硬基网柄菌Dictyostelium firmibasis生活史的研究

本文研究了网柄细胞状黏菌中国新记录种硬基网柄菌Dictyostelium firmibasis从孢子-黏变形体-细胞集群-假原质团-成熟孢堆果的无性生活循环过程。结果表明：该种细胞状黏菌孢子萌发至少需要8h,孢子萌发释放出具有不规则形状的黏变形体,黏变形体无色并进行不规则的高速运动;黏变形体细胞集群为典型的辐射状;孢堆果对光极为敏感,在成熟发育期微弱的光刺激便会导致子实体生长畸形或停止生长并死亡;从集群开始形成到孢堆果成熟持续约12-14h,完成一个完整的生活史循环约需36-38h。     

大网柄菌Dictyostelium magnum生活史的研究

为了了解网柄菌生活史循环的整个过程,显微观察了大网柄菌Dictyostelium magnum在双凹载玻片及水琼脂培养基上的完整生长循环,记录了大网柄菌生活史中各阶段,即孢子、黏变形体、集群、假原质团、拔顶、孢堆果及再次释放孢子的显著特征,整个生活循环历时2－3d。     

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis.

Proteinases of the cellular slime mould Dictyostelium discoideum have been analysed using electrophoresis on polyacrylamide gels containing gelatin (gelatin/PAGE). Multiple proteinase forms were apparent in vegetative myxamoebae, but the presence of individual enzyme forms depended on the manner in which the cells were grown. Axenic cells had a characteristic A-pattern of proteinases consisting of six bands, the most active enzymes having apparent Mr values of 51,000 and 45,000 (these have been named ddCP51 and ddCP45, respectively). Some of the proteinases were also present in the medium, the major extracellular form was ddCP42, a 42,000-Mr enzyme. Cells grown in association with bacteria had a distinct B-pattern with three main enzymes that had apparent Mr values of 48,000, 43,000 and 38,000. All of the A- and B-pattern proteinases were most active at acid pH in the presence of dithiothreitol and were inhibited by various agents such as trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), leupeptin and chymostatin, which inactivate cysteine proteinases. One of the enzymes, ddCP30, was identified as cysteine proteinase B which had been purified and characterized previously [North, M.J. & Whyte, A. (1984) J. Gen. Microbiol. 130, 123-134]. During starvation of axenic cells in shaken suspensions some of the vegetative proteinases disappeared, ddCP42 was released from the cells and one new enzyme with an apparent Mr of 48,000 appeared. Addition of cyclic AMP had little effect on these changes. When the axenically grown myxamoebae underwent development on filters, similar changes in band pattern were observed and the aggregation stage was characterized by the presence of three cysteine proteinase bands (apparent Mr values of 48,000, 45,000 and 43,000). Proteinases, especially ddCP42, were released from the cells and could be collected from the buffer-saturated pads which supported the filters. The results demonstrate that cysteine proteinases are present throughout growth and development of D. discoideum and that the forms present are subject to nutritional and developmental regulation.     

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.     

Carnitine function and requirements during the life cycle.

L-Carnitine has been described as a "conditionally essential" nutrient for humans. Segments of the human population suggested as having a requirement for carnitine include infants (premature and full-term), patients on long-term parenteral nutrition, and perhaps children. The evidence to support these claims includes 1) low circulating carnitine concentrations; 2) abnormal (or at least different) circulating metabolite concentrations (free fatty acids, triglycerides, ketone bodies), and 3) very limited and inconsistent growth data. A number of subjective observations and anecdotal case reports have been offered in support of a requirement for carnitine. Exogenous carnitine is required to maintain "normal" (in the epidemiologic sense) plasma or serum carnitine concentrations in humans of all ages. But "functional carnitine deficiency," defined by abnormal clinical presentation correctable by carnitine administration, has not been demonstrated in an otherwise normal (nonpathologic) population. On the other hand, nutritional or pharmacological intervention with carnitine or its esters may be beneficial for very premature infants, infants and children with various clinical conditions associated with low circulating carnitine concentrations, and in some chronic diseases associated with the aging process.     

Inheritance of extrachromosomal ribosomal DNA during the asexual life cycle of Dictyostelium discoideum: examination by use of DNA polymorphisms.

Wild-type isolates of Dictyostelium discoideum exhibited differences in the size of restriction fragments of the extrachromosomal 88-kilobase ribosomal DNA (rDNA) palindrome. Polymorphisms in rDNA also were found among strains derived solely from the NC4 wild-type isolate. These variations involved EcoRI fragments II, III, and V; they included loss of the EcoRI site separating fragments II and V and deletion and insertion of DNA. More than one rDNA form can coexist in the same diploid or haploid cell. However, one or another parental rDNA tended to predominate in diploids constructed, using the parasexual cycle, between haploid NC4-derived strains and haploid wild-type isolates. In some cases, most if not all of the rDNA of such diploids were of one form after ca. 50 generations of growth. Segregant haploids, derived from diploids that possessed predominantly a single rDNA allele, possessed the same allele as the diploid and did not recover the other form. This evidence implies that replication does not proceed from a single chromosomal or extrachromosomal copy of the rDNA during the asexual life cycle of D. discoideum.     

Serological investigations of cell adhesion in the slime molds,Dictyostelium discoideum,Dictyostelium purpureum,and Polysphondylium violaceum

1. Antibodies to slime molds were produced by injecting D. discoideum and D. purpureum amebas from 48 hour cultures into rabbits. 2. Anti-D. discoideum and anti-D. purpureum sera caused agglutination of homologous amebas from 24 to 26 hour cultures, agglutination of certain heterologous amebas from 30 to 36 hour cultures, and agglutination of all heterologous amebas from 43 to 48 hour cultures. 3. The data show that new surface antigens are formed in cultures after 26 hours and it is suggested that the new antigens are concerned with cell adhesion. 4. The probable role of surface antigens in the interaction of cells of different species of slime molds was discussed.     

Esterase activity during the life cycle of Blastocladiella emersonii.

Total esterase activity was measured in extracts on Blastocladiella throughout its life cycle by the degradation of alpha-naphthyl acetate. A fivefold incease in activity, apparently due to the synthesis of new enzymes, was found during sporulation.     

Association of a cGMP-binding activity with nuclei isolated from amoebae of Dictyostelium discoideum and Polysphondylium violaceum.

When vegetative and early slug stage amoebae of Dictyostelium discoideum or Polysphondylium violaceum were lysed by filter breakage in a nuclear isolation buffer not containing detergents, substantial levels of a cGMP-binding activity with slow-dissociation kinetics were detected. After fractionation by centrifugation, 50% or more of this binding activity was associated with isolated nuclei. In addition, with Polysphondylium cells, the fraction of stable, nuclear-associated binding activity appeared to increase during cell aggregation. These results support the idea that cGMP might function in the nucleus during early development.     

Conservation and variation of ribosomal proteins in several species of the cellular slime molds Dictyostelium and Polysphondylium

Genus- and species-specific composition of ribosomal proteins was investigated in four species of the genus Dictyostelium (D. discoideum, D. purpureum, D. murcoroides and D. giganteum) and two species of the genus Polysphondylium (P. pallidum and P. violaceum). Ribosomal proteins were resolved by a high-resolution, two-dimensional gel method. In general, the numbers and distributions for the majority of ribosomal proteins were similar within the species of each genus, although some differences were detected. More differences were observed between Dictyostelium and Polysphondylium than among the individual species within each genus. Stage-specific ribosomal proteins previously demonstrated in D. discoideum were found to be developmentally regulated in other Dictyostelium species, and in both Polysphondylium species. The study shows that ribosomal proteins may be a potentially useful new biochemical parameter for the molecular taxonomy of the cellular slime molds.     

Macrocyst formation in three dictyostelid species,Dictyostelium monochasioides, Polysphondylium candidum, andP. pseudo-candidum

Macrocyst formation in the sexual cycle was found in three dictyostelid species:Dictyostelium monochasioides, Polysphondylium candidum, andP. pseudo-candidum. Mating tests suggested thatD. monochasioides andP. pseudocandidum were heterothallic andP. candidum was homothallic. The primary walls of macrocysts had partially or fully degenerated, while the inner walls, believed to be tertiary walls, showed an undulate structure.     

The cyclic adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum. Binding characteristics of aggregation-competent cells and variation of binding levels during the life cycle.

Both cyclic guanosine 3':5'-monophosphate and dithiothreitol stimulate binding of cyclic adenosine 3':5'-monophosphate (cAMP) to aggregation-competent amoebae. Both compounds appear to function solely by preventing the hydrolysis of cAMP by the cell-bound phosphodiesterase. The dissociation constant for binding of cAMP is 36 nM. Both cAMP binding and membrane-bound phosphodiesterase activities increase dramatically as cells develop aggregation competence, reach a maximum at about 11 hours, and remain at high levels for up to 48 hours if cells are maintained in shaken suspension. When amoebae are allowed to aggregate and develop naturally, binding of cAMP increases during aggregation, decreases during tip formation, and disappears during culmination. Phosphodiesterase activity parallels binding activity except that the decreased level after tip formation is retained throughout culmination. Two N-6-modified cAMP derivatives compete with cAMP for binding sites. One derivative is fluorescent (1,N-6-etheno-cAMP); the other is photolyzable [N-6(ethyl-2-diazomalonyl)cAMP]. This result opens the possibilities of using fluorescence quenching for assay of in vitro binding and of affinity labeling of binding sites. Competition by the derivatives is only partial, indicating possible heterogeneity of binding sites. Both compounds inhibit hydrolysis of cAMP by the membrane-bound phosphodiesterase.     

An anticarbohydrate monoclonal antibody inhibits cell-cell adhesion in many species of Dictyostelium but not of Polysphondylium.

The anticarbohydrate monoclonal antibody d-41 inhibits the adhesion of aggregating cells, as measured by an in vitro assay, in every species of Dictyostelium tested but in none of the species from the genus Polysphondylium. Although d-41 binds significantly to the surface of cells from both genera, the ability to inhibit adhesion correlates with the binding of the antibody to a few, mostly developmentally regulated, membrane-associated proteins in each of the species affected. Previous work in D. discoideum and D. purpureum have shown that the major d-41-b binding proteins from these species at this time in development are directly involved in the adhesion process. Therefore, the presence of the epitope on these proteins in the other species of Dictyostelium implicates them in the adhesion mechanism. The function of the carbohydrates containing the epitope is yet to be determined.     

Distribution of lectin during the life cycle of Phaseolus vulgaris L.

Phaseolus vulgaris L. cv. ‘garrafal encarnada’ plants have been utilized to study the distribution of lectins accumulated in the seeds and through the life cycle of the plant.The distribution of both, total proteins and lectins was studied in aqueous and saline (1 M NaCl) extracts from different parts and organs in four distinct stages of plant development.Our results showed that lectin concentration decreases sharply during the first weeks of the plant growth, reaching the lowest value in trifoliate leaf stage and increasing during the following phases of plant development. However, the presence of lectins have been detected in all the plant tissues through every phase of the life cycle.The observed differences on lectin levels (RIA) and lectin activity (hemagglutination), suggest the presence of different molecular forms of the lectin in aqueous and saline extracts of plant tissues.These results, as well as the observation about the fixation of lectin on the bacterial surface, support the idea that the function of lectins in the plant may not be limited to storage proteins, but may be involved in specific host-parasite recognition.     

The tricarboxylic acid cycle in Dictyostelium discoideum. A model of the cycle at preculmination and aggregation.

A preliminary model of tricarboxylic acid-cycle activity in Dictyostelium discoideum is presented. Specific-radioactivity labelling patterns of intra- and extra-mitochondrial pools are simulated by this model and compared with the experimental data. The model arrived at by this method shows the following features. (1) The cycle flux rate is approx. 0.4 mM/min. (2) Both fumarate and malate are compartmentalized at approx. 1:5 between cycle pools and non-cycle pools. These may represent mitochondrial and cytoplasmic pools. Citrate is compartmentalized at 1:10. Succinate appears to exist in three compartments, two of which become labelled by [14C]glutamate and only one by [14C]aspartate (3) Two pools of aspartate with two associated pools of oxaloacetate are necessary for simulation. (4) Exchange between the cycle and non-cycle pools of both citrate and fumarate occurs at very low rates of about 0.003 mM/min, whereas exchange between the malate pools is about 0.004 mM/min. The exchange reaction glutamate in equilibrium 2-oxoglutarate runs at approx. 15 times the cycle flux. (5) A reaction catalysed by "malic" enzyme is included in the model, as this reaction is necessary for complete oxidation of amino acid substrates. (6) Calculation of the ATP yield from the model is consistent with earlier estimates of ATP turnover if the activity of adenylate kinase is considered.