Binary fission of Pneumocystis carinii trophozoites grown in vitro

Trophozoites grown in vitro were shown to undergo binary fission by transmission electron microscopy (TEM). Standard fixation with subsequent embedding in Spurr was employed using 3% glutaraldehyde and 1% osmium tetroxide with 5% sucrose added to both fixatives and 0.1 M cacodylate buffer washes. Trophozoites were grown on WI-38 cells in vitro. Trophozoites were found in various stages of fission. The dividing trophozoite has daughter cells that are rounder than the pleomorphic, non-dividing trophozoites. Tubular forms external to the dividing trophozoites were decreased in number; tubular forms when present were concentrated around the forming septa. Nuclear material was sometimes, but not always, well defined in both daughter cells. There was no concentration of nuclear material at the poles. Vacuoles without membrane were present in the dividing forms. Separate nuclear regions were sometimes found in the dividing trophozoites. These observations suggest that binary fission does occur in culture; however, the significance of binary fission to the life cycle of Pneumocystis carinii (Pc) is not yet clear.     

Giardia muris: scanning electron microscopy of in vitro excystation

A recently developed in vitro excystation procedure results in almost total excystation of Giardia muris, an intestinal parasite of mice. The present experiment examines the G. muris cyst morphology by scanning electron microscopy and the efficacy of the excystation procedure. Untreated cysts of G. muris were elliptical and displayed a distinctive surface structure. Excystation began almost immediately after incubation had begun and most trophozoites emerged within 30 min. Excystation appears to involve flagellar action of the encysted trophozoite. A tear of the wall occurred at one pole. This opening was subsequently enlarged, presumably by flagellar action. Trophozoites emerged, posterior end first, and an associated mucoid-like material was extruded. Newly emerged trophozoites were nearly oval in shape. Trophozoites quickly became flattened, elongate, and underwent cytokinesis resulting in two daughter trophozoites. Few organisms not excysted were seen after 30 min incubation.     

Is actin involved in the nuclear division in Amoeba proteus?

During semi-open mitosis of Amoeba proteus the nuclear envelope is not dispersed and nucleus divides by fission. The presence of actin layer close to nuclear envelope was demonstrated in interphase and telophase nuclei of that amoeba stained with rhodamine labelled phalloidin. In telophase, an accumulation of actin arises in the space between the future daughter nuclei. It appears to be comparable with the contractile ring of dividing cells. This suggests that actin associated with the nuclear envelope of Amoeba proteus may be involved in final separation of the daughter nuclei, forming a constriction ring at the middle of dividing nucleus.     

The Ultrastructure of Trophozoites of Entamoeba histolytica with Particular Reference to Spherical Arrangements of Osmiophilic Cylindrical Bodies

SYNOPSIS. Trophozoites of Entamoeba histolytica were cultured under xenic, monoxenic and axenic conditions. Some of the monoxenically cultured trophozoites were grown in the presence of Bacteroides symbiosus and others in the presence of Trypanosoma cruzi (Mexican strain). Other trophozoites were obtained from experimentally produced amebic liver abscesses in hamsters. The trophozoites were centrifuged and prepared for study by electron microscopy. Acid phosphatase activity in the parasites was determined by cytochemical reactions.
The various elements of the trophozoites were described and special attention was given to the spherical arrangements of electrons-dense cylindrical units surrounding finely granular material. Their presence was independent of the strain studied and of the nutrient elements in the culture medium. The cylindrical units probably arise from endoplasmic reticulum elements and their possible function in the digestive processes and aggression mechanisms of the trophozoites is discussed.
Acid phosphatase activity was found in round non-branching intranuclear bodies in trophozoites cultured in various media. Whether these bodies represent part of the lysosomal system of the parasite is unknown.
In preliminary work on the action of some amebicidal drugs, a special arrangement of cytomembranes morphologically similar to a Golgi complex was frequently seen in trophozoites of E. histolytica.     

Development and proliferation of Trypanosoma musculi in the presence of adherent splenic cells

The development and proliferation of Trypanosoma musculi parasites were studied in vitro in the presence of adherent splenic cells. The parasites grew and proliferated only when attached by their flagellar tips to adherent splenic cells. Analyses of excretory-secretory products of the adherent cells-parasites did not indicate any detectable soluble growth factor that might be responsible for the growth of these trypanosomes. During the proliferation, the kinetoplast migrated toward the nucleus, and once in the vicinity of the nucleus, nuclear division was triggered. The nucleus and kinetoplast divided at the same time Trypanosoma musculi parasites started dividing from their flagellar ends, and daughter cells were formed within 48 hr. In the absence of adherent splenic cells in vitro, the parasites were transformed into round nonviable forms.     

A New Saurian Malaria Parasite Plasmodium clelandi sp. n. from Ceylon*

Plasmodium clelandi sp. n. is described from Varanus cepedianus of Mannar District, Ceylon. The earliest asexual stages were seen as a chromatin dot. Trophozoites have a vacuole with chromatin material spread around its periphery. The trophozoites divide and mature to form the schizont. The maximum number of merozoites observed in a schizont was 8. Pigment was scanty and sometimes absent. The gametocytes had a characteristic elongate form and tend to encircle the host cell nucleus.     

The formation of primary and secondary lysosomes in Balantidium coli, Ciliata

Trophozoites, vegetative forms of Balantidum coli isolated from pigs affected by acute and asymptomatic balantidiasis were studied. Lysosomes and food vacuoles were revealed by cytochemical detection of lysosomal marker, acid phosphatase. The cytoplasm of all the B. coli trophozoites examined was found to contain numerous structures which differed widely in shape, size and location in the cells. One of them was located among the rough endoplasmic reticulum membranes and another one in the vicinity of endosomes. Those structures were regarded as the primary lysosomes. The two types of vesicular structures most probably represent two stages of the primary lysosome formation. Trophozoites were also found to contain secondary lysosomes which are formed by fusion of several primary lysosomes with phagosomes. The ultrathin sections of B. coli trohozoites showed the presence of two types of phagosomes. They were divided, based on their contents, into auto- and heterophagosomes.     

Comparative positions of kinetoplasts in Trypanosoma musculi and Trypanosoma lewisi during development in vitro

Abstract. The development of Trypanosoma musculi and Trypanosoma lewisi were studied in vitro in the presence of adherent splenic cells. Both parasites developed only when attached by their flagellar tips to adherent splenic cells. During the proliferation of T . musculi , the kinetoplast migrated towards the nucleus, and once in the vicinity of the nucleus, the nuclear division was triggered. The kinetoplast of T . lewisi did not migrate towards the nucleus, but remained at its original location. The nucleus and kinetoplast divided at the same time in both parasites, and parasites started dividing from their flagellar ends and T . musculi and T . lewisi daughter cells were formed within 48 h. The unavailability of the adherent splenic cells in vitro led the parasites to transform into round/oval nonviable forms.     

Effect of iron on the virulence of Trichomonas vaginalis

The role of iron was evaluated with respect to the virulence of Trichomonas vaginalis in mice. Iron-supplemented and iron-depleted Diamond's trypticase-yeast extract-maltose (TYM) media were prepared by adding 360 microM of ferrous sulfate and 100 microM of 2,2'-dipyridyl. Trophozoites cultivated from normal TYM and iron-supplemented TYM media produced subcutaneous abscesses; however, trichomonads grown in an iron-deficient TYM medium failed to produce any pathology. In addition to the increased virulence of trophozoites in mice, iron affects the level of adherence and the cytotoxicity of trichomonads to HeLa cells, which are significantly reduced in trophozoites grown in iron-deficient medium. In conclusion, it is suggested that under iron-depleted conditions such as that induced by 2,2'-dipyridyl the virulence of T. vaginalis is reduced.     

Basic biology of Pneumocystis carinii: a mini review

Basic aspects of cell biology of Pneumocystis carinii are reviewed with major emphasis on its life cycle and the structural organization of the trophozoites and cyst forms. Initially considered as a protozoan it is now established that Pneumocystis belongs to the Fungi Kingdom. Its life cycle includes two basic forms: (a) trophozoites, which are haploid cells that divide by binary fission and may conjugate with each other forming an early procyst and (b) cysts where division takes place through a meiotic process with the formation of eight nuclei followed by cytoplasmic delimitation and formation of intracystic bodies which are subsequently released and transformed into trophozoites. Basic aspects of the structure of the two developmental stages of P. carinii are reviewed.     

Entamoeba histolytica: inflammatory process during amoebic liver abscess formation involves cyclooxygenase-2 expression in macrophages and trophozoites

It has been demonstrated that expression of cyclooxygenase-2 (COX-2) isoform is induced by Entamoeba histolytica in macrophages and polymorphonuclear cells during amoebic liver abscess (ALA) formation in hamsters. Trophozoites present in the lesion were also positive for COX-2 signal. However, no cross reactivity of the anti-COX-2 antibody with protein extract of cultivated trophozoites was found. To clarify if trophozoites are involved in PGE(2) production during ALA development, COX-2 expression was detected by in situ hybridization and RT-PCR in liver tissue from intrahepatically infected hamsters. COX-2 mRNA was in polymorphonuclear cells since 4h postinfection, and subsequently, local macrophages expressed COX-2 mRNA in a similar way. Additionally, a positive signal for COX-2 mRNA expression was detected in E. histolytica trophozoites, suggesting that, in vivo, parasite COX expression may be an important mechanism to promote inflammation.     

Relative susceptibility of Giardia muris trophozoites to killing by mouse antibodies of different isotypes.

The aim of this work was to examine the ability of mouse IgA, IgG, and IgM anti-Giardia antibodies to kill Giardia muris trophozoites in the presence and absence of complement. Using a 2-color flow cytometry assay, binding of antibody to trophozoites was assessed with fluorescein-conjugated anti-mouse immunoglobulin, and percentages of killed trophozoites were quantified by staining with propidium iodide. Trophozoites were killed in the presence of complement by IgG3 and IgM anti-trophozoite monoclonal antibodies. Anti-trophozoite IgA, obtained from the intestinal lumen of G. muris-infected BALB/c mice, became bound to trophozoites in vitro but did not kill these organisms in the presence or absence of complement. The results suggest that clearance of G. muris infection by intestinal IgA directed against G. muris trophozoites does not involve antibody-dependent killing of trophozoites in the intestinal lumen.     

Cryptobia cataractae from the blood of Semotilus atromaculatus: structure and division in the fish

A cryptobiid was found in the blood of 2 of 9 Semotilus atromaculatus from a tributary of the Saugeen River in Ontario, Canada. Blood inoculation produced an infection in 2 uninfected S. atromaculatus but not in any Oncorhynchus mykiss, Catostomus commersoni, or Carassius auratus. The flagellate was identified as Cryptobia cataractae, based on host restriction. Cryptobia cataractae occurred as slender and broad forms (body width 3.0-8.7 microns). The length of the anterior flagellum was equal to body length, whereas that of the free recurrent flagellum was half body length. Cryptobia cataractae divided by equal binary fission that produced elongate, slender daughter cells.     

Mesosomes in Escherichia coli

When Escherichia coli was grown in a synthetic medium and fixed with osmium, sections of the cells revealed clearly defined mesosomes. These mesosomes appeared to develop, in dividing cells, as coiled infoldings of the cytoplasmic membrane. Mature mesosomes formed a link between the cytoplasmic membrane and the nucleus of the cell. The arrangement of the mesosomes in dividing cells led to the hypothesis that division of the nucleus in these cells is accomplished by two separate polar mesosomes. One mesosome is derived from the parent cell and is present at one pole of the daughter cell. The other is freshly synthesized at or near the newly forming pole of the daughter cell. While the old mesosome remains attached to the chromosome received from the parent cell, the newly synthesized mesosome becomes attached to and initiates replication of the new chromosome. As the cell grows and elongates, the two mesosomes, attached to their respective chromosomes move apart, thus effecting nuclear division.     

DEVELOPMENT OF THE EMBRYONIC CHICKEN THYMUS III. UNEQUAL DIVISION OF LYMPHOID CELLS

Cell division of thymus lymphoid cells from 11- to 17-day old embryonic chickens, as well as chickens just after hatch was investigated on cell smears stained with Giemsa. Unequally dividing cells were observed in the developmental stage of thymocytes. At the telophase of such cells, the cytoplasm of one of two future daughter cells was apparently larger in amount and was sometimes stained deeper than the cytoplasm of its counterpart. Unequal division was also observed in pro-, meta- and anaphase; sometimes a dividing cell had a large cytoplasmic process belonging to one hemisphere, suggesting that only one of the two daughter cells would receive the cytoplasmic process through cell division.
The incidence of unequal division calculated by a rough estimation was around 10% of the total cell division between 11 and 13 days of embryonic development, and decreased progressively thereafter.     

Molecular subdivision of the cortex of dividing Tetrahymena is coupled with the formation of the fission zone.

In contrast to a mitotic-spindle-associated bipolar cytokinesis, the cytokinesis of polarized ciliates is preceded by a reorganization of the cortex into dual metameric patterns for prospective daughter cells and then separated by a transverse fission line. This study concerns relations between the generation of cortical metamery and the formation of the fission line in an amicronuclear (i.e., without mitotic spindle) ciliate, Tetrahymena pyriformis. The fission line appears in the division of T. pyriformis as a transverse line formed by equatorial gaps in the meridional ciliary rows, with the second oral structure (OA2) formed posterior to it. It was found that the metamery of cortical morphogenesis is expressed by the appearance of increased MPM2 antibody binding in dividing cells in an apical area and posterior to the fission line gaps, including patterned changes of this binding in both oral apparatuses (OA1 and OA2), and by a reciprocal decrease of binding of an anti-epiplasm antibody. These tested antigens are localized to different cortical structures, but in predividing cells both uniformly show formation of the fission line contrast of labeling. A serine/threonine kinase inhibitor, 6-dimethylaminopurine (6-DMAP), was applied to dividing T. pyriformis at specific stages: (1) if 6-DMAP was added to early dividing cells, it prevented cells from initiating cytokinesis. (2) If 6-DMAP was added to cells at stages close to the physiological transition point of cell division, it yielded either (i) a partial formation of the fission line on the ventral side, combined with modified growth of undivided cortex adjacent to the fission line, with abnormal cytokinesis, or (ii) variable anterior displacement of the complete fission line, which contracted slowly but uniformly. (3) If 6-DMAP was applied during cytokinesis, it did not delay cell division, but daughter cells become abnormal and underwent an incomplete oral reorganization. These results suggest that the generation of metamerism in the cortex of T. pyriformis involves differentiation of the asymmetric fission zone. At least four stage-dependent 6-DMAP-sensitive effects jointly control the progress of cell division and the mutual spatial relations between the generation of metamery and the appearance, completeness, and position of the fission zone in the cortex of polarized T. pyriformis.     

Ultrastructural morphology and staining characteristics of Pneumocystis carinii in situ and from bronchoalveolar lavage

The ultrastructure of Pneumocystis carinii obtained from rats by bronchoalveolar lavage (BAL) was compared with organisms in situ. All developmental forms of the organism as seen in situ were present in the lavage fluid. Trophozoites in situ were adhered to type I epithelium, had smooth surfaces, and were interdigitated with the underlying epithelium. Nonadherent trophozoites in situ and trophozoites in lavage fluid were more pleomorphic and irregular in shape with tubular projections extending from all surfaces. Microtubular and nuclear details not reported elsewhere were observed. To enhance the ultrastructural detail of P. carinii obtained by lavage, phosphotungstic and tannic acid fixation, uranyl acetate en bloc staining, and acid phosphatase staining were performed. These techniques enhanced the visibility of membranes, mitochondria, nuclei, and vacuoles. With tannic acid, increased contrast of the organism's cell coat was obtained and differences in staining intensity and thickness related to developmental stages were observed. In lavage samples with few pneumocystis organisms or those specimens heavily contaminated with macrophages, erythrocytes, or other cellular debris, tannic acid allows for easier recognition as other lung materials do not show the same distinctive staining reaction. Lung sections observed after BAL showed intact but damaged epithelial surfaces devoid of organisms. No intracellular organisms were observed. BAL removes organisms from the alveolar lumen as well as adhered organisms and is a useful method for concentrating the various morphologic forms of P. carinii.     

Cell morphology and life history of Dictyocha octonaria (Dictyochophyceae,Ochrophyta) from Wellington Harbour,New Zealand

Even though many aspects of Dictyocha fibula and D. speculum have long been studied, very little is known about D. octonaria. For the first time a clonal culture derived from a single cell of D. octonaria from Wellington Harbour was studied in detail. In the skeleton‐bearing stage three morphotypes were observed – skeleton bearing, mucocyst‐bearing and amoeboid, while in the naked stage only the naked form was studied. In this study the mucocyst‐bearing form was described as a new morphotype. Vegetative reproduction of the skeleton‐bearing form in the exponential growth phase was by both direct binary fission and by first forming a doublet and then two separate daughter cells, while that of naked form was by simple binary fission. Occasionally double skeletons were observed as end products of both the vegetative and sexual reproductions. In sexual reproduction all three forms in the skeleton‐bearing stage exhibited the same polymorphic life history involving a multinucleate stage. The newly formed daughter cells of all three forms developed individual siliceous skeletons prior to being released from the parent cell. The naked form in the naked stage, however, exhibited a separate polymorphic life history that produced only skeleton‐free daughter cells. For the first time both vegetative and sexual reproduction of D. octonaria were documented.     

The price of independence: cell separation in fission yeast

The ultimate goal of cell division is to give rise to two viable independent daughter cells. A tight spatial and temporal regulation between chromosome segregation and cytokinesis ensures the viability of the daughter cells. Schizosaccharomyces pombe, commonly known as fission yeast, has become a leading model organism for studying essential and conserved mechanisms of the eukaryotic cell division process. Like many other eukaryotic cells it divides by binary fission and the cleavage furrow undergoes ingression due to the contraction of an actomyosin ring. In contrast to mammalian cells, yeasts as cell-walled organisms, also need to form a division septum made of cell wall material to complete the process of cytokinesis. The division septum is deposited behind the constricting ring and it will constitute the new ends of the daughter cells. Cell separation also involves cell wall degradation and this process should be precisely regulated to avoid cell lysis. In this review, we will give a brief overview of the whole cytokinesis process in fission yeast, from the positioning and assembly of the contractile ring to the final step of cell separation, and the problems generated when these processes are not precise.