Calcium-cadmium competition in ovarian vitellogenic follicles of Galleria mellonella as revealed by X-ray microanalysis.

Dissected ovarioles of Galleria mellonella were exposed for 60 min to cadmium, supplied to insect culture medium in amount equalling that of calcium therein. The sections of ovarioles treated with a fixative precipitating both elements and embedded in epon were examined using energy-dispersive X-ray microanalysis system. Analysis of the digital maps obtained suggests a free inflow of Ca2+ and Cd2+, in approximately equal amounts. Calcium-cadmium competition was revealed within the whole sample as well as in the zoomed fragments comprising ooplasm filled with yolk and trophocyte nucleolus.

The role of vacuolar digestive system in preventing cadmium poisoning in Acanthamoeba castellanii.

Constituents showing cadmium affinity were found in digestive residues of the vacuolar digestive system in Acanthamoeba. Cadmium was detected by means of X-ray analysis in complexes appearing both in residues included within the digestive vacuoles and in those egested outside the cell. The digestive vacuolar system in Acanthamoeba castellanii is characterized by a particularly high turnover rate (4). Confronted with this fact, results of the present study suggest that the system is able to egest rapidly the ligand-bound cadmium. In this way protects the amoeba against serious toxic effects of the Cd-polluted environment.

Replacement of calcium by cadmium ions from Ca-affinity sites localized in different cytoplasmic compartments of Acanthamoeba cells.

In Acanthamoeba cells both Ca and Cd may be precipitated in different cytoplasmic compartments forming electron-opaque deposits, as shown in cells treated with glutaraldehyde supplied with either Ca or Cd respectively. It was found by semiquantitative X-ray microanalysis that the transfer of cells containing Ca-deposits to glutaraldehyde supplied with Cd causes a considerable replacement of Ca by Cd: in deposits formed at cell membrane, in cytoplasm, and in mitochondria the total weight percentage of Cd amounted to over 90, only in deposits formed in vacuoles the value was about 80. The replacement was not prevented by the presence of Ca in the transfer medium. When cells containing Cd-deposits were transferred to Ca-supplied medium, Cd predominated as well, its total weight percentage also amounting to over 90 in all the examined deposits. The results suggest that calcium bound in different cell structures may be easily replaced by cadmium, but not conversely, which suggests that Cd is more firmly than calcium linked to many cell constituents well preserved by fixation.

Ultracytochemical localization and microprobe quantitation of calcium stores in the insect oocyte.

Detection of calcium in the follicles of Galleria mellonella (Lepidoptera) was performed using two cytochemical methods. Calcium precipitation was obtained either with ammonium oxalate (AO) or with N,N-naphtaloylhydroxylamine (NHA). In both cases the X-ray "on line" analysis monitored the presence of calcium in the oocytes, which was correlated with the accumulation of yolk spheres. Concentration of calcium in oocytes filled with yolk and treated with AO amounted to 9 mmoles per 1,000 g tissue wet weight. This value is similar to that calculated previously for follicles untreated with any reagent and prepared for the analysis by the freeze-drying technique (Przelecka et al. 1980). Examination of the ultrastructure of oocytes treated with NHA revealed calcium precipitate at the follicular epithelium/oocyte interface, in endocytotic canaliculi and vesicles formed by the oocyte plasma membrane, in ooplasm, and in yolk spheres. In oocytes treated with AO, the calcium-precipitate intermingled with the precipitate produced by the osmium alone. The presumed cause of this phenomenon is discussed.

Displacement of cell-surface associated calcium inhibits phagocytosis and Ca-ATPase activity in amoeba.

Displacement of calcium from the cell surface region was observed in cells treated with either chlorpromazine or reserpine with chlorotetracycline being used as a calcium-fluorescent probe. The drugs also significantly inhibit the intensity of phagocytosis and Ca-ATPase activity. The possible role of Ca associated with the cell surface region in regulation of both phagocytosis and Ca-ATPase was discussed.

Ultrastructural alterations of the egg chambers of Galleria mellonella L. (Lepidoptera) developing after reserpine administration.

In the egg chambers of the wax moth Galleria mellonella L., developing after application of reserpine, a multilayered epithelium was observed through all the developmental stages. This epithelium consisted of "light" cells loaded with intracellular organelles. No "dark" cells, characteristic of the control material, were observed. All cells of the egg chambers of reserpine-treated insects were characterized mostly by an abundance of microtubules, strong vacuolation of cytoplasm, and dilation of intercellular and perinuclear spaces. Moreover, changes, such as malformation of nuclei and nucleoli, appearance of large glycogen deposits, occurrence of widened cisternae and canaliculi of rough endoplasmic reticulum (RER) connected with lipid droplets, and appearance of nonhomogeneous protein yolk spheres, were noted in the oocyte. The observed disturbances in the ultrastructure of the developing ovary are discussed in the aspect of a direct action of reserpine through the insect neurohormonal system, and of a possible indirect one by an impairment of calcium homeostasis.

Calcium binding sites in the insect polytrophic egg vesicles as possible markers of the route of inflow of calcium.

Calcium binding sites were revealed in the polytropic egg vesicles of Galleria mellonella (Lepidoptera) by fixation of the ovariole with glutaraldehyde in the presence of excess of calcium. Calcium dependent deposits appeared in the mitochondrial matrix in cells of the epithelial sheath and of the follicular epithelium. The deposits were also seen in the extracellular space around the follicular epithelial cells and around the trophocytes , at the oolemma, and in the coated vesicles of the oocytes at the stage of vitellogenesis. On the other hand, the deposits were not detected in the mitochondria of the trophocytes and the oocytes. Considering the high content of calcium in the insect oocyte ( Przel ecka et al. [17]) the possible route of entry of calcium to the oocyte is discussed on the basis of distribution of the visualized calcium binding sites.

Growth phase dependent alterations in the surface coat of Acanthamoeba castellanii.

Application of ruthenium red, cationized ferritin and concanavalin A to exponentially growing trophozoites reveals on their plasma membrane negatively charged surface coat bearing sugar residues. In the coat of trophozoites from advanced stationary growth phase no sugar residues can be visualized. In mature cysts the external layer of their wall is negatively charged, however, on their protoplast surface no terminals reacting with the 2 polycations, or with concanavalin A can be revealed, even though the penetration of the reagents has been ensured by enzymatic impairing of the cyst wall. The results are confronted with the known facts concerning alterations of physiological properties of plasma membrane occurring during the life cycle of Acanthamoeba.

Developmental changes in the localization of calcium binding sites in Acanthamoeba castellanii.

Vegetative cells of Acanthamoeba castellanii have the ability to bind calcium on the plasma membrane in form of the electron-dense deposits. The appearance of the deposits depends on the age of Acanthamoeba culture. In 24-h-old culture the deposits are very small, with diameter of 26 nm. During aging of culture, at both logarithmic and stationary growth phases, the diameter of deposits is larger (70-80 nm), while the deposits are localized only on the plasma membrane. During differentiation of Acanthamoeba cells into cysts electron-dense deposits with a diameter of about 170 nm appear in the mitochondria, whereas no deposits are observed on the plasma membrane. However, at the first stage of differentiation electron-dense material together with extruded membraneous fragments are also observed outside of some newly-formed young cysts. These results suggest that in Acanthamoeba cells, depending on the stage of life cycle, either plasma membrane or mitochondria may be involved in storage of excess cellular calcium.

Surface coat of plasma membrane of L-1210 lymphoid leukemia cells. A cytochemical study.

In the plasma membrane surface coat of lymphatic cells L 1210 the negatively charged residues are distributed all over the membrane and are due mainly to neuraminic acid, as revealed by neuraminidase digestion followed by cationized ferretin (CF) binding. They bind eagerly to CF but not all of them bind ruthenium red (RR). In these cells RR seems to reveal rather hyaluronic acid residues, as suggested by hyaluronidase digestion. Basing on the known characteristics of CF one can calculate that neuraminic acid residues are grouped in assemblies including no more than 32 of them and spaced from 20 to 60 nm. The hyaluronic acid residues seem to be dispersed more irregularly. The membrane constituents bearing concanavalin A receptor are distributed in assemblies as well the distances between which may range up to 350 nm. After applying osmium-ferrocyanide and tannic acid reactions the surface coat appears as a continuous layer what indicates that the contrasted molecules are distributed at distances below the practically achieved resolving power of the microscope used.

Seasonal changes in ultrastructure of brown adipose tissue in the common shrew (Sorex araneus l.).

Seasonal changes have been detected in the ultrastructure of brown adipose tissue (BAT) of the common shrew, Sorex araneus (a true nonhibernator) living under natural conditions and collected at the time when the most representative growth phase of the animal for the given season could be expected. In summer and autumn, BAT is characterized by the presence of large, regular, spherical lipid droplets and mitochondria closely adhering to one another. During winter, mitochondria possess densely packed cristae and are dispersed in the cytoplasm, sometimes invaginating into lipid droplets; the latter are diminished and often irregular in contour. The BAT in winter specimens is distinguished also by a large amount of blood capillaries penetrating the tissue. In spring, mitochondria of BAT are found more frequently adhering to each other, and are characterized by loosely arranged cristae. In addition to the spherical lipid droplets, agglomerations of lipid material may be found in the cytoplasm. The observed seasonal fluctuations in the ultrastructure of BAT in the shrew correspond to the metabolic rhythm of this animal. The latter point is discussed.

Visualization of calcium-binding sites at plasma membrane of shock-frozen Acanthamoeba cells.

Electron-dense deposits appear at the protoplasmic side of plasma membrane in Acanthamoeba log-phase cells when fixed either with glutaraldehyde or formaldehyde supplemented with Ca2+. Similar deposits appear when the cells are preloaded with Ca2+ and thereafter shock-frozen and prepared for electron microscopic examination by freeze-substitution technique. This suggests that their formation reflects the presence of a physiologically active system involved in capturing excess of inflowing calcium.

Calcium content and distribution in egg vesicles of Galleria mellonella (Lepidoptera) as determined by X-ray microanalysis.

In egg vesicles of Galleria mellonella (Lepidoptera) electron microprobe analysis reveals calcium in concentrations of 9 and 3 mmoles per 1,000 g tissue wet weight in oocyters and accompanying trophic cells, respectively. This high average level of calcium characterizes both pre- and postvitellogenic oocytes, but the distribution of calcium is not uniform. In postvitellogenic vesicles the central area of the ooplasm shows a higher content of Ca than peripheral one, what may be correlated with the distribution of mature yolk platelets within the ooplasm.

Effect of a membrane-stabilizing compound on calcium binding to the plasma membrane of Acanthamoeba castellanii.

Binding of calcium ions at the plasma membrane was studied in Acanthamoeba cells pretreated with ZIMET 3164, a benzimidazole nitrogen mustard derivative, which is known to show a potent immunosuppressive action combined with a membrane-stabilizing effect in mice. For reference, 2 compounds were applied: ZIMET 3393 (Cytostasan¿), another benzimidazole mustard derivative, which exerts only a moderate membrane effect and acts as a strong cytostatic, and ZIMET 176/68, a barbituric acid derivative, which acts as an inhibitor of humoral immune responses but without membrane-stabilizing effect. Application of any of the 3 compounds does not reduce the appearance of calcium binding sites, visualized by means of ultracytochemical reaction, notwithstanding their different action in the mammalian organism. On the contrary, it was estimated by morphometric analysis that the number of Ca-dependent deposits was augmented after treatment with low doses of any of the 3 compounds, what seems to be connected with the induced metabolic disturbances in low molecular phosphates level. High doses and/or prolongation of treatment of the cells resulted in diminution of the number of deposits and induces profound disturbances in cell ultrastructure, probably due to the toxic action of the applied doses. In these cases, band-like structures crosslinking the two leaflets of the plasma membrane may be observed; it is suggested that they represent integral membrane proteins.

Morphometric analysis of macronuclei and macronuclear fragments in autogamous cultures of Paramecium aurelia.

The general pattern of ultrastructural organization of macronuclear fragments which appear during autogamous changes of the nuclear apparatus of Paramecium aurelia is similar to that observed in undisturbed macronuclei. Morphometric analysis of electron-micrographs reveals, however, that only the nucleolar relative surface area remains unchanged in macronuclear fragments as compared with macronuclei, this suggesting the same intensivity of rRNA synthesis in them. Moreover, there exists, between the relative surface area covered by heterochromatin and the nucleoli in macronuclear fragments, an inversely proportional, statistically significant correlation, different than that calculated for macronuclei. The relative surface area of heterochromatin in macronuclear fragments is smaller than in macronuclei, what may be a sign either of enhancement of chromatin activity or of proceeding nuclear lysis.