhinus cyanocephalus) draw sophisticated inferences about their own dominance status relative to that of strangers that they have observed interacting with known individuals. These results directly demonstrate that animals use transitive inference in social settings and imply that such cognitive capabilities are widespread among social species.