Inhibition of Candida albicans adhesiveness to human buccal and vaginal cells by sub-inhibitory concentrations of rilopirox.

Candida albicans is an opportunistic dimorphic pathogenic yeast which is present on the human mucosal epithelial cell surface. Its adhesion is considered to be an important first step in colonization and in the subsequent symptomatic or asymptomatic infection of buccal or vaginal mucosa. Because the ability to adhere is an important element of the pathogenicity of Candida we investigated in this study the compared effects of sub-inhibitory concentrations (sub-MICs) of rilopirox (CAS 104153-37-9) with those of ciclopirox olamine (CAS 41621-49-2) in inhibiting Candida adhesion to human buccal (BEC) and vaginal cells (VEC). Rilopirox is a new hydroxypyridone antimycotic agent with strong activity, especially against Candida albicans. There was a significant reduction in the mean number of Candida adhering to both buccal and vaginal cells with up to 1/8 MIC rilopirox for buccal and 1/16 MIC for vaginal cells, while for ciclopirox olamine reduction was significant up to 1/16 MIC for buccal and 1/8 MIC for vaginal cells. There were no significant differences in the dose-effect curves for BEC and VEC with either rilopirox and ciclopirox olamine, but on a molar basis, rilopirox was more active than ciclopirox olamine. The present in-vitro results support the developmental concept of an oropharyngeal and vaginal preparation of rilopirox. It can be expected that even sub-inhibitory concentrations of rilopirox exert an important additional effect in the treatment of oral and vaginal candidosis by impairing the pathogenic adhesion process of the fungus.

Effects of rilopirox on rabbit blastocysts in a protein-free in vitro culture under different culture conditions.

The effect of rilopirox (Hoe 351, CAS 104153-37-9) on rabbit blastocysts in vitro was studied. Blastocysts of day 6 post coitum were cultured in Ham's F 10 medium supplemented with polyvinylpyrrolidone using different concentrations of rilopirox with and without serum proteins. In culture conditions without serum proteins there were no differences in the growth rates of the blastocysts after 24 h of culture in the presence of 1 microgram/ml of rilopirox. However, 5 micrograms/ml of rilopirox resulted in a significant reduction of the mean growth rate as well as in a serious morphological damage of the treated blastocysts. Purified human serum albumin could partly prevent this toxic effect, while complete human serum and foetal calf serum did not show this characteristic. Analyses of the protein synthesis of the blastocysts treated with rilopirox by two-dimensional gel electrophoresis and subsequent fluorography did not show any differences in comparison to the untreated controls, despite of obvious morphological alterations.

Ultrastructural investigations of Candida albicans after in vitro treatment with the new fungicidal agent rilopirox.

Candida albicans was maintained in various culture media and incubated with different concentrations of the antifungal agent rilopirox. After fixation, dehydration and embedding in Spurr's medium, the cells were analysed at the ultrastructural level to investigate morphological aspects of the antifungal mode of action of this new hydroxpyridone compound. All untreated or sham-treated control cells exhibited a normal ultrastructural appearance. The cells were surrounded by a multilayered cell wall of typical structure, and the plasmalemma was in close contact with the cell wall. Also, the cell organelles of the protoplast corresponded well with the findings of other authors. After treatment with rilopirox, a variety of ultrastructural changes were seen, and the extent of damage was dependent on the specific culture condition, drug concentration and incubation time. After only 6 h and 1-10 micrograms ml-1 rilopirox, the plasmalemma exhibited elongated invaginations, the number and size of the lipid droplets had increased and greatly enlarged mitochondria containing electron-dense deposits became visible. The vacuolar system was strongly expanded and occupied nearly the whole cell. Exposure to higher concentrations of the antifungal agent and prolonged incubation times resulted in complete cytoplasmic autolysis and membrane breakdown, while the fungal cell wall remained unaffected. After treatment with 0.5% rilopirox suspension gel on agar cultures, the extent of cellular damage was clearly enhanced and included all cell types of a treated yeast colony, i.e. single blastospores and pseudohyphae.

Uteroglobin in the developing rabbit conceptus in vivo and in vitro.

Uteroglobin (UGL) was measured in day-4 to day-10 rabbit conceptuses by a competitive ELISA. Levels in blastocyst fluid, tissues, coverings and in the early fetus were determined separately. The total amount of UGL increased from 18.4 ng to 6.8 micrograms per conceptus. The UGL content of individual day-6 blastocysts was studied in vitro. Culturing was carried out up to 60 h in Ham's F10 medium with polyvinylpyrrolidone as macromolecular component, with and without progesterone, and with progesterone plus estradiol. UGL was determined in the blastocyst fluids, tissues with coverings and in the culture media. After labelling with [35S]-methionine, protein patterns of total blastocysts and of culture media were analysed by two-dimensional gel electrophoresis and fluorography. The morphology of cultured blastocysts was examined by electron microscopy. During 60 h of culture, the blastocysts expanded in diameter by 84%, and released 19% of their initial UGL content into the medium, independent of the hormonal substitution. Neither de novo synthesis, nor degradation of UGL was found: the protein remained unlabelled in fluorography, and its total quantity was not significantly different from that of non-cultured controls. Trophoblast, endoderm and embryoblast cells showed well preserved cell organelles and intercellular junctions, while the morphological differentiation of the germ layer was inhibited.

Nephrocytes of harvestmen, Leiobunum limbatum and L. rotundum.

Nephrocytes are said to be able to take up substances from the hemolymph. In Opiliones, which were examined electron microscopically three different types of nephrocytes were found. Numerous large nephrocytes lie clustered between the muscles in the anterior region of the body. Smaller nephrocytes occur scattered throughout the opilionid body, often affixed to tracheoles. The third group, pericardial cells, are always attached to the heart wall by connective ligaments. All nephrocytes are surrounded by a thick basement membrane and their plasma membrane forms pedicels. Junctional complexes link the adjacent pedicels to form diaphragm-like slit-membranes, which form the entrance to an extracellular compartment. The cytoplasm of the nephrocytes contains many pinocytotic vesicles and tubular elements. Different types of large electron-dense and electron-lucent vesicles can be distinguished. Occasionally a large bundle of unmyelinated nerve fibers is enclosed by a pericardial cell. Morphological differences between the types of nephrocytes are described and the ultrastructural characteristics of the nephrocytes of harvestmen are compared with those of other arthropods. Functional aspects are discussed with respect to their ultrafiltration structures.

Uptake and accumulation of tritiated uteroglobin by day-6 rabbit blastocysts.

Uptake of uteroglobin (UGL) by day-6 rabbit blastocysts and the intracellular fate of this protein were studied by light- and electron-microscopic autoradiography, immunocytochemistry and acid-phosphatase cytochemistry. UGL, labelled with N-succinimidyl-(2-3-3H)-propionate, was administered to embryos in vitro for 25 min to 4 h. The kinetics, determined from light-microscopic autoradiographs, showed a continuous uptake of the labeled protein over a 4-h period of incubation. At the ultrastructural level, increasing numbers of silver grains and an intense UGL immunoreaction in protein vacuoles and crystalloid bodies of trophoblast cells indicated that 3H-UGL had accumulated in these organelles. The presence of crystalloid inclusions in protein vacuoles suggests their origin by a condensation of the protein content, including UGL. Lysosomes containing radioactivity were rarely found, suggesting a very low degradation rate of the 3H-UGL. Protein vacuoles and crystalloid bodies exhibited no acid phosphatase reaction. The enzyme was mainly found outside the basal and lateral cell membranes of trophoblast cells, and on the rough endoplasmic reticulum of endoderm cells.

Uptake of tritiated uteroglobin by the endometrium of the rabbit during peri-implantation.

Uteroglobin, labelled with N-succinimidyl-(2-3-3H)-propionate, was applied in vivo for 3 h to pregnant rabbit uteri 7 and 9 days after mating. Light- and electron-microscopic autoradiographs showed that the endometrial epithelium, both ciliated and non-ciliated cells, is able to take up 3H-uteroglobin, however, with differing intensity. Large areas of labelling were found in the luminal epithelium, whereas the glandular epithelium contained fewer silver grains. Moreover, intensively labelled single cells or symplasms occurred in both luminal and glandular epithelium. They were identified as degenerating or dead cells. After internalization by pinocytosis or phagocytosis, the tritiated uteroglobin was observed in multivesicular bodies or in lysosomes with floccular content. Later, radioactivity was either found within residual bodies or distributed throughout the entire epithelium and the subepithelial stroma, i.e., the silver grains could no longer be assigned to specific cell organelles.

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium.

Day-6 rabbit blastocysts were cultured in Ham's F10 medium supplemented with polyvinylpyrrolidone as a macromolecular component, for 4 to 12 h. The integrity of the blastocyst cells was demonstrated by electron microscopy. Expansion and biosynthesis of proteins and of DNA were studied after culturing in the presence of 35S-methionine and 3H-thymidine. Polyvinylpyrrolidone did not interfere with the subsequent protein analysis, which was performed by two dimensional gel electrophoresis followed by silver staining and fluorography. More than 600 labelled proteins were found in the blastocyst tissue, many of them were also present in the blastocyst fluid and in the blastocyst coverings. Several proteins seemed to be produced for incorporation into the blastocyst coverings; others, only detected in the culture medium, might have been synthesized for secretion into the environment.

Purification of uteroglobin using monospecific antibodies coupled to divinylsulphone-activated agarose.

As a model for the isolation of a labile or trace protein, the purification of uteroglobin (UGL) by immunoaffinity chromatography is described. Antibody was isolated from sheep antiserum by immunoprecipitation, and coupled to divinylsulphone-activated agarose (Mini Leak). For the immunoabsorption stage rabbit uterine mucosal scrapings were defatted and incubated directly with the immunosorbent. After washing and desorption, the UGL preparation contained relatively few high molecular weight impurities and these were removed by gel chromatography. Purification was monitored at each step by two-dimensional SDS polyacrylamide gel electrophoresis and immunoelectrophoresis. Furthermore, affinity-purified UGL was tritiated with N-succinimidyl[2,3-3H]propionate and assayed by fluorography. In order to determine absolute UGL concentrations a competitive ELISA was developed.

Hemocytes of Leiobunum limbatum and two other species of harvestmen (Arachnida, Opiliones): Morphological classification and functional aspects.

The hemocytes of Leiobunum limbatum, Mitopus morio, and Opilio ravennae number from about 8,000 (juveniles) to 41,000 (pregnant females) per microliter of hemolymph. Five different types of hemocytes occur in all three species and both sexes. According to their ultrastructural appearance and their similarities to other arthropod hemocytes these five types are designated as prohemocyte, plasmatocyte, granulocyte, coagulocyte, and spherulocyte. From the ultrastructural point of view the prohemocytes are interpreted as stem cells for plasmatocytes which on their part differentiate into granulocytes. Transitional stages which would indicate the origin of coagulocytes and spherulocytes could not be found. Granulocytes and spherulocytes are interpreted as being storage cells; coagulocytes burst when hemolymph is transferred to a microscopic slide. Plasmatocytes are involved in the removal of dead cells or cell fragments. Plasmatocytes are demonstrated as being able to phagocytize and digest bacteria.

Ultrastructure and function of the circulatory organs of Leiobunum limbatum and two other species of harvestmen (arachnida: Opiliones).

The ultrastructure of the circulatory organs of Leiobunum limbatum, Mitopus morio, and Rilaena triangularis (Arachnida: Opiliones) has been investigated. We studied the organization of the heart, the myocardial contractile elements, its tubular system, organelles, and cell-junctions. The epicardium exhibits a large number of longitudinally arranged microtubules and is attached to the myocardium by special membrane complexes. Because of these structures, the epicardium is interpreted as the elastic antagonist to the heart muscle. At both ends of the heart the epicardium continues into a nonbranching aorta. Consequently the ultrastructure of the aortal wall, containing numerous microtubules, is similar to the ultrastructure of the epicardium. At the posterior end of the heart is a muscular slit-valve, while at the anterior end is a lobe-valve that lacks contractile elements. The heart is innervated by a dorsal ganglion. It covers the entire length of the heart and contains neural and glial cells. Two different types of neurosecretory granules and both electron-dense and electron-lucent synaptic vesicles are described.