Effect of internal deletion of Myosin Ⅱ on growth and development of Dictyostelium discoideum

Four deletion mutant Dictyostelium myosin Ⅱ heavy chain genes, MyΔ824-941 (Δ1/ 3S2), MyΔ934-1454 (ΔS2), MyΔ934-1194 (ΔS2-1) and MyΔ1157-1454 (ΔS2-2), were transformed by standard electroporation into mhcA- cells (T-null), a mutant Dictyostelium cell devoid of endogenous myosin Ⅱ heavy chain gene. The growth, development and formation of fruiting bodies of cells expressing those mutant myosin Ⅱ s under suspension culture were investigated by comparison with the wild type cell. The results indicate that internal deletion of myosin Ⅱ affects the growth and development of Dictyostelium. Furthermore, the longer the length of deletion , the more serious the defect in phenotype.     

盘基网柄菌(Dictyostelium discoideum)KAX-3和AK127细胞形态发生和同工酶的比较研究

比较了盘基网柄菌(Dictyostelium discoideum)野生型细胞KAx-3和突变型细胞AK127在多细胞发育过程中的形态发生.两者有明显的区别;突变细胞的发育只能停留在细胞疏松聚集阶段,而野生型细胞能顺利完成整个发育过程.利用SDS-PAGE电泳比较分析了两者在重要发育阶段的三种同工酶:醋酶(EST)、苹果酸脱氢酶(MDH)和谷氨酸脱氢酶(GDH).结果发现AK127细胞发育阶段含有两种醋酶成分a和p,而KAX-3细胞中只有一种醋酶a;而且AK127细胞中苹果酸脱氢酶(MDH)含量在发育后14 h和16 h均高于KAX-3细胞;谷氨酸脱氢酶(GDH)同工酶在发育后14 h时相对含量高于KAX-3细胞,而在发育后16 h含量却低于KAX-3细胞.这些数据显示了两种类型细胞的同工酶有明显的差异;表明突变细胞基因的表达发生了一定的改变,由此说明了gp150分子在盘基网柄菌细胞发育中有重要作用.     

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.     

Identification of an actin-binding protein from Dictyostelium as elongation factor 1a

Indirect evidence has implicated an interaction between the cytoskeleton and the protein synthetic machinery. Two recent reports have linked the elongation factor 1a (EF-1a) which is involved in protein synthesis, with the microtubular cytoskeleton. In situ hybridization has, however, revealed that the messages for certain cytoskeletal proteins are preferentially associated with actin filaments. ABP-50 is an abundant actin filament bundling protein of native relative molecular mass 50,000 (50K) isolated from Dictyostelium discoideum. Immunofluorescence studies show that ABP-50 is present in filopodia and other cortical regions that contain actin filament bundles. In addition, ABP-50 binds to monomeric actin in the cytosol of unstimulated cells and the association of ABP-50 with the actin cytoskeleton is regulated during chemotaxis. Through complementary DNA sequencing and subsequent functional analysis, we have identified ABP-50 as D. discoideum EF-1a. The ability of EF-1a to bind reversibly to the actin cytoskeleton upon stimulation could provide a mechanism for spatially and temporally regulated protein synthesis in eukaryotic cells.     

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.     

A mutation altering the function of a carbohydrate binding protein blocks cell-cell cohesion in developing Dictyostelium discoideum.

In Dictyostelium discoideum, carbohydrate binding proteins (CBPs) or lectins have been implicated in the molecular basis of cellular cohesion. To determine the role of these CBPs, we have attempted to isolate structural gene mutants in which the CBPs have a defective affinity for carbohydrate ligands. We now report the isolation of a spontaneous, cross-reacting material (CRM) mutant which is non-cohesive and fails to develop. The mutant seems to have a defect in the structural gene for one of the two developmentally regulated carbohydrate binding proteins (CBP-26), which renders it unable to bind to galactose-containing ligands. The fact that wild-type cells interact with the mutant and carry it through development strongly supports a model of cell-cell interaction in which cohesion is mediated by complementary molecules.     

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.     

Peptide selection by MHC class I molecules.

Synthetic peptides have been used to sensitize target cells and thereby screen for epitopes recognized by T cells. Most epitopes of cytotoxic T lymphocytes can be mimicked by synthetic peptides of 12-15 amino acids. Although in specific cases, truncations of peptides improves sensitization of target cells, no optimum length for binding to major histocompatibility complex (MHC) class I molecules has been defined. We have now analysed synthetic peptide captured by empty MHC class I molecules of the mutant cell line RMA-S. We found that class I molecules preferentially bound short peptides (nine amino acids) and selectively bound these peptides even when they were a minor component in a mixture of longer peptides. These results may help to explain the difference in size restriction of T-cell epitopes between experiments with synthetic peptides and those with naturally processed peptides.     

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.     

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.     

不同介质中分化逆转对细胞粘菌mRNA稳定性影响

细胞粘菌(Dictyostelium discoideum)发育后期细胞被重新分散于营养培养基或缓冲液后,mRNAs稳定性在这二种介质中的变化相似。一些mRNAs稳定性下降,快速降解,另一些mRNAs不受影响。环状AMP选择性地保护其中一些受快速降解的mRNAs。放线菌酮有稳定mRNA作用。受检mRNAs在不同分化逆转介质中的相同效应表明,营养条件不是引起分化逆转细胞mRNA稳定性变化的因素。     

盘基网柄菌野生型KAx-3和突变型AK127细胞mRNA差异显示分析

为研究gp150蛋白调控盘基网柄菌发育相关基因的功能,用mRNA差异显示技术比较分析盘基网柄菌发育14 h的KAx-3细胞和AK127细胞.结果显示两种类型细胞基因表达有明显差异,突变细胞不能表达275 bp片段.对其测序分析后发现差异片断与编码组氨酸激酶、STATc蛋白及同源框蛋白等的基因中的一段序列有很高的相似性.推测gp150蛋白的缺失引起某些调控因子表达异常,从而使突变细胞的基因表达不正常,最终导致细胞不能完成多细胞发育.     

Characterization of a naturally processed MHC class II-restricted T-cell determinant of hen egg lysozyme

Compelling evidence indicates that T cells recognize complexes formed by major histocompatibility complex-encoded molecules and antigenic peptide fragments. This is based largely on the ability of small synthetic peptides to substitute for naturally processed antigen in stimulating T cells. Naturally processed fragments of exogenous antigen are thought to arise by limited proteolytic degradation of native antigen inside acidic compartments of antigen-presenting cells, but until now no physiologically processed antigen has been directly analysed. Here we report the characterization of physiologically processed antigen eluted from mouse class II major histocompatibility complex I-Ed molecules. The antigenic material corresponds to a previously described antigenic determinant of hen egg lysozyme (HEL 107-116) and has a relative molecular mass Mr of about 2,000. HPLC analysis identified at least two or three separate molecular species, suggesting limited, albeit significant, heterogeneity of naturally processed peptides. Finally, under our experimental conditions, it was calculated that a substantial proportion (10-40%) of I-Ed molecules were occupied by these HEL-derived antigenic determinants.     

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.     

Phenotypic changes induced by a mutated ras gene during the development of Dictyostelium transformants

The ras proto-oncogene, found in all eukaryotes so far examined, encode s a protein with guanine nucleotide-binding and GTPase activity. Gene disruption experiments in yeast indicate that ras is essential for cell growth. Anit-sense mutagenesis approaches suggest that this is also true for Dictyostelium. Most mutations causing an amino-acid substitution for Gly 12 result in decreased GTPase activity and produce a transforming phenotype. In yeast, a Gly 19---- Val 19, missense mutation (Gly 19 is similar to Gly 12 in mammalian and Dictyostelium ras proteins) causes a series of dominant phenotypes, including elevated adenylate cyclase activity. In mammalian cells there is no evidence that ras activates adenylate cyclase activity. D. discoideum contains a single ras gene (Dd-ras) that encodes a protein very similar to the mammalian ras protein and identical to c-ras at the potentially transforming positions. Dd-ras is expressed in vegetative cells and later in development in prestalk cells whereas ras protein is found in vegetative and developing cells. In the migrating pseudoplasmodium, ras protein is found in prestalk but not prespore cells, suggesting it is involved in the function and/or differentiation of the anteriorly localized prestalk cells. In this report we examine the effects of expression of a Dd-ras gene carrying a Gly-12----Thr 12 missense mutation.     

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.     

An embryo protein induced by SV40 virus transformation of mouse cells

A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.     

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.     

Synthetic design of crystalline inorganic chalcogenides exhibiting fast-ion conductivity

Natural porous solids such as zeolites are invariably formed with inorganic cations such as Na(+) and K(+) (refs 1, 2). However, current research on new porous materials is mainly focused on the use of organic species as either structure-directing or structure-building units; purely inorganic systems have received relatively little attention in exploratory synthetic work. Here we report the synthesis of a series of three-dimensional sulphides and selenides containing highly mobile alkali metal cations as charge-balancing extra-framework cations. Such crystalline inorganic chalcogenides integrate zeolite-like architecture with high anionic framework polarizability and high concentrations of mobile cations. Such structural features are particularly desirable for the development of fast-ion conductors. These materials demonstrate high ionic conductivity (up to 1.8 x 10(-2) ohm(-1) cm(-1)) at room temperature and moderate to high humidity. This synthetic methodology, together with novel structural, physical and chemical properties, may lead to the development of new microporous and open-framework materials with potential applications in areas such as batteries, fuel cells, electrochemical sensors and photocatalysis.