5-Azacytidine accelerates yeast-mycelium conversion in Candida albicans.

The effect of the DNA demethylating agent 5-azacytidine (5-azaC) on the morphological development of Candida albicans blastospores has been investigated by microscopic observations. It was found that this compound does not produce any morphogenetic effect when the cells are not induced to mycelial form. By contrast, on the induced cells, 5-azaC markedly accelerates the process of germ tube formation. In addition in the treated cells, yeast-mycelium conversion develops synchronously, whereas it is asynchronous and slow in the normal cells. These data indicate that, together with phenotypic modifications, modulation of gene activity by DNA demethylation occurs in Candida albicans during morphogenetic changes.

Aberrant development of Trichophyton mentagrophytes hyphae cultured in the presence of Congo red.

When Trichophyton mentagrophytes colonies were placed on a medium containing 150 micrograms/ml of Congo red, a dye which prevents chitin fibrillogenesis, their growth rate was reduced. The newly formed mycelium, examined under an ultraviolet microscope, consisted of thick, curled and branched hyphae endowed with swollen tips and subapical bulges. Short-time exposure revealed that the major sites of dye accumulation were the extension zones. Normally structured hyphae arose from aberrant mycelia when they were transferred onto a dye-free medium. The phenomena observed suggest that Congo red alters the wall properties of the extension zone, by inhibiting the gradual conversion of chitin chains, synthesized at the extreme tip, into microfibrils of increasing size and density.

Exocytosis in Saccharomyces cerevisiae treated with congo red.

When dividing cells of Saccharomyces cerevisiae were exposed to the polysaccharide-binding dye Congo red, the walls and septa became sites of chitin accumulation. In addition, the cytoplasm showed many vesicles that were different from those accumulating in the growing bud and from the lytic vacuoles of the untreated yeasts. To obtain information about these membranous structures, living cells were observed under phase contrast and UV light microscopes. Furthermore, ultrathin sections of Congo red-treated cells were processed by cytochemical techniques to reveal the chitin areas. Observations suggest that the aberrant vesicles were involved in a secretory process, and that pre-assembled chitin was not among the components transported to the cell periphery.

The effect of monensin on chitin synthesis in Candida albicans blastospores.

Monensin, a monovalent cation ionophore, was used to investigate some steps of the wall synthesis and morphogenesis in Candida albicans blastospores. In the presence of the drug, the pathogenic yeast developed enormous wall and septum thickenings that reacted intensely and specifically with wheat germ agglutinin and chitinase coupled to colloidal gold and fluorescein isothiocyanate. Therefore, the aberrant zones are interpreted as sites of chitin accumulation. The increased production of this homopolymer, also demonstrated by the chemical analysis of cell wall preparations, implies that monensin interferes in some way with the regulatory factors that normally control, in space and time, chitin synthetase activity.

Effects of a new pyrazolo [3, 4-d]pyrimidine on growth and morphology of Candida albicans.

A new pyrazolo [3, 4-d]pyrimidine derivative was synthesized and its antifungal activity evaluated in vitro against mycelial and yeast cells of Candida albicans. The most striking ultrastructural changes following treatment with 10-30 micrograms/ml (mycelia) and 25-75 micrograms/ml (yeasts) consisted in the deterioration of the organelle membranes and in aberrant thickenings of the cell wall. The complete disorganization of the cytoplasmic structures seemed to be the final event.

Morphological anomalies induced by Congo red in Aspergillus niger.

Aspergillus niger germ tubes were exposed for 6 h to 0.15 mg/ml of Congo red, a stain which prevents chitin microfibril assembly. The most evident alterations, detected under ultraviolet light and by transmission and scanning electron microscopy, concerned the hyphal tips which burst or, most frequently, expanded into bulges. In the latter structures, new hyphal tips appeared which, after giving rise to more or less developed hyphae, were themselves converted into new bulges. Therefore, segments derived from isotropic and polarized growth alternated in the organisms exposed to the dye. An interpretation of these abnormalities is advanced based upon the assumption that the maintainance of a regular gradient of wall viscosity in the hyphal extension zone depends primarily on the capability of glycan chains to form crystalline aggregates of increasing complexity.

Autophagic vacuole development in Trichophyton mentagrophytes exposed in vitro to miconazole.

Trichophyton mentagrophytes was treated in vitro for 24 h with an inhibitory concentration of miconazole (10(-5) M). The youngest, apical hyphal cells showed alterations of the ultrastructure of their membranes and cell wall as well as several vacuoles of an autophagic nature. Cellular autophagy appeared as a multi-step process beginning with swelling of the endoplasmic reticulum, whose membranes later fused and gave rise to large vacuoles that entrapped previously demarcated portions of the cytoplasm. Sequestered materials, initially unaltered, were ultimately degraded. The initial event in vacuolation was interpreted as entry of water into the cells, especially into the luminal space of the endoplasmic reticulum, in response to a decrease of wall pressure on the cytoplasm.

Degeneration and regeneration of chloroplasts in Euglena gracilis grown in the presence of acetate: ultrastructural evidence.

When green cells of Euglena gracilis, strain Z, were light-grown for several months on a solid medium containing an excess of sodium acetate (1.0% instead of the normal 0.1%), some 30% of the cells were colourless. The 'acetate-bleached' organisms, isolated by plating methods and subsequently incubated in the light in a liquid medium, regained the capacity to form chlorophyll in a few days in the absence of any organic carbon source, and within 1-2 weeks in the presence of 0.1% acetate. A number of bleached colonies, however, gave rise to populations in which the delay in pigment synthesis initiation was at least 2 months. Besides numerous paramylum granules and lipid inclusions, the acetate-bleached cells exhibited variably shaped and sized plastids, apparently lacking in ribosomes and showing a deeply disorganized membrane system. In the alga greened in the presence of 0.1% acetate, the pattern of plastidome reorganization was altered; the thylakoids were often unpaired and vesiculated in different degrees, owing primarily to the swelling of the lumen. A complete recovery of normal chloroplast structure occurred only after several weeks of exponential growth. The entire population greened in the absence of acetate constantly showed normal chloroplasts with perfectly reassociated thylakoids and clear partitions.

Antifungal properties of some pyrazolyl-alkyl-sulfides. Electron microscopic evidence in two dermatophytes and Candida albicans.

Four 5-amino-4-alkylthio-pyrazoles were synthesized and their antifungal activity was evaluated in vitro in Trichophyton mentagrophytes, Microsporum cookei and Candida albicans. The compounds slightly influenced the growth kinetics of the yeast, but at concentrations ranging from 20 to 40 micrograms/ml completely prevented the mycelial growth of the two dermatophytes cultivated on Sabouraud's agar medium. An electron microscopic study, undertaken by using the most active compound, showed that in C. albicans mitochondria were the only cell targets affected whereas in the dermatophytes cell wall, plasmalemma and the main cytoplasmic organelles were damaged in various degrees. Since the most remarkable alterations were connected with membrane abnormalities, the cytological changes observed were tentatively interpreted as a consequence of the compound intrusion into the lipid bilayer of the membranes, since the drug is lipophilic in nature.

Coumarin-induced disturbances of morphological development and cell wall formation in Trichophyton mentagrophytes.

When Trichophyton mentagrophytes thalli are placed on a medium containing 300 microgram/ml of coumarin, their growth rate is drastically reduced and the newly formed mycelium consists of curled and branched hyphae showing subapical bulges and swollen tips. Under the electron microscope, the most relevant abnormalities concern the cell walls which are often thickened by aberrantly shaped zones that are usually smeared unevenly over the surface of the "primary" wall instead of being deposited in discrete ridges. An ultracytochemical analysis suggests that the irregular thickenings are formed of chitin. Arguments suitable to explain the phenomena observed are discussed.

Electron microscopic evidence of Phosfon D-induced alterations in the membranes of the dermatophytic fungus Microsporum cookei.

The dermatophytic fungus Microsporum cookei was cultivated for 24 h in the presence of subinhibitory and inhibitory concentrations (50 and 100 micrograms/ml) of Phosfon D, a growth retardant for higher plants also affecting fungal development, and its toxic effects were examined at the ultrastructural level. In both treatments, Phosfon D attacked the membranes, whose structural integrity was clearly compromised with damage of particular severity to mitochondria, nuclei and endoplasmic reticulum. In the instance of fungal growth suppression, the compound also caused plasmolytic and autolytic phenomena, sometimes accompanied by plasma membrane breakages. The submicroscopic effects observed confirm that Prosfon D is an antifungal compound which displays its toxic effects in the area of lipid metabolism, probably preventing the synthesis of fundamental components of the cellular membranes, such as unsaturated fatty acids and sterols.

Fungal toxicity of phosfon D and its reversion by lipids.

The growth retardant Phosfon D inhibits the growth of some yeasts and human pathogenic filamentous fungi. The toxic effects of the compound on the dermatophyte Microsporum cookei were completely reversed by adding ergosterol and oleic acid to the medium. The significance of these findings is discussed.

Submicroscopic morphology of Trichophyton mentagrophytes grown at different temperatures.

Several modifications were observed in Trichophyton mentagrophytes cultivated at 19 degrees and 37 degrees C, i.e. nine degrees below and above the optimum of 28 degrees C. The phenomena included inhibition of the growth rate, changes in the gross aspects of the cultures as well as of the microscopic and submicroscopic morphology of the hyphal cells. At the ultrastructural level, in particular, it was shown that, at the suboptimal temperature, although the organelle structure in both young and aged hyphal cells remained nearly unchanged, unusual bodies of probable storage significance and plasmalemmasomes were formed. At the supraoptimal temperature, the youngest cells showed a normal organization but were richer in glycogen clusters and enveloped by a cell wall thicker than the ones at the optimal condition. In the cells far from the apex, the endomembrane integrity was lost and consequently an autolytic activity occurred. Degradation phenomena were detectable also at cell wall level. The cytological changes observed were tentatively correlated with a possible different sensitivity of the membrane system at the experimented temperature conditions.

Plasmalemmasome development in Trichophyton mentagrophytes grown at suboptimal temperature.

When the dermatophytic fungus Trichophyton mentagrophytes is cultivated at the suboptimal temperature of 19 degrees C, the plasmalemma of the youngest cells appears to be associated with a variety of membranous bodies, the number of which is much greater than at the optimum temperature of 28 degrees C. These structures can be indicated as plasmalemmasomes both for morphological characteristics and cytochemical reactivity. A possible correlation between the suboptimal condition and plasmalemmasome ontogeny and function is proposed.

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: a comparative study on the effects of coagulant and non-coagulant fixatives.

The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulant fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.

On the fungitoxicity of some new thiocyanatopyrazole derivatives: electron microscopical study in Trichophyton mentagrophytes.

Four thiocyanatopyrazole derivatives were synthesized and their fungistatic activity was demonstrated in vitro against a number of dermatophytic fungi. In Trichophyton mentagrophytes, the most active compound induced as unusual increase of the plasma membrane with production of intra and extracytoplasmic complexes, a deterioration of nuclear and mitochondrial membranes and a formation of autophagic-like vacuoles. Plasmolysis, accompanied by an almost complete disorganization of cytoplasmic structures, seemed to be the final event. A possible mechanism of action of the compounds were discussed.

Fine structural characterization of microbodies and Woronin bodies in Trichophyton mentagrophytes.

Microbodies and Woronin bodies, organelles surrounded by a single unit membrane, were identified in the hyphal cells of Trichophyton mentagrophytes by employing a fixative containing TAPO. The fine structure of the organelles is described and their possible significance discussed.