SUBCELLULAR EFFECTS OF DIMETHYLSULFOXIDE ON EUGLENA-GRACILIS

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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

Anomalous morphogenetic events in yeasts exposed to the polysaccharide-binding dye Congo red

When exposed to Congo red, Rhodotorula glutinis halts cell proliferation, whereas Saccharomyces cerevisiae continues to bud; however, the separation between mother and daughter cells is impeded. Both yeasts develop diverse wall and septum abnormalities due to the accumulation of amorphous materials instead of chitin microfibrils. In Saccharomyces, the original wall frequently breaks and numerous vesicles, probably of secretory nature, are visible in the cytoplasm. The microorgamisms employed react differently to Congo red exposure due to the different roles that chitin plays in their walls. To explain why Congo red forces chitin synthase to form excessive amounts of N-acetylglucosamine chains with a random distribution, a model is proposed in which multienzyme complexes dissociate into single units since the polymers under synthesis, with the dye attached, can not assemble regularly. Each enzyme subunit continues to form glucan chains while it moves freely and randomly along the plasma membrane

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

Alterations induced by dimethyl sulfoxide in Euglena gracilis with emphasis on the side effects

In a previous study, we demonstrated that 5% dimethyl sulfoxide (DMSO) alters the contractile system responsible for cell motility (euglenoid movements) and cytokinesis in Euglena gracilis. However, the nucleus continued to divide and most cells were larger than normal and binucleated. The present study reveals that DMSO, besides altering the cell functions requiring microfilaments, also affects other cell parts. More precisely, the materials normally covering the plasma membrane detach from it; the nucleus shows an irregular outline and aberrations in the nucleolus and chromosomes; the chloroplasts decompose the internal structures and, in a number of cells, transform into proplastid-like organelles. Also, the development of the proplastid into chloroplast in etiolated algae exposed to the light in the presence of DMSO is highly disturbed. These results show that DMSO has remarkable side effects like all the cytoskeletal poisons experimented up to now. An interpretation of the nuclear and chloroplast alterations is advanced

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

Cytoskeletal structures in Euglena gracilis after Triton X-100 extraction and dry cleaving

A three-dimensional network of structural filaments was visible with common electron microscopes in the cytoplasm of Euglena gracilis green cells extracted with buffers containing the nonionic detergent Triton X-100. A similar filamentous web was detected at the periphery of critical point dried cells cleaved on grids by means of an adhesive tape. SDS-polyacrylamide gel electrophoresis of the detergent-resistent cytoskeleton showed that actin or actin-like proteins of molecular weight in the range of 43–45 K are not among the components having a structural role inEuglena. The significance of these findings was discussed in relation to the capability of the alga to change the cell shape

ALTERATION OF CHITIN BIOGENESIS IN FUNGI EXPOSED TO A POLYSACCHARIDE BINDING DYE

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Ultrastructural effects of tetracaine in Euglena gracilis

Euglena gracilis green cells were treated for 1-5 min with 0.5-1 mM tetracaine (TC), a local anaesthetic known to displace membrane-bound calcium. One or 2 min exposure was sufficient to stop cell motility, induce cell rounding and alter the main organelles, except for the nucleus. The chloroplasts became irregularly shaped and displayed dilated locula; the mitochondria and Golgi apparatus lost their normal morphology; the reservoir microtubules (MTs) disappeared, whereas pellicular and flagellar MTs were unmodified. After 5 min treatment, mitochondria and plastids aggregated in clusters. In the nucleus, the chromatinic masses were fewer and smaller than normal and the nucleoplasm was infiltrated by inclusions of uncertain nature. The observed alterations suggest that TC alters the network which motility and scaffolding to both cytoplasmic and nuclear structures. Such effects may be interpreted in terms of increased intracellular Ca2+ even if additional mechanisms of action of the molecule cannot be excluded

Rhodamine 123, as a vital stain for mitochondria of plant cells

Rhodamine 123 (Rh 123), a relatively new mitochondrial marker, little used in the study of plant cells, was tested on excized leaves of Elodea canadensis Michx. and on suspension-cultured cells of Ranunculus serbicus Vis. In both preparations, the dye accumulated rapidly and selectively in the mitochondria whose number, morphology and cell distribution could be easily observed. In the presence of Rh 123, cytoplasmic movements could also be perceived and the spatial arrangement of the mitochondria with respect to that of the auto-fluorescent chloroplasts was studied in connection with a normal or altered cytoskeletal framework. The specific uptake of Rh 123 by the organdies seemed to be potential-dependent since it was influenced by cations, ionophores and inhibitors of electron transport. Short exposures to the stain were practically non-toxic, whereas prolonged treatments (6–20 h) provoked specific alterations in structure of the mitochondria. The data reported here indicate that Rh 123 may be an excellent vital stain to study the morphology, function and dynamics of the mitochondria in living plant cells