Regulation of levels of actin threonine phosphorylation during life cycle of Physarum polycephalum

Under various environmental stresses, the true slime mold Physarum polycephalum converts into dormant forms, such as microcysts, sclerotia, and spores, which can survive in adverse environments for a considerable period of time. In drought-induced sclerotia, actin is threonine phosphorylated, which blocks its ability to polymerize into filaments. It is known that fragmin and actin-fragmin kinase (AFK) mediate this phosphorylation event. In this work, we demonstrate that high levels of actin threonine phosphorylation are also found in other dormant cells, including microcysts and spores. As the threonine phosphorylation of actin in microcysts and sclerotia were induced by drought stress but not by other stresses, we suggest that drought stress is essential for actin phosphorylation in both cell types. Although characteristic filamentous actin structures (dot- or rod-like structures) were observed in microcysts, sclerotia, and spores, actin phosphorylation was not required for the formation of these structures. Prior to the formation of both microcysts and sclerotia, AFK mRNA expression was activated transiently, whereas fragmin mRNA levels decreased. Our results suggest that drought stress and AFK might be involved in the threonine phosphorylation of actin.     

Diphospho-myo-inositol phosphates during the life cycle of Dictyostelium and Polysphondylium.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.     

Trehalase expression during the germination of three resting stages of the dictyosteliaceae

Three resting stages (spores, microcysts, and macrocysts) of the Dictyosteliaceae were found to possess low levels of trehalase activity. A significant increase in the activity of trehalase was detected upon germination. The increase in activity appeared to be dependent on protein synthesis.     

Polysphondylium multicystogenum sp. nov., a new dictyostelid species from Sierra Leone, West Africa

Polysphondylium multicystogenum, a new heterothallic species of dictyostelids, is described based on three isolates collected from soils in Sierra Leone, West Africa. This species is characterized by sorophores with a combination of clavate base and ovoid to oblong tip cell, smaller spores and abundant microcyst production under the usual culture conditions for sorocarp formation at 20 C. This is the first report of Polysphondylium producing such abundant microcysts.     

Microcyst Germination in Myxococcus xanthus

Germination of glycerol-prepared microcysts of Myxococcus xanthus was studied. The sequence of morphological events during germination resembled that of germinating fruiting body-microcysts. The turbidity drop of a culture of germinating microcysts could be described by McCormick's formula derived for germinating Bacillus spores. The rate of uptake of labeled glycine and acetate did not change during germination. Temperature, aeration, and pH optima for germination were the same as for vegetative cell growth. Germination was induced by protein hydrolysates and the individual amino acids glycine, alanine, valine, aspartic acid, and glutamic acid. A number of organic compounds, including sugars, alcohols, aldehydes, ketones, organic acids, and chelating agents, did not induce germination. The inorganic ions HPO(4) (2-), Mg(++), Ca(++), and NH(4) (+) induced germination, although ionic strength was not a factor. Microcysts incubated in distilled water at concentrations greater than about 10(9) cells/ml germinated; supernatant fluid from such suspensions (germination factor) induced germination of less concentrated suspensions. The activity of germination factor was resistant to boiling, but was lost on charring and dialysis. Germination of microcysts and growth of vegetative cells was equally sensitive to a variety of metabolic inhibitors, including penicillin and chloramphenicol. Germination was more resistant than vegetative growth to inhibition by antibiotics of the streptomycin family and by actinomycin D.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.     

The fate of the cell envelopes of Myxococcus xanthus during microcyst germination

Summary By using an improved method for processing germinating microcysts for electron microscopy it was shown that no breakdown and resynthesis of the cell wall occured in M. xanthus during germination of microcysts. A third, dense cell wall layer, not described before for any species of myxobacteria, could be discerned when cells were fixed and embedded by a method described in Materials and Methods.     

Stable Messenger Ribonucleic Acid and Germination of Myxococcus xanthus Microcysts

We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.     

Glucan common to the microcyst walls of cyst-forming bacteria.

Chemical analysis indicated that D-glucose is tha major neutral monosaccharide present in the microcysts of a range of gram-negative bacteria. Varying amounts of other neutral sugars were found. The glucose was mainly present as a glucan that could be extracted from microcysts of representative strains with alkali or mild acid treatment. The glucan could be identified as an alpha-1,3-linked polymer on the basis of (i) periodate resistance of the extracted polymer and the material present in microcysts; (ii) lectin agglutination of the microcysts; (iii) lectin precipitation of the extracted glucans; and (iv) susceptibility of the glucan either in the walls or after extraction to a specific alpha-1,3-glucanase from Aspergillus nidulans, yielding glucose as the sole hydrolysis product. The galactosamine found in microcysts of Myxococcus xanthus by other workers is clearly a component of another polymer, distinct from the glucan. The presence of an alpha 1,3-linked glucan, common to microcyst walls of various bacterial genera, probably contributes to the rigidity of the walls of these forms and, inter alia, to their resistance to ultrasonic treatment. Preliminary experiments indicate that the gulcan is discarded on germination of the microcysts rather than being broken down by specific enzymes.     

In vitro metabolism or testosterone by breast microcysts

Breast microcysts are considered to be a normal findings in the adult female breast without any increased risk of developing carcinomatous change. Breast cysts fluid contains steroid but not studies have been reported on the ability of breast microcysts to metabolise steroid hormones. It was, therefore, the aim of this study to identify the metabolites formed on incubation of radiolabelled testosterone with microcysts. In all instances dihydrotestosterone and androstenedione were formed. Oestrogens were not identified. Tis study, therefore, provides evidence for th presence of 5-alpha-reductase and 17-oxidoreductase enzyme systems in breast microcysts.     

Induction of encystment of Polysphondylium pallidum amoeba

The induction of microcyst formation could be triggered in washed amoebae of the cellular slime mold Polysphondylium pallidum (strain-2) by the addition of 2 mM ethionine. Methionine at a ratio of 2: 1 with ethionine would inhibit microcyst induction by ethionine. The involvement of polyamines in morphogenesis was also shown. Putrescine (0.02 to 0.1 M) induced the formation of microcysts, whereas spermidine (2 to 4 mM) was capable of causing a fourfold reduction in 0.05 M putrescine-induced microcysts but incapable of inhibiting microcyst induction by 0.08 M itrescine. Glycerol (0.5 M or 0.4 mM) was also found to be an effective inducer of microcysts.     

Comparative Intermediary Metabolism of Vegetative Cells and Microcysts of Myxococcus xanthus

Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2). In addition, microcysts and vegetative cells showed a different distribution of (14)C-label from incorporated acetate.     

True divergent differentiation in a cellular slime mold, Polysphondylium pallidum.

Three abstract models of divergent cell differentiation to multiple cell types are presented. These differ primarily in the proportion of developmental events common to the pathways leading to each cell type. Two experimental approaches are outlined to determine which type best describes divergent differentiation occurring in a particular organism. The first technique is to describe and compare changes in labeling of specific polypeptides which characterize development to the several cell types. The second is to observe the ability of mutants which are blocked in one pathway to develop along alternate pathways. These approaches are applied to the case of Polysphondylium pallidum, where amebae develop into stalk cells, spores, or microcysts. It is concluded that cell differentiation in P. pallidum is of the truly divergent type in which developing cells show identical sequences of events until a branch point, and thereafter very different sequences of events.     

True Divergent Differentiation in a Cellular Slime Mold, Polysphondylium pallidum

Three abstract models of divergent cell differentiation to multiple cell types are presented. These differ primarily in the proportion of developmental events common to the pathways leading to each cell type. Two experimental approaches are outlined to determine which type best describes divergent differentiation occurring in a particular organism. The first technique is to describe and compare changes in labeling of specific polypeptides which characterize development to the several cell types. The second is to observe the ability of mutants which are blocked in one pathway to develop along alternate pathways. These approaches are applied to the case of Polysphondylium pallidum , where amebae develop into stalk cells, spores, or microcysts. It is concluded that cell differentiation in P. pallidum is of the truly divergent type, in which developing cells show identical sequences of events until a branch point, and thereafter very different sequences of events.     

Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique

The aim of this work was the achievement of microcysts formation from protoscoleces of E. granulosus of cattle origin using the in vitro vesicular culture technique. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. On day 20, some microcysts with a complete laminated layer were observed. By day 48, microcysts completely developed could be observed. This is the first study where microcysts formation was obtained using protoscoleces of E. granulosus of cattle origin.     

Association of Microcyst Formation in Spirillum itersonii with the Spontaneous Induction of a Defective Bacteriophage

Mitomycin C and ultraviolet light were found to induce the formation of microcysts in Spirillum itersonii. These forms, as well as spontaneously occurring microcysts in this species, were found to contain phage tail parts, rhapidosomes, and a granular substance not seen in normal cells. It is suggested that microcysts are formed as the result of the induction of a defective phage. The production of phage lysozyme within the cell could lead to the formation of spherical forms as the cells lose their structural mucopeptide layer. Complete virus particles were not seen, nor was any biological activity demonstrated when the induced cultures were tested against two other strains of S. itersonii. The other strains of this bacterium also formed microcysts and phage tail parts when induced with mitomycin. Attempts to isolate an organism lacking the defective phage have been unsuccessful.     

Tyrosine phosphorylation of actin during microcyst formation and germination in Polysphondylium pallidum

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. The tyrosine phosphorylation state of actin does not appear to influence the long-term viability of dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.     

Microcyst germination in the cellular slime mold, Polysphondylium pallidum: Effects of actinomycin D and cycloheximide on macromolecular synthesis and enzyme accumulation

Germination of microcysts of Polysphondylium pallidum is characterized by an immediate rapid increase in incorporation of [³H]leucine into protein which is cycloheximide-sensitive but unaffected by actinomycin D. Significant RNA synthesis, as measured by [³H]uridine incorporation, does not begin until approx. 2 h after the onset of germination. The increase in [³H]uridine incorporation is prevented by actinomycin D. Germination and the increase in alkaline phosphatase and β-glucosidase enzyme activities are prevented by cycloheximide but unaffected by actinomycin D. The data strongly imply the presence of stable RNA in dormant microcysts and indicate a requirement for a discrete period of protein synthesis for germination of microcysts of P. pallidum.     

Caractérisation combinée par TEP au 18F-FDG et par IRM des lésions hépatiques dans l’échinococcose alvéolaire

Positron emission tomography (PET) with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) can be used to evaluate parasitic activity in alveolar echinococcosis (AE) as visualization of host–parasite immune reaction around the lesions. We performed a retrospective interpretation of PET and MR (magnetic resonance) images in 24 patients with AE to compare the results of metabolic imaging to Kodama et al. anatomical classification. All patients underwent hepatic MRI and dual-time point FDG-PET (1 hour and 3 hours after injection), improving sensitivity of standard FDG-PET in AE. The comparison of the images showed perilesional ¹⁸F-FDG uptake in all seven patients with type 1 (multiple microcysts without a solid component) and type 2 (multiple microcysts with a solid component) lesions and in ten of 12 patients with type 3 lesions (a solid component surrounding cysts and microcysts). No abnormal perilesional uptake was observed at standard and delayed PET acquisitions in two of 12 patients with type 3 lesions and in all five patients with type 4 (a solid component without microcysts) and type 5 (a large liquid cavity without microcysts) lesions. Our study demonstrates a strong correlation between anatomical “microcystic” aspect and metabolic activity of AE lesions in 89.5% of cases. Thus, microcysts and metabolic perilesional activity seem correlated to parasite activity, which suggests an important role to combined follow-up of AE patients, through multimodality imaging using MRI and ¹⁸F-FDG-PET scan.