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The dark-grown unicellular flagellate alga Euglena gracilis (Euglenophyceae) contains rudimentary proplastids, whose development can be stimulated in darkness by DNA-demethylating drugs. In this study, proplastid organization was examined in dark-grown cells treated with a non-competitive inhibitor of DNA-methyltransferase. With 10 U ml-1 inhibitor, the activating effect was observed in 15-20% of the cell population, with a peak after 20 h of exposure. Fluorescence, confocal laser scanning, and transmission electron microscopy revealed the conspicuous enlargement of the plastid system. The inner proplastid membranes consisted of several prothylakoids connected with enlarged and compact prolamellar bodies. A strong accumulation of protochlorophyll(ide) was shown with 77 K fluorescence emission spectra. Although the protochlorophyll(ide)-rich prolamellar bodies recalled a condition typical of etiolated higher plants, the absence of changes in protochlorophyll(ide) spectral forms and the lack of competence for building up crystalline membrane networks suggest that in Euglena the prolamellar body is simply a “generic” storage of membrane materials. The role of DNA methylation in light-triggered plastid development is discussed. [ABSTRACT FROM AUTHOR]

Thylakoid dismantling is one of the most relevant processes occurring when chloroplasts are converted to non-photosynthetically active plastids. The process is well characterised in senescing leaves, but other systems could present different features. In this study, thylakoid dismantling has been analysed in dividing cells of the unicellular alga, Euglena gracilis, cultured in darkness. Changes in photosynthetic pigments and in the abundance of LHC and PSII core proteins (D2 and CP43) showed that: (i) during the 0–24 h interval, the decline in LHCII was faster than that in the PSII core; (ii) during the 24–48 h interval, PSII and LHCII were strongly degraded to nearly the same extent; (iii) in the 48–72 h interval, the PSII core proteins declined markedly, while LHCII was maintained. These changes were accompanied by variations in room temperature fluorescence emission spectra recorded from single living cells with a microspectrofluorimeter (excitation, 436 nm; range 620–780 nm). Emission in the 700–715 nm range was proposed to derive from LHCI-II assemblages; changes in emission at 678 nm relative to PSII matched PSII core degradation phases. Overall, the results suggest that, in degreening E. gracilis, thylakoid dismantling is somewhat different from that associated with senescence, because of the early loss of LHCII. Moreover, it is proposed that, in this alga, disruption of the correct LHCI-II stoichiometry alters the energy transfer to photosystems and destabilises membrane appression leading to the thylakoid destacking observed using transmission electron microscopy. [ABSTRACT FROM AUTHOR]

Rhodamine 123 (Rh 123), a relatively new mitochondrial marker, little used in the study of plant cells, was tested on excized leaves of Elodca 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 autofluorescent chloroplasts was studied in connection with a normal or altered cytoskeletal framework. The specific uptake of Rh 123 by the organelles 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. [ABSTRACT FROM AUTHOR]

The changes in the pigment pattern and composition occurring in the Arum italicum berry during the various steps of maturation (ivory to deep-green stages) and ripening (yellow and red-orange stages) were studied and correlated to the ultrastructural modifications of plastids. Transmission electron microscopy showed that each stage was characterized by a specific plastidial type following the unusual sequence amyloplast-->chloroplast-->chromoplast. Plastidial transitions were accompanied by profound modifications in the pigmental composition, in particular, in the pattern of carotenoids and their precursors. The HPLC analysis of the carotenoids showed that, besides the two usual all-trans metabolic pathways leading to lutein through alpha-carotene and to auroxanthin through beta-carotene, an additional cis-isomeric biosynthetic pathway leading to cis-neoxanthin through cis-beta-carotene exists. During the pre-ripening stages, the three pathways were present even if with qualitative and quantitative variations. When complete ripening was reached, a block occurred at the cyclization level causing the accumulation of both all-trans (i.e. gamma-carotene and neurosporene) and cis-isomer (i.e. lycopene and zeta-carotene) carotene precursors. Because of the occurrence of unusual pigments and the presence of the three main plastidial types, the fruit of A. italicum may constitute a most instructive model for the study of carotenogenesis.

Spermine (Sp) produces growth inhibition and wall malformation in Saccharomyces cerevisiae in response to oversynthesis of beta-glucans and chitin. The effect is related to the polycation nature of the molecule. In the present work, to verify this hypothesis, the yeast was treated with the abiogenic polycation ruthenium red (RR). The strict analogy observed between the RR- and Sp-induced alterations reinforced our earlier assumption that Sp interacted with the anionic sites of the plasmalemma determining a spurious activation of the two inserted enzymes beta-glucan and chitin synthases. This view was further confirmed by the aberrant accumulation of beta-glucans in Schizosaccharomyces pombe and of chitin in Rhodotorula glutinis treated with Sp and RR. In these micro-organisms spermidine, which bears three amino groups instead of the four encountered in Sp, was ineffective. It is inferred that at least four cation sites must be present in a compound in order to affect wall morphogenesis in yeasts.

Monensin, an exocytosis perturber, was assayed on the dimorphic fungus Candida albicans in order to study its effects on wall synthesis and morphogenesis. In the presence of the drug, both germ-tube formation and hyphal elongation were markedly inhibited according to the dose and exposure time. The effect was accompanied by morphogenetic malformations which could be detected under UV light and by transmission electron microscopy. Enormous wall and septa thickenings revealed by cytochemical tests indicated an abnormal deposition of chitin. Since chitin synthase is inserted in the plasma membrane in a latent state and its activation depends on regulatory factors reaching the cell periphery through an orderly exocytosis, it is assumed that monensin affects the vesicular traffic leading to a prevalence of activators over inhibitors of the enzyme.

Blastospores of Candida albicans were exposed in vitro to fluconazole, a bis-triazole which inhibits ergosterol biosynthesis in fungi by interfering with the cytochrome P-450 dependent 14 alpha-demethylase. Electron microscope examination revealed that a low dose (3 micrograms/ml), short treatment (1-3 h) with this compound greatly stimulated autophagic activity not accompanied by alterations of the cell organelles. Only rarely, wall thickenings and some damage to the membrane system, except for the plasma membrane, was noted. This unusual phenomenon suggests that in the presence of fluconazole, due to the depletion of ergosterol and the consequent accumulation of 14 alpha-methylsterols, changes are induced in the properties of the tonoplast. It seems that in the presence of the molecule the membrane is not able to distinguish what is to be degraded from what is still useful for intracellular metabolism with the consequent disintegration of cell compartmentation. Fluconazole may be useful for indicating the mechanism for controlling lytic activity and the homeostatic role of the vacuole in yeasts.

The effect of polyamines (PA) on the synthesis and deposition of wall constituents in Saccharomyces cerevisiae was investigated. Spermidine (Spd) in various doses was ineffective whereas spermine (Sp) in the same concentrations caused a marked inhibition (25-60%) of cell proliferation accompanied by evident morphogenetic malformations. Sp-treated cells were elongated, grouped in small clusters, and showed malformed septa and aberrant wall thickenings. The PATAg technique revealed that the aberrations consisted of an abnormal accumulation of both reactive materials, like 1,3-beta-glucans, and unstained chitin components. Since 1,3-beta-glucan synthases and chitin synthases are inserted in the plasma membrane, whose anionic sites interacted with the cation groups of Sp, it is assumed that the molecule determines a condition resulting in an unregulated activation of the two enzymes. The fact that Spd, which contains three cationic groups instead of the four contained in Sp, is without effect suggests that a compound must have at least four cation sites in order to affect the cell division and wall morphogenesis of S. cerevisiae.

Alpha-terthienyl (alpha-T) in the presence of UV-A irradiation reduced the growth rate of Microsporum cookei. In the dark, alpha-T accumulated in small diffuse vacuoles within the hyphae. After UV-A treatment, alpha-T caused damage to the membranes of the nucleus, mitochondria and endoplasmic reticulum. Plasmolytic and autolytic changes occurred resulting in plasma membrane breakage and cell wall aberrations. UV-A activated alpha-T would appear to target membrane proteins.

Protoanemonin (PrA) is an antimicrobial agent whose mechanism of action has not been clearly described. Some evidence exists which indicates that the compound inhibits growth by interacting with microtubules (MTs). In order to confirm this hypothesis, an ultrastructural study was undertaken in vitro on Epidermophyton floccosum and Trichophyton mentagrophytes, two dermatophytes which proved to be sensitive to the drug. A concentration of 1.25 x 10(-4) M (half-MIC dose) for 48 h, caused a series of ultrastructural alterations which were detected by scanning and transmission electron microscopy. Wave-like hyphae with distorted apical tips were frequently observed. Wall formation was variously affected as revealed by the deposition of incomplete septa and the accumulation of lomasome-like infoldings. An extrusion of extraparietal material binding adjacent filaments of the mycelia together was also produced. The nucleoplasm was characteristically crossed by MT-like structures. The possibility exists that PrA exerts its antifungal activity by MT interaction.

Based on the main physiological and ultrastructural effects induced in Euglena gracilis by erythromycin, one of the least toxic of the commonly used antibiotics that specifically inhibit protein synthesis on 70S ribosomes of prokaryotic organisms, a symptomatologic picture is outlined which could be useful for the preliminary evaluation of the toxicity of antibacterial agents.

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.

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.

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.

The antipyretic and antibacterial activities of the ethanolic extract of the flowering tops of Teucrium polium (L.) were been studied. The extract was effective against both yeast and carrageenin pyrexia in rats. It also exhibited a marked antibacterial action against both gram positive and gram negative organisms and was found to be non toxic in acute studies.

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.