Vacuolar Space Research Articles

Development of protein bodies in oat starchy endosperm

The development of protein bodies in the starchy endosperm of oat (Avena sativa L.) was studied by an ultrastructural examination of the subaleurone layer at various stages of maturation. In caryopses of 11 mg fresh weight, cells were highly vacuolate with little evidence of protein deposition. By 18 mg, vacuoles were smaller, rough endoplasmic reticulum (RER) was evident, and some vacuoles contained protein deposits. As the caryopsis matured, protein deposits filled a greater proportion of the vacuolar space. Small protein deposits were observed within the cistemae of the RER, and direct connections between the RER and the tonoplast of vacuoles were evident. Dictyosomes were infrequently observed at any stage. The electron micrographs are consistent with the hypothesis that protein synthesized on the membrane-bound ribosomes is transported within the ER cisternae to the vacuoles, where it condenses, forming protein bodies.

The quantitative relationship of the fibrillar centres and other nucleolar components to changes in growth conditions, serum deprivation and low doses of actinomycin D in cultured diploid human fibroblasts (strain MRC-5).

ABSTRACT The proportions of the 4 components of nucleoli: namely, granular component, fibrillar component, vacuolar space and the fibrillar centre material, were calculated by electron microscopical stereological procedures for human diploid fibroblasts (strain MRC-5) under 6 different culture conditions. Using nucleolar volumes obtained by light microscopy of unsectioned cells, estimates of the volume of each constituent per nucleolus were obtained. From the size and number of fibrillar centres encountered it was possible to estimate approximately their numbers per nucleolus. This ranged from an average of 42 ±4 to 234 ±25, depending on the level of cell activity, the number rising with increasing cell activity. Their volumes were inversely proportional to their number per nucleolus, indicating a possible fusion with cell inactivation. The number of fibrillar centres exceeds the number of nucleolus organizers in man (which is ten) and is nearer the number of ribosomal genes, which has been quoted at between 100 and 400 for diploid cells. The volumes of granular and fibrillar components also reflect changes in cell activity. A different response follows drug-induced inactivity when compared with the less artificial inactivation resulting from confluence or serum starvation. There was less fibrillar component in the actinomycin D-inactivated nucleoli. It is suggested that the nature of the fibrillar component may not be the same in cells in different states and that the simple interpretation that this is the transcriptional component may need to be revised. The change to fewer larger fibrillar centres upon nucleolar inactivation may be a consequence of 3 simultaneous processes. First, that the organizers increase in size by the condensation of previously active organizer chromatin, which is withdrawing from its transcriptional configuration. Secondly, this process may be accompanied by the fusion of the resultant larger nucleolar organizer regions. And finally, the increase in sizes of fibrillar centres may be further affected by an accretion of some non-chromatin material, possibly matrix or skeletal protein material, onto the organizers.

Nutrient uptake and accumulation by sugarcane cell cultures in relation to the growth cycle

Growth characteristics and nutrient changes in medium and cells of batch-grown sugarcane cultures were investigated over a period of 14 days. Amino acids, PO 4 3− and K+ were substantially removed from the medium during the first seven days of culture; a strong preference for uptake of organic nitrogen over inorganic nitrogen was observed. Sodium uptake increased during the time when K+ was becoming deficient in the medium. The main anions taken up were SO 4 2− and PO 4 3− . Strong acidification and a virtually total extracellular hydrolysis of sucrose in the medium during the first seven days of culture were also observed. Tapering off of the rapid growth phase was accompanied by an increase of intra-cellular sucrose and a decrease of intracellular protein. As cells went from rapid growth into stationary phase, cytoplasmic space of the cells decreased slightly in favor of vacuolar space. Overall cell volume stayed constant throughout the growth cycle, except during a short period before onset of rapid growth. Transport of the glucose analog 3-O-methyl glucose remained constant in terms of Km value but the Vmax was slightly higher in rapidly growing cells.

Leishmania donovani. Hamster macrophage interactions in vitro: cell entry, intracellular survival, and multiplication of amastigotes.

An in vitro system was developed for studying host-parasite cellular interactions in visceral leishmaniasis with amastigotes isolated from infected spleens of hamsters and their peritoneal macrophages maintained by an improved method. The culture system supports the growth of Leishmania donovani amastigotes with different parasite/macrophage ratios for up to 2 wk, yielding results more consistent and reproducible than previously possible. Results indicated that the "forms" of the amastigotes (with or without adherent host membranes) and the "state" of the macrophages (with or without stimulation in vivo by thioglycollate or in vitro by aging) had no effect on the growth rate of the parasites, which, however, seems to vary with the macrophage subpopulations. An electron microscope study suggests that amastigotes are ingested through phagocytosis by the macrophages and become lodged in loose phagosomes. Additional evidence with quantitative data is presented to support the earlier findings that phagosome-lysosome fusion occurs after the interiorization of the parasites and that they not only survive but multiply in these vacuoles. During the postinfection periods, reorientation of amastigotes in vacuolar space results in the appearance of three types of parasitophorous vacuoles (parasites in loose vacuoles, in tight-fitting vacuoles or abutting in part against the inner lining of vacuoles). The last category may be the predominant type giving rise to the variations observed. Exogenously introduced dense marker accumulated in these parasitophorous vacuoles of the macrophages infected for several days indicating a continuous accessibility of amastigotes to the ambient mestruum via phagosome-lysosome vacuolar system of the host cells. This finding may have significant implications in parasite nutrition, host immunity, and chemotherapy of leishmaniasis.

Ultrastructure of the Cyst of Sarcocystis muris

Cysts of Sarcocystis muris develop within muscle cells and each is bounded by a parasitophorous vacuole membrane. Closely spaced spherical blebs formed from this membrane extend into the muscle cell cytoplasm. A dense substance fills the cavity of the bleb and occupies the vacuolar space immediately adjacent to the membrane. The remainder of the vacuole is filled with a moderately dense matrix within which the parasites develop. At 40 days after infection only metrocytes are present, characterized by their ovoid shape, lightly stained cytoplasm, amylopectin-like granules, and lack of micronemes. Metrocytes divide by a process resembling endodyogeny and eventually produce bradyzoites. By 78 days after infection, at which time the cyst is infective for cats, the few remaining metrocytes are located at the cyst periphery but most organisms are elongated and contain organalles characteristic for bradyzoites including micronemes, dense granules, and amylopectin. Structures indicative of division were not seen in bradyzoites. Rhoptries are few in number. Numerous vesicles of smooth endoplasmic reticulum accumulate in the cytoplasm of muscle cells adjacent to the periphery of the enlarging cyst but significant destruction of muscle fibers containing cysts with viable organisms was not seen in specimens fixed between 40 and 325 days after infection. Unusual lamellar structures were seen in some parasitized muscle cells and intracystic tubules occurred in some cysts.

An electron microscopic study of Babesia microti invading erythrocytes.

Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of the other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.

Etude ultrastructurale et cytochimique de la différenciation des bactéroïdes de Rhizobium trifolii Dangeard dans les nodules de Trifolium repens L.

An ultrastructural and cytochemical study of the mature nodules from Trifolium repens L. indicates that bacteroid formation may be considered as a differentiation of the bacteria Rhizobium trifolii which, after invading the leguminous cell cytoplasm, undergo significant modifications including essentially: (a) progressive increase of the volume (about fortyfold), (b) modification of the shape of the bacteria which are successively short, rod-shaped, filamentous, pear-shaped, then spherical, and sometimes polyhedric, (c) dispersion of the initial nucleoid and appearance of granular bodies, (d) formation of a plasma membrane derived vesicular system, bearing or bounding oxydoreductive enzymes, which react with diaminobenzidine (DAB) at pH 6, 8, and 9. Bacteria to bacteroid transformation is related to host-cell differentiation which is mainly characterized by organelles distributed in concentric layers (peripheral starch-containing plastids, mitochondria, bacteroids, nucleus, and central vacuole) and by leghaemoglobin formation in the vacuolar space surrounding the bacteroids. Leghaemoglobin is detected by its electron density and positive reaction with DAB after incubation at alcaline pH in the presence of H2O2. The cytological events preceding or following the functional state of differentiation are also illustrated and discussed: infection of the host-cells by bacteria released from the threads, senescence of the bacteroids with formation of voluminous drops of poly-β-hydroxybutyrate (PHB), concomitant death of the host-cells, and the bacteroids at the base of the nodule.

Studies of Blastocystis hominis.

SYNOPSISBlastocystis hominis, grown in Boeck‐Drbohlav culture medium, modified by the omission of rice starch and the addition of 20% human serum and mineral oil cover to the Locke's solution overlay, can assume 3 morphologic forms. In the absence of human serum the vacuolated form, which divides by binary fission predominates. In medium with high serum content the granular form appears, with 3 types of granules. Spheroid or more elongate cytoplasmic granules predominate. In older organisms, lipid granules are found either in the peripheral cytoplasm or in the central vacuolar space. In occasional cells, variable numbers of reproductive granules develop in the central vacuolar space. These latter granules are released from the organism and give rise to typical B. hominis cells. The 3rd form, the ameba form, appears in small numbers in older cultures and in those treated with antibiotics. Ameba forms feed on bacteria and have slow pseudopodial activity. Exposure to oxygen causes rapid damage to cell membrane, with resulting leakage and collapse.

Filamentous particles in human alveolar cell carcinomas: electron microscopy studies of six cases (preliminary report).

Over a 10-month period we observed 6 cases of human alveolar cell carcinoma, a relatively uncommon human tumor. Electron microscopy study of freshly excised tumor tissue revealed intracytoplasmic vacuoles with dense osmophilic inclusions containing filaments in 4 cases. The filamentous structures were about 90 nm in diameter, with a central core about 15 nm in diameter. Spherical bodies about 90–130 nm in diameter in the vacuolar space had a double membrane, and electrondense particles randomly dispersed in their matrices.