Pneumocystis carinii: Freeze-fracture study of stages of the organism

The morphology and life cycle of Pneumocystis carinii were studied in cortico-steroid-treated rats by ultrathin section and freeze-fracture electron microscopy. The following stages of P. carinii were noted: trophic, precyst, and cyst. The crescent-shaped cysts appeared to be intermediate forms between precyst and cyst. The cell wall of the trophic stage showed membrane structures suggestive of protozoan endocytosis, whereas the surface of the precyst stage was smooth. The cell wall of the cyst lacked the specialized structural differentiation of yeasts and resembled that of Plasmodium spp. We conclude that P. carinii belongs to the Protozoa, and is presumably Rhizopoda.     

The Fine Structure of the Cyst Wall of the Ciliated Protozoon Didinium nasutum1

SYNOPSIS. Electron microscope observations of the complex cyst wall of Didinium nasutum are reported. The cyst wall is composed of 3 major coats. The outermost coat, the ectocyst, consists of short strands of filamentous material which forms a diffuse, amorphous layer approximately 8–9 μ thick. Culture debris, bacteria and unidentified inclusions have been observed adhering to the outer coat. The mesocyst, approximately 2.5 μ thick, has 2 distinct regions. The outer region is differentiated into several slightly thicker layers which in face view have a honeycomb appearance. The deeper region of the mesocyst consists of compact lamellae lacking the obvious honeycomb appearance of the layers of the outer region. Finally, the endocyst (0.3 μ thick), which arises in the mature cyst in the space that develops between the pellicle and the mesocyst, consists of delicate fibrils in a compact matrix. Both mesocyst and endocyst may be undulant and folded. The structure, origin and possible relationships of the various coats composing the cyst wall are discussed. The present study also contributes information on the role and fate of mucocysts and other cytoplasmic structures during the formation of the cyst wall.     

Mirex and Aroclor® 1254: Effect on and Accumulation by Tetrahymena pyriformis Strain W*

Effects of 2 toxicants, Mirex and Aroclor 1254, on Tetrahymena pyriformis strain W in axenic cultures were investigated. Mirex is a chlorinated hydrocarbon effective against the fire ant, and Aroclor 1254 is a compound structurally related to DDT and used extensively in various industrial processes. Both toxicants reduced growth rates and population densities of T. pyriformis grown at 26 C generally in proportion to concentrations of the chemicals, their effects becoming statistically significant (P < 0.05) at 0.9 μg/liter for Mirex and 1.0 and 10.0 μg/liter for Aroclor 1254. Ciliates exposed to the toxicants for 7 days concentrated Mirex 193 × and Aroclor 60 × as compared to the initial concentrations of these compounds. It is suggested that the chief effect of the 2 toxicants on populations of T. pyriformis and of similarly responding ciliates in nature would be to reduce the availability of these protozoa as food organisms and nutrient regenerators. The ability of the ciliates to concentrate the tested compounds would permit the toxicants to enter into and to be translocated through aquatic food chains. In this manner the compounds could exert toxic effects at higher trophic levels.     

Characterization of Deciliation-Regeneration Process of Tetrahymena Pyriformis for Cellular Robot Fabrication

Artificial magnetotactic Tetrahymena pyriformis GL (T. pyriformis) cells were created by the internalization of iron oxide nano particles and became controllable with a time-varying external magnetic field. Thus, T. pyriformis can be utilized as a cellular robot to conduct micro-scale tasks such as transportation and manipulation. To complete these tasks, loading inorganic or organic materials onto the cell body is essential, but functionalization of the cell membrane is obstructed by their motile organelles, cilia. Dibucaine HCl, a local anesthetic, removes the cilia from the cell body, and the functional group would be absorbed more efficiently during cilia regeneration. In this paper, we characterize the recovery of artificial magnetotactic T. pyriformis after the deciliation process to optimize a cellular robot fabrication process. After sufficient time to recover, the motility rate and the average velocity of the deciliated cells were six and ten percent lower than that of non-deciliated cells, respectively. We showed that the motile cells after recovery can still be controlled using magnetotaxis, making T. pyriformis a good candidate to be used as a cellular robot.     

IDENTIFICATION OF LECTINS IN THE KINETIDS OF TETRAHYMENA PYRIFORMIS

Previously we described lectin-like molecules in the ciliate Tetrahymena pyriformis; by application of synthetic neoglycoconjugates it is now shown that T. pyriformis contains considerable amounts of both a β-d-glucose- and a lactose-specific lectin. No evidence for the presence of α-d-mannose-, α-d-galactose- or of α-l-fucose-specific lectins could be obtained. The two lectins, identified in T. pyriformis, are associated with the kinetids. During cell division the lectins disappear or become masked in the fission furrow. Therefore, we assume that these lectins are involved in the organization of the distribution pattern of the kinetids during cell division perhaps due to lectin—glycoprotein interactions.     

The outer cyst wall ofBdellovibrio bdellocysts is made de novo and not from preformed units from the prey wall

Bdellovibrio sp. strain W bdellocysts were produced inEscherichia coli using three sources of³H-diaminopimelic acid (DAP) for incorporation into the cyst wall peptidoglycan: (a) labeledE. coli peptidoglycan, (b) labeledBdellovibrio peptidoglycan, and (c) exogenous³H-DAP in the encystment medium. After cysts were produced, they were either sonicated to remove the prey cell wall, or germinated to solubilize the cyst wall. The results show that label was incorporated into the cyst wall preferentially from the exogenous DAP in the medium, and not from the bdellovibrio or bdelloplast peptidoglycan. The encysting bdellovibrio does not therefore incorporate existing peptidoglycan units from the bdelloplast for synthesis of the cyst wall.     

The Fine Structure of Acanthamoeba astronyxis,with Special Emphasis on Encystment1

ABSTRACT. The fine structure of the trophozoite, encysting cells, and the cyst of Acanthamoeba astronyxis has been examined. In the trophic form a microtubule organizing center was associated with a well developed Golgi complex. During encystment the organelles of the amoeba changed considerably. The profiles of rough endoplasmic reticulum elongated and were often arranged in circles of multilayered concentric systems, enclosing mitochondria, the nucleus, or other inclusions. The mitochondria showed a tendency toward elongation and constriction. One or two nucleolus-like bodies appeared in the nucleus. Lipid droplets increased considerably in amount and were distributed individually or as aggregates. The mature cyst was star-shaped and surrounded by an almost circular exocyst and an endocyst that was closely apposed to the cell membrane. Both walls differed in their thickness and granulation. The exocyst was continuous over the entire cyst, while the endocyst was interrupted by gaps, ostioles. in the region of the rays. Within the ostioles was a bell-shaped structure, the operculum. The latter was composed of a granular material comparable in electron density to that of the endocyst.     

Structure of Cyst Wall Precursors and Kinetics of Their Appearance During the Encystment of Laurentiella acuminata (Hypotrichida,Oxytrichidae)1

The encystment of Laurenliella acuminata was divided into five stages: stage A (precystic semitransparent cell with dark-globules), stage B (precystic transparent cell), stage C (precystic pigmented cell), stage D (spherical shape without cyst wall) and stage E (young resting cyst), on the basis of observations of changes in morphology and pigmentation during encystment. The duration of these stages was also established. Observations by electron microscopy confirmed that the cyst wall, composed of four layers, is derived from different kinds of precursors which are synthesized “de novo.” The ectocyst precursors are composed of stacks of between 5 and 12 small thin plates or discs; these stacks are about 0.9 μm in length and 0.06 μm in height. The mesocyst precursors are fibrillar bodies of variable shapes, about 2.4 μm in maximum length and 0.12–0.16 μm in diameter. These precursors appear in the cytoplasm of the precystic cell during the first precystic stage (stage A). The endocyst precursors are rounded bodies surrounded by a fine membrane, and their contents appeared similar to the endocyst. The granular layer precursors are spherical bodies about 0.1–0.2 μm in diameter, surrounded by a double membrane presenting ribosomes adhering to its outer membrane. Both endocyst and granular layer precursors are observed in the precystic cytoplasm from stage B. On the basis of ultrastructural studies, a formation and growth model of the cyst wall of the hypotrichous ciliate Laurentiella acuminata is proposed.     

The Polychlorinated Biphenyls,Aroclors 1248 and 1260: Effect on and Accumulation by Tetrahymena pyriformis*

SYNOPSIS. Effects of 2 polychlorinated biphenyls, Aroclor 1248 and 1260, on axenic Tetrahymena pyriformis strain W were investigated and compared with published data on Aroclor 1254. Aroclors 1248 and 1260 at 1 mg/liter in the presence of 0.1% (v/v) polyethylene glycol 200 reduced significantly (P < 0.005) growth rates and 96-hr populations of T. pyriformis grown at 26 C. Both toxicants were ～ 0.001 as toxic as Aroclor 1254. Ciliates were exposed for 7 days to concentrated Aroclors 1248 40X, 1254 60X, and 1260 79X over initial concentrations in the media. Accumulation of Aroclors increased with increased chlorination. It is suggested that if levels in the environment reached those used in these studies, the chief ecologic effect of Aroclor 1254 would be reduction of availability of the ciliates as food and as nutrient regenerators, but with Aroclors 1248 and 1260, this effect would be secondary to accumulation of the toxicants by the ciliates. Accumulation of polychlorinated biphenyls by ciliates would permit the toxicants to enter aquatic food chains. Thus the compounds could exert toxic effects at higher trophic levels.     

The different morphologies of yeast and filamentous fungi trigger distinct killing and feeding mechanisms in a fungivorous amoeba

Size and diverse morphologies pose a primary challenge for phagocytes such as innate immune cells and predatory amoebae when encountering fungal prey. Although filamentous fungi can escape phagocytic killing by pure physical constraints, unicellular spores and yeasts can mask molecular surface patterns or arrest phagocytic processing. Here, we show that the fungivorous amoeba Protostelium aurantium was able to adjust its killing and feeding mechanisms to these different cell shapes. Yeast-like fungi from the major fungal groups of basidiomycetes and ascomycetes were readily internalized by phagocytosis, except for the human pathogen Candida albicans whose mannoprotein coat was essential to escape recognition by the amoeba. Dormant spores of the filamentous fungus Aspergillus fumigatus also remained unrecognized, but swelling and the onset of germination induced internalization and intracellular killing by the amoeba. Mature hyphae of A. fumigatus were mostly attacked from the hyphal tip and killed by an actin-mediated invasion of fungal filaments. Our results demonstrate that predatory pressure imposed by amoebae in natural environments selects for distinct survival strategies in yeast and filamentous fungi but commonly targets the fungal cell wall as a crucial molecular pattern associated to prey and pathogens.     

The Life Cycle of Ganymedes oaklandi n. sp., an Acephaline Gregarine of Gammarus fasciatus (Say)*

SYNOPSIS. Ganymedes oaklandi n. sp. (Ganymedidae) from the intestine of Gammarus fasciatus (Say) is described, and its life cycle is given. In the trophic stage the gregarine is acephaline, with an elongated cylindrical body slightly tapered anteriorly, and is up to 298 × 64 μ. The gametocysts are up to 121 × 99 μ, and are covered with a secreted thick sticky gelatinous coat. The spores are released from the gametocyst by rupturing of the cyst wall. The spore body is spherical, approximately 6 μ in diameter, and has 4 episporal rays about 20 μ long.     

Effects of natural amino acids and sugars on activity of infusiorian cyclases

Natural amino acids and sugars in unicellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, tyramine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis rose in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of the infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large specter of comparatively simple natural molecules has a hormone-like action.     

The structure and composition of the cyst wall of the metacercaria of Cloacitrema narrabeenensis (Howell & Bearup, 1967) (Digenea: Philophthalmidae).

The mstacercarial cyst of Cloacitrema narrabeenensis which is formed in the open is composed of four layers: an outermost layer of acid mucopolysaccharide, a layer of protein which is presumed to be tanned, a layer of neutral mucopolysaccharide and an innermost layer of keratinized protein. The two layers which together form the outer cyst wall can be split off by slight pressure from the two remaining layers which together form the inner cyst wall. In the centre of the ventral side of the inner cyst wall, the keratinized layer is incomplete and this ventral plug region is composed of neutral mucopolysaccharide. The cyst wall is therefore very similar to that of Fasciola hepatica, the main difference being that the order of the two layers of the outer cyst is reversed. General evolutionary and functional relationships of metacercarial cysts are discussed.     

A Freeze-Fracture Study of the Microcyst Wall,Plasma Membrane,and Intramembrane Components of Physarum polycephalum1

ABSTRACT. Freeze-cleave replicas of encysted cells of the myxamoebae of Physarum polycephalum were examined to determine the intrawall substructure and to compare the intramembrane structural components. Cleaved areas of the cyst wall revealed a laminated substructure devoid of the macromolecular interruptions (intramembrane particles or IMP) visible in the cleaved cell membranes. The cyst wall adjacent to the cell membrane appears laminated, and a loose branching network of fibers and fibrils occurs at the wall periphery. The absence of intrawall particles is interpreted as a lack of protein or polypeptide components, thus suggesting additional support for the conclusion that polysaccharides are the major wall constituents. When cleaved cell membranes of encysted cells were examined, more intramembrane particles per unit area were observed on the extracellular membrane leaflet than on the protoplasmic membrane leaflet. In addition, homogeneous as well as aggregated particle distributions were visible on cleaved membrane leaflets. Moreover, the presence of aggregated and unaggregated particles on the same membrane leaflets similarly suggests asynchrony of the cell population. This paper examines and compares biological processes involving the cell membrane that may be related to different stages in the cell cycle or to periods of temporary stasis during the cell cycle.     

ULTRASTRUCTURE OF CYST FORMATION IN OCHROMONAS TUBERCULATA (CHRYSOPHYCEAE)1

The cysts (statospores) of Ochromonas tuberculata Hibberd are produced within a cytoplasmic silica deposition vesicle (SDV) whose membrane (silicalemma) appears to be formed by the coalescence of golgi vesicles. Silica is first deposited as small nodules and the collar and spines develop by centrifugal growth only after a complete but still thin wall has been laid down. Small vesicles appear to be attached to the SDV only in the region overlying the developing collar; a cap of radially arranged, moderately electron-dense material occurs at the tip of the growing spines. The cyst pore is formed at the anterior end of the flagellate cell, by lack of silica deposition over a small region of the SDV and rupture of the SDV and other membranes crossing this region. When the cyst wall is complete, an extracystic plug is formed in the pore, resulting in the loss of some extracystic cytoplasm and the plasmalemma, and the inner SDV membrane becomes the functional plasmalemma. The plug develops first by coalescence with the cell membrane of golgi-derived vesicles containing dense but apparently nonsiliceous spicules surrounded by amorphous material. During later stages of plug formation only fibrous material is deposited, some of which may be extruded through the pore forcing out some of the spiculate component. Scanning electron micrographs of the mature wall show it is smooth except for the concentrically wrinkled inner face of the flared collar and that the real pore diameter is only ca. half that of the collar. At germination the plug completely disappears in an unknown way and a single cell, similar to a normal vegetative cell emerges through the pore. Chrysophycean cyst formation generally resembles cell wall formation in diatoms, but differs in some details.     

Structure and Elemental Composition of the Cyst Wall of Echinosphaerium nucleofilum Barrett (Heliozoea,Actinophryida)1

The structure of the cyst wall of the heliozoon Echinosphaerium nucleofilum has been investigated using light microscopy, scanning and transmission electron microscopy, and X-ray microanalysis. The cyst wall is a composite structure of seven or eight layers. These are: an enveloping gelatinous layer; a layer of siliceous spheroidal bodies; an electron-dense supporting membrane; a broad electron-lucent zone; an electron-dense layer; a layer of helicoidally packed material; and one or two layers with a granular appearance lying next to the plasma membrane of the encysted organism. The structure of the cyst wall closely resembles that of Actinophrys sol, confirming the close relationship of these actinophryid heliozoa while emphasizing their distinctiveness from other amoeboid protista.     

梨形四膜虫衰老过程中DNA含量的变化

应用一种新型的细胞核内DNA含量测定方法──图像分析法,测定真核细胞梨形四膜虫衰老过程中DNA含量的变化.根据Beer-Lambert定律,以细胞核在不同生长期内的积分光密度的水平表示核内DNA含量的变化.该方法具有测量速度快,重复性好,操作简单,结果可靠等优点.实验结果表明:四膜虫在进入对数生长期时,DNA含量逐渐达到高峰,随着细胞逐渐老化,细胞分裂次数及核内DNA含量逐步减少.     

Ultrastructure of the Ameboid Cells of Protostelium zonatum (Mycetozoa)*

SYNOPSIS. The ameboid cells of Protostelium zonatum typically have filose pseudopodia that contain almost exclusively microfibers. Microfibers, scattered or arranged parallel in bundles, are observed in great abundance and often closely associated or directly connected with other organelles in the cytoplasm. Mitochondria typically have an expanded, undulate outer membrane and vesicular cristae containing electron-dense material which may appear denser than the mitochondrial matrix in the same section. The nucleus, as in other protostelids, contains only one centrally located nucleolus. Other organelles are essentially similar in ultrastructure to those of another nonflagellate protostelid, Protosteliopsis fimicola. Protostelium zonatum differs, however, in lacking a distinct anterior zone of hyaloplasm during migration of the ameboid cells.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.