Presence and localization of vinculin in Giardia.

A requisite element of pathogenicity in Giardia infections is the parasites' ability to adhere to the intestinal epithelial brush border. The presence of vinculin in Giardia was studied because this protein is known to link the cytoskeleton to the plasma membrane and is localized at adhesion foci in many cell-cell and cell-substrate contact sites. Actin, alpha-actinin, and vinculin were identified in Giardia by western blot analysis. Giardia trophozoites attached to glass substrates were examined by interference reflection microscopy (IRM) and immunofluorescence. The IRM defined the lateral crest, bare area, and overlap region of the ventral disk, as well as the ventrolateral flange and lateral shields as close contact areas between parasite and substrate. These close contact regions were then correlated with immunofluorescence localization of actin, alpha-actinin, and vinculin. Actin was seen in the lateral crest, while alpha-actinin was observed in the ventral disc periphery and lateral shields. Vinculin was viewed at the bare and overlap areas of the ventral disc and portions of the lateral crest, as well as the ventrolateral flange and lateral shields. The correspondence of close contact sites with vinculin localization suggests a role for vinculin in Giardia attachment and adherence.

Trypanosoma cruzi infection of BSC-1 fibroblast cells causes cytoskeletal disruption and changes in intracellular calcium levels.

The disruption of vimentin and actin filaments of host BSC-1 fibroblast cells by Trypanosoma cruzi was investigated using a mouse monoclonal anti-vimentin antibody and rhodamine phalloidin, respectively. Indirect immunofluorescence microscopy demonstrated that infection of BSC-1 cells by T. cruzi caused disruption of both cytoskeletal components. The disruption was greater as infection progressed. Mechanisms other than mechanical ones may play a role in the disruption since disrupted cytoskeletal elements were well removed from the parasites. In the determination of intracellular calcium concentrations using Fura-2 AM, infected and uninfected cells both showed an initial increase in intracellular calcium levels. At later times of infection (3 to 5 days), intracellular calcium levels of infected cells were significantly lower than those of control cells. There was no specific localization of intracellular calcium in the infected host cells as determined by image analysis.

Reconstruction of the chondriome of the amastigote form of Trypanosoma cruzi.

The chondriome of the amastigote form of Trypanosoma cruzi was studied by fluorescent microscopy using rhodamine 123 and conventional transmission electron microscopy of serial thick sections. The 3-dimensional models of the chondriome generated from the serial sections using manual or computer-assisted analysis revealed a single circular or horseshoe-shaped mitochondrion in each amastigote studied. The morphology of the amastigote chondriome does not conform to that seen in the epimastigote or trypomastigote forms of T. cruzi. Cell shape and mitochondrial form are comparable for a given morphotype, but they differ between cyclical forms.

Immunochemical studies on the FAD-dependent NADPH-cytochrome c reductase from Trypanosoma cruzi.

The cytosolic flavin enzyme from Trypanosoma cruzi was isolated by a modification of the previously reported method (T. Kuwahara, R. A. White, Jr., and M. Agosin (1985) Arch. Biochem. Biophys. 239, 18-28). In the present study, rabbits were inoculated with the purified enzyme and antibodies were purified from the sera. Ouchterlony double-diffusion analysis indicated that the antibodies reacted specifically with the flavoenzyme and not with other T. cruzi proteins. At the equivalence point, 1 ml of antibody neutralized about 4 nmol of enzyme. The IgG fraction had a small inhibitory effect on the catalytic activity of the enzyme as measured by cytochrome c reduction but only at IgG concentrations well above the equivalence point. Immunotitration of the enzyme in T. cruzi cultures showed that the enzyme corresponds to about 1% of the total protein during the logarithmic phase of growth, but this value decreases to about 0.6% during the stationary phase. Among various trypanosomatids tested, T. cruzi had the highest enzyme concentration; whereas, in other species it ranged from 0.25 to 2.4 micrograms/mg protein. These marked differences suggest that the antibody may be suitable for taxonomic purposes. The presence of the enzyme in amastigotes maintained in tissue culture cells was demonstrated by indirect immunofluorescence. The enzyme was found localized in the periphery of the cell, just beneath the subpellicular microtubules. However, distribution of the enzyme in epimastigotes was more diffuse. As immunofluorescence could be detected only in amastigotes and not in the tissue culture cells, it is suggested that the antibody may be suitable for histopathological diagnosis of Chagas' disease.

A monoclonal antibody to alpha tubulin recognizes host cell and Trypanosoma cruzi tubulins.

A mouse monoclonal anti-alpha-tubulin antibody was used to investigate the disposition of the cytoskeletal microtubules of three tissue culture cell lines--J774 macrophages, BSC-1, and Vero cells--infected with the Brazil strain of Trypanosoma cruzi. Indirect immunofluorescence light microscopy was used to demonstrate the antigenic response in host cells and parasites, simultaneously. In all morphotypes of T. cruzi, the monoclonal antibody reacted with all subpopulations of microtubules, inclusively, the subpellicular, flagellar, cytopharyngeal, and mitotic. The host cell cytoskeletal microtubule framework was revealed and the redistribution and destruction of the microtubular lattice in response to parasite infection over a 120 h period recorded. Our results show that after the initial inoculation of tissue cultures with trypomastigotes, the parasites penetrate the cells and locate in the perinuclear region of the cell where they multiply. The number and distribution of host cell microtubules were altered during the infection. The normal radial distribution of microtubules extending from the center of the cell to the periphery was destroyed. The remaining microtubules were observed at the periphery encircling, but well removed from the proliferating parasites. The complete transformation of the parasites was monitored throughout the infection with the end result being the liberation of parasites and the near complete destruction of the microtubular framework of the host cell. A residual population of dividing spheromastigotes was observed in cells liberating trypomastigotes. Colloidal gold labeling of thin sections as seen in the electron microscope affirmed the specificity of our monoclonal antibody to all subpopulations of microtubules in T. cruzi.

The cellulolytic enzyme complex of Clostridium thermocellum is very large.

The cellulolytic enzyme system bound to cellulose during the early stages of growth of C. thermocellum on this substrate was resolved into two major complexes. These complexes, as viewed by electron microscopy, are spherical particles with diameters of 210 A and 610 A and calculated molecular weights of 4.2 million and 102 million daltons, respectively.

Ultrastructural modifications during the metabolism of metronidazole by Trypanosoma cruzi.

Trypanosoma cruzi epimastigotes incubated in the presence of [14C] metronidazole are capable of a rapid uptake of the drug as shown by timecourse experiments and by autoradiography of the cells. The drug is metabolized to a more polar compound which has the chromatographic behavior of 2-methyl-5-nitroimidazole-1-yl-acetic acid. Mass spectral analysis of the metabolite shows diagnostic mass values (185, 184, 126) which are compatible with the 2-methyl-5-nitroimidazole-1-yl-acetic acid derivative. Flavone dramatically increases the production of the metabolite both in control and cells pretreated with phenobarbital. The cells show the presence of vesicles whose number is not significantly increased by phenobarbital. Metronidazole, on the other hand, significantly increases the number of vesicles in both control and cells grown in phenobarbital. The vesicles do not contain acid phosphatase and/or polyphosphates. It is concluded that the vesicles may correspond to a marked proliferation of the endoplasmic reticulum. A secondary effect of flavone is the proliferation of the mitochondrial membranes.

Ultrastructural features of Allantosoma intestinalis, a Suctorian ciliate isolated from the large intestine of the horse.

Allantosoma intestinalis, a suctorian ciliate isolated from the intestine of the horse, was studied utilizing light and electron optical methods. These small sausage-shaped organisms have a varying number of tentacles (between one and 14) located at each extremity of the body. The microtubular axoneme of each tentacle in cross-section consists of two files of microtubules arranged in a daisy-like configuration. Haptocysts occur in the tentacle shaft, abutted to the plasma membrane of the knob of the tentacle, and in the cell body. The haptocysts are bottle-shaped, with prominent annular striations around their midportion. The cell is covered by three membranes, an outer plasma membrane, an outer alveolar, and an inner alveolar membrane. A thin epiplasmic layer is found beneath the inner alveolar membrane, and a single row of microtubules underlies the epiplasm. The subpellicular microtubules are arranged parallel to each other forming a corset around the cell along the long axis; such a system is not characteristic of suctorians. A field of diminutive kinetosomes (each 180 mm long, max, of 15 per field), lacking cilia, was found below the cortex. The function of these prokinetosomes is unknown. A ciliated swarmer has not been observed, only the nonciliated adult. The characteristics of Allantosoma are compared with those of other suctorian genera.

Scanning electron microscopy of budding and metamorphosis in Discophrya collini (Root).

Budding and metamorphosis in the suctorian ciliate, Discophrya collini, have been investigated by scanning electron microscopy. The adult body form, tentacles, stalk, and attachment disk are described. A field of depressions or small pits was observed in the pellicle of adult suctorians in the early stages of bud formation. These pits deepen and coalesce until one large pore, the birth pore, remains. Cilia protrude through the pore, and as eversion of the bud proceeds the meridional arrangement of the larval ciliation is evident. After eversion is completed, a pronounced division furrow is found between the adult and soon-to-be-released swarmer. The stalk-forming region is seen on swarmers. Metamorphosing swarmers produce tentacles upon settling before any indication of ciliary resorption. Resorption of cilia and change in body form occur progressively with the production of the attachment disk and stalk.

Macronuclear differentiation during oral regeneration in Stentor coeruleus.

The moniliform macronucleus of Stentor coeruleus coalesces and renodulates during division, reorganization and regeneration. These nuclear events are spatially and temporally synchronized with oral primordium development occurring at stages six and seven of membranellar morphogenesis. Coalesced, elongating and early renodulating macronuclei at states six and seven contained microtubules within double membrane-bound channels, passing through the nucleus parallel to the long axis. The number of microtubules per channel varied between 4 and 23. Microtubules were also found in the perinuclear cytoplasm at these stages, forming a loose network around the nucleus. The microtubules and channels are absent in control cells and macronuclei of regenerating cells prior to stage six. These transient microtubules and channels appearing in late stage six and stage seven may provide the axial plane on which elongation of the macronucleus proceeds.

The chondriome of selected trypanosomatids. A three-dimensional study based on serial thick sections and high voltage electron microscopy.

The unitary nature of the chondriome of two species of trypanosomatids, Blastocrithidia culicis and Trypanosoma cruzi, has been demonstrated by utilizing serial thick-sectioning techniques combined with high voltage electron microscopy. Profiles of mitochondrial elements seen in thin sections and suspected to be parts of a continuum were confirmed by serial thick sectioning (0.25-0.50 mum thick) and stereopair analysis to be parts of the same mitochondrion. Three-dimensional models obtained from tracings of mitochondrial profiles on cellulose acetate reveal the mitochondrion of B. culicis to consist of a posterior mass with six tubular extensions extending upward and terminating in the anterior apex. The kinetoplast was found suspended between two of the tubular extensions, or less frequently, protuding as a nodule from one of the extensions. A bifurcation of one of the extensions was found in some specimens. The mitochondrion of T. cruzi consists of a triangular-shaped convoluted tubule, the base being the kinetoplast portion while the apex is directed posteriorly. The mitochondrion bifurcates behind the flagellar pocket, lateral to the kinetoplast, sending two entwined extensions into the tenuous anterior apex. Whether the mitochondrion of T. cruzi is unitary in the trypomastigote form was not determined in this study, since only epimastigote forms were used.