A. Bonora

A simple colorimetric method for detecting cell viability in cultures of eukaryotic microorganisms

The dye methylene blue can be taken up by dead or severely damaged cells, but not by living cells. Based on this fact, a method was devised which permits quantitative determinations of injured cells in populations of microorganisms such asSaccharomyces cerevisiae, Rhodotorula glutinis, andEuglena gracilis. The percentage of damaged cells was determined by measuring, at 664 nm, the optical density of cell suspensions pretreated with 0.15 mM methylene blue for 6 min, a condition that does not affect cell integrity as determined by oxygen consumption and release of potassium ions. This technique is faster and simpler than the classical dye-exclusion and plate-counting methods.

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.

Growth Response of Sphagnum capillifolium to Nighttime Temperature and Nutrient Level: Mechanisms and Implications for Global Change

Individuals of Sphagnum capillifolium were cultured for 2 mo under six different combinations of nighttime temperature and nutrients. Low nighttime temperature caused a five-fold reduction of growth. Growth was also reduced when the plants were cultured without any addition of nutrient in the growing medium, but only when nutrient deficiency was coupled with high nighttime temperature. Growth reduction was associated with decreased rates of net photosynthesis, but was not accompanied by a degradation of photosynthetic pigments and/or variations in the pigment ratios, nor was the ultrastructure of chloroplasts significantly altered. The decline in the net photosynthetic rate may be due to a limitation in the enzymatic reactions at unfavorable temperatures. Nitrogen and, especially, phosphorus appeared to limit growth of Sphagnum capillifolium at optimal temperatures. A nighttime temperature of 50C was above the lower threshold triggering the synthesis of red wall-pigments, known to be promoted by nighttime chilling. Climate warming is expected to increase the growth rates of Sphagnum, but the consequences on the carbon balance of peatlands cannot be predicted because temperature rise may also enhance breakdown of peat.

The Vertical Pattern of Pigment Concentrations in Chloroplasts of Sphagnum capillifolium

The concentrations of chloroplast pigments were determined in current-year tissues of Sphagnum capillifolium (Ehrh.) Hedw. harvested at the end of the growing season in a bog in the southern Alps of Italy. Concentrations of both chlorophylls were highest in the midsummer segment. Concentrations of carotenoids were fairly stable, except in the spring segment, whereas chlorophyll derivatives (pheophytin, pheophorbide, and chlorophyllide) accumulated in the top segment, corre- sponding to the autumnal capitulum. This indicates a rapid degradation of chlorophyll, probably caused by nightly chilling, at the end of the growing season.

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.

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.

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.

Effects of Amitrole and Norflurazon on Carotenogenesis in Barley Plants Grown at Different Temperatures

A carotenoid deficiency can account for serious alterations of thylakoids, due to the essential roles played by these pigments in membrane protection against photo-oxidative damage (1,2). In spite of carotenoid importance in preserving the photosynthetic apparatus, information on their biosynthesis in green tissues is rather scarce, and the knowledge of carotenogenesis and its regulation mainly comes from studies on chromoplasts of ripening fruits (3,.4). Previous research on the lycopinic tigrina o 34 mutant of barley (5) and on barley plants treated with amitrole (6), a herbicide inhibiting lycopene cyclization (7), showed that plant growth temperature could affect carotenoid biosynthesis in leaf chloroplasts, suggesting the existence of thermo-modulated steps bypassing the block of mutation or herbicide. Recently, in tomato (4) and Arabidopsis (8) alternative reactions, which allow s-carotene and xanthophylls to be synthesized without involving lycopene as intermediate, have been proposed. In order to better define the relationship between temperature and carotenoid synthesis in chloroplasts, barley plants have been grown at two rather close temperatures. They have been treated with two herbicides, to analyze whether interruption of the carotenogenic pathway at distinct points would cause differentiated responses to the change in plant growth temperature. The two chemicals used were norflurazon (NF) and amitrole (AM), which inhibit phytoene desaturation and lycopene cyclization, respectively (7). In leaves of plants grown in different experimental conditions, the total and relative quantities of carotenoids synthesized and the ratios between the pigment forms were analyzed. The ultrastructural organization, chlorophyll contents and photosynthetic activity of chloroplasts were also investigated, to verify the photooxidative damage caused by the disturbed carotenoid biosynthesis.

Callus Formation, Cell Suspension Culture and Plant Regeneration in Ranunculus serbicus Vis.

Summary A procedure is described for the regeneration of whole plants of Ranunculus serbicus Vis. from cell suspension cultures through somatic embryogenesis. The best response for callus induction and growth was obtained from petioles on Murashige and Skoogs medium (MS) with hormones. Two different types of calli were obtained: (i) a friable white-yellow and fast growing callus with naphthaleneacetic acid (NAA) and benzyladenine (BA) and (ii) a compact, yellow-brown and slow growing callus with 2,4- \dichlorophenoxyacetic acid (2,4-D) and BA or kinetin (KIN). Cell suspension cultures arised from friable calli kept in liquid MS plus NAA and BA. They contained both single, vacuolated, non-embryogenic cells and densely cytoplasmic cell aggregates having the ultrastructural characteristics of embryogenic cells. Somatic embryos developed from most of the cell clusters plated on agarised MS plus NAA and BA or 2,4-D and KIN. When embryoids from NAA+BA containing MS media were transferred onto MS (half-strength of salt solution) without hormones, they developed plantlets that became mature plants.

Separation and quantitative determination of imipramine and desipramine from rat biological samples by high pressure liquid chromatography.

Imipramine and its metabolite, desipramine, have been determined in several rat biological samples by high pressure liquid chromatography. The method allows one to detect 15 ng in column of both drugs with a lower sensitivity limit of 20 μg l−1.