G. L. Vannini

Effects of Congo red on wall synthesis and morphogenesis in Saccharomyces cerevisiae

Abstract Congo red, a stain which prevents polyglycan microfibril assembly, was assayed on growing cells of Saccharomyces cerevisiae. In the presence of 0.25 mg/ml of the chemical, yeast developed aberrant wall thickenings and malformed septa, and underwent several budding events without the occurrence of the separation between mother and daughter cells. It is suggested that Congo red may uncouple polymerization and crystallization of the wall constituents, primarily chitin, that normally form microfibrils. It is also propsed that the regular assembly of the chains into microfibrils is needed to give a correct rate and direction to the process of polyglycan synthesis.

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.

Binucleation and abnormal chromosome distribution inEuglena gracilis cells treated with dimethyl sulfoxide

SummaryThe addition of 5% of dimethyl sulfoxide (DMSO) to cultures ofEuglena gracilis in the logarithmic phase of growth caused an immediate inhibition of cell multiplication and motility without completely blocking nuclear division. Importantly, some 50% of the cells were 2–3 times larger than normal and were also binucleate after 24–48 hours of treatment. Evidently binucleation resulted from the lack of cytokinesis after mitosis was induced. Transmission electron microscopy, using serial sections, showed the occurrence of nuclei either with a normal or a reduced number of chromatin masses. Solvent withdrawal led to a rapid recovery of all the normal cell activities.On the contrary, 2.5% of DMSO produced no effect during the entire period of treatment (48 hours), whereas a 1-hour exposure to 10% of the solvent was sufficient to provoke aspecific and irreversible cellular damage.Since DMSO is known to produce alterations in actin-containing structures in a wide variety of cells types, an involvement of microfilaments in cell motility, cytokinesis and chromosome separation during mitosis inEuglena is proposed and discussed.

A cytochemical approach to the wound repair mechanism inUdotea petiolata (Siphonales)

SummaryWhen injured, the thalli of the coenocytic algaUdotea petiolata undergo a rapid sealing process mainly due to the extrusion of two successive plugs. In the first, external and transitory plug, sulphated polysaccharides are the predominant components. In the second, permanent and internal plug, roundish bodies having a complex polysaccharidic composition are embedded in a fibrillar matrix of still unknown nature. The sulphated sugars were identified and located by means of Alcian Blue staining and X-ray microanalysis. A periodic acid-thiocarbohydrazide-silver proteinate technique proved useful especially in the study of the roundish bodies and in the compositional and structural comparison of the siphon wall with the wound wall. Phosphotungstic acid at low pH was used to evidentiate an extensive plasma membrane activity in the repairing cytoplasm.

Distribution of the receptors for concanavalin A on the surface of Euglena gracilis as revealed by fluorescence microscopy

Abstract The binding of concanavalin A (Con A) to the cell surface of Euglena gracilis was explored in various conditions by fluorescence microscopy. In both living and glutaraldehyde-fixed cells, the membrane of the emergent flagellum and of the reservoir are intensely and uniformly labelled by the fluorescein isothiocyanate-conjugated lectin. In contrast, the pellicular surface shows an alternation of reactive and unreactive strips. The streaks of binding, weak in brightness, pass helically around the cell body, probably at the level of the grooves and/or of the posterior margin of each ridge. Neither temperature variations nor exposure to the fluoresceinated ligand over a 30-min period consistently modify the binding pattern which in all experimental conditions occurs almost immediately at the flagellum level and subsequently at the reservoir and pellicle surface. Two main indications arise from the results: (i) concanavalin A receptors are especially localized in membrane areas possessing an unstable structure and numerous external glycosylated components; (ii) the distribution pattern of the lectin along the pellicle is similar to that known for the antipellicular antibodies.

Physiological and Ultrastructural Changes Induced in LightGrown Cells of Euglena gracilis by Myomycin Treatment

Summary The effect of myomycin, a new member of the family of water soluble, basic cyclitol antibiotics, the mechanism of action of which is not yet known, was tested on light-grown cells of Euglena gracilis. At the concentrations of 10, 20, 35 and 50 ,µg/ml the drug exhibited a permanent bleaching activity, which approached or attained the 100 % level after plating. In spite of the great bleaching efficiency, the formation of chlorophyll was retarded but not arrested during a five day treatment period and normal cell division was maintained. A fluorescence and electron microscopy study indicated that the chloroplast structure was affected to a high degree. Ultrastructural observations carried out on bleached cells demonstrated that plastids did not disappear, but remained present as poorly differentiated bodies strictly resembling the proplastids of dark-grown cells. On the basis of these results and the structural analogies of myomycin with two groups of antibiotics which strongly bleach Euglena by acting as protein synthesis inhibitors at the prokaryotic level, a possible mechanism of action of the drug is proposed and discussed in the light of the existing literature on bleaching agents.

Structural and developmental aspects during the greening process ofEuglena gracilis treated with myomycin

SummaryThe effects of myomycin (MM), a new bleaching antibiotic forEuglena gracilis, were investigated during the greening process of the alga. Dark-grown cells, after preincubation in the dark for 21 hours, were exposed to continuous light for 72 hours in both growing and resting conditions in the presence of 20, 35, 50, 100, 150 μg/ml of the antibiotic. In dividing cells, chlorophyll synthesis was strongly inhibited and practically suppressed with the two highest concentrations of MM, while in non-dividing cells, the process was only partially influenced. The cell division rate was also lowered, although in lesser degree than chlorophyll formation, but in any case a normal level of cell viability was maintained. Fluorescence and electron microscopic observations showed that the decrease in pigment synthesis corresponded to several stages of inhibition of the light-induced proplastid-to-chloroplast transformation. In particular, the plastids of the cells treated with the three lowest concentrations of MM showed an abnormal outline and a reduced number of thylakoids. In the presence of 100 and 150 μg/ml of the antibiotic, the thylakoids either did not form or were present as perforated or fragmented structures inside small plastids sometimes containing non-crystalline prolamellar bodies. In the permanently bleached cells, the plastids persisted as poorly differentiated bodies strictly resembling the proplastids of dark-grown cells. In any case, in spite of the profound plastidial alterations, the remnant cell was not apparently affected by the antibiotic. On the basis of the results obtained and the literature on bleaching agents, it can be inferred that the action of MM is due to a permanent block of protein synthesis on prokaryotic type plastidial ribosomes.

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

SummaryThe new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiffs reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulatn fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenkers 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.

Possibilità d'impiego dell'arancio di acridina e dell'acriflavina nella ricerca istocliimica dei polisaccaridi insolubili della cellula vegetale

Abstract Possible use of acridine orange and acriflavine in histochemistry staining of plant cell insoluble polysaccharides. – It is suggested to use acridine orange and acriflavine for staining insoluble polysaccharides in plant histochemistry. In fact the two dyes can give, under certain conditions, specific metachromatic effects. A discussion of the results and a comparison with those obtained by PAS follow.

Coumarin as a Cytostatic Drug for Euglena gracilis A Clue to Cell Cycle Study

Summary Concentrations of coumarin ranging from 50 to 250 , µg /ml were tested on non-synchronous etiolated cells of Euglena gracilis maintained under constant illumination for 72 h. Proportionally to the doses employed, the lacton depressed cell division rate without affecting plastid replication, so that chloroplast number per cell tended to increase. During the first 48 h, chlorophyll synthesis and oxygen evolution increased, but both of these processes and also respiration subsequently declined owing to coumarin toxicity. The most striking morphological effects induced by the compound consisted of odd-shaped, incompletely developed chloroplasts and giant mitochondria. Since with respect to the nuclear ultrastructure, taken as a cell cycle marker, most of the coumarin treated cells were recognized in the last period of interphase, it is suggested that the compound prevents the formation of mitotic spindle microtubules, probably by interacting with the microtubule organizing centers (MTCO s ).