G. Dall'Olio

An hplc screening of some italian ranunculaceae for the lactone protoanemonin

Abstract An HPLC study of the distribution and quantitation of protoanemonin was carried out on several Italian Ranunculaceae species. The results indicated that protoanemonin may be a useful chemical marker in elucidating systematic relationships within this family.

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.

Cell Suspension Cultures of Cassia didymobotrya: Optimization of Growth and Secondary Metabolite Production by Application of the Orthogonal Design Method

Summary The optimization of both the cell growth and the accumulation of secondary metabolites in cell suspension cultures of Cassia didymobotrya Fres. was achieved for the first time by applying an orthogonal design method. The influence of the concentration of 2,4-D, kinetin, and sucrose as well as of the amount of the inoculum was studied.

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.

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.

Quaternary alkaloids in rhizomes of Ranunculus serbicus

The isolation, identification and quantification of four alkaloids from the quaternary alkaloid fraction of rhizome extracts of Ranunculus serbicus is described. The alkaloids isolated were the protoberberine-type palmatine, berberine and columbamine, and the benzylisoquinoline-type magnoflorine. This is the first reported isolation of quaternary alkaloids in the genus Ranunculus.

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 ).

5-Azacytidine-Removal of the Dark Repression in Plastid Development of Euglena gracilis Klebs

Summary Asynchronous, etiolated cells of Euglena gracilis, a light-dependent organism for plastid differentiation, were grown in complete darkness for 72 h in the presence or absence of 5 μg/mL 5-azaC, an inhibitor of DNA methylation. Plastid differentiation was followed by fluorescence and electron microscopy while the formation of Chl(ide) was ascertained by fluorimetric and HPLC analyses. It was found that in the presence of the compound, 20–40 % of the cells was induced to form differentiated plastids, with kinetics greatly comparable to that of the wild-type dark-grown Euglena exposed to light. Based on the immediate or light-induced red-fluorescence of the organelles viewed under UV light it was established that in about 10 % of the mentioned cells plastidogenesis reached the stage of Chl(ide) synthesis whereas in the other ones only the Chl(ide) precursors formed and a brief photoinduction was necessary for their assemblage. The removal of the dark repression of plastidogenesis operated by 5-azaC points to an involvement of DNA de(hypo)methylation in the expression of the genes responsible for plastid development as it occurs in other biological processes in both animals and plants. For the first time, Euglena gracilis was induced to form differentiated plastids in darkness.

Cytoskeletal structures inEuglena gracilis after triton X-100 extraction and dry cleaving

SummaryA three-dimensional network of structural filaments was visible with common electron microscopes in the cytoplasm ofEuglena 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.