Cultivation modes affect the morphology, biochemical composition, and antioxidant and anti-inflammatory properties of the green microalga Neochloris oleoabundans.

Microalgae are considered promising sustainable sources of natural bioactive compounds to be used in biotechnological sectors. In recent years, attention is increasingly given to the search of microalgae-derived compounds with antioxidant and anti-inflammatory properties for nutraceutical or pharmacological issues. In this context, attention is usually focused on the composition and bioactivity of algae or their extracts, while less interest is driven to their biological features, for example, those related to morphology and cultivation conditions. In addition, specific studies on the antioxidant and anti-inflammatory properties of microalgae mainly concern Chlorella or Spirulina. The present work was focused on the characterization of the Chlorophyta Neochloris oleoabundans under two combinations of cultivation modes: autotrophy and glucose-induced mixotrophy, each followed by starvation. Biomass for morphological and biochemical characterization, as well as for extract preparation, was harvested at the end of each cultivation phase. Analyses indicated a different content of the most important classes of bioactive compounds with antioxidant/anti-inflammatory properties (lipids, exo-polysaccharides, pigments, total phenolics, and proteins). In particular, the most promising condition able to prompt the production of antioxidant algal biomass with anti-inflammatory properties was the mixotrophic one. Under mixotrophy, beside an elevated algal biomass production, a strong photosynthetic metabolism with high appression of thylakoid membranes and characteristics of high photo-protection from oxidative damage was observed and linked to the overproduction of exo-polysaccharides and lipids rather than pigments. Overall, mixotrophy appears a good choice to produce natural bioactive extracts, potentially well tolerated by human metabolism and environmentally sustainable.

Cyto-histological and morpho-physiological responses of common duckweed (Lemna minor L.) to chromium.

Along with cadmium, lead, mercury and other heavy metals, chromium is an important environmental pollutant, mainly concentrated in areas of intense anthropogenic pressure. The effect of potassium dichromate on Lemna minor populations was tested using the growth inhibition test. Cyto-histological and physiological analyses were also conducted to aid in understanding the strategies used by plants during exposure to chromium. Treatment with potassium dichromate caused a reduction in growth rate and frond size in all treated plants and especially at the highest concentrations. At these concentrations the photosynthetic pathway was also altered as shown by the decrease of maximum quantum yield of photosystem II and the chlorophyll b content and by the chloroplast ultrastructural modifications. Starch storage was also investigated by microscopic observations. It was the highest at the high concentrations of the pollutant. The data suggested a correlation between starch storage and reduced growth; there was greater inhibition of plant growth than inhibition of photosynthesis, resulting in a surplus of carbohydrates that may be stored as starch. The investigation helps to understand the mechanism related to heavy metal tolerance of Lemna minor and supplies information about the behavior of this species widely used as a biomarker.

Degreening of the unicellular alga Euglena gracilis: thylakoid composition, room temperature fluorescence spectra and chloroplast morphology.

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.

Responses of Trapa natans L. floating laminae to high concentrations of manganese.

The present study focuses on the responses of floating laminae of the Mn-tolerant hydrophyte Trapa natans L. to 1 mM Mn and their ability to accumulate the metal. Studies were carried out first on young floating laminae belonging to the second verticil of 30-day-old plants which originated from fruits that had been maintained in a 1 mM Mn-treated environment and again on the young floating laminae after 10 days of further treatment with 1 mM Mn. Mn storing was observed from the first days after germination, but only 10-day-treated laminae showed the capability to hyperaccumulate the element inside specialised cells (>20000 microg/g [dry weight]). Electron microscopy and the Folin-Ciocalteu reaction for phenolics revealed deposits of chelated material inside vacuoles of the first palisade layer and of idioblasts in the spongy tissue. X-ray microanalysis indicated that the deposits were Mn chelated with phenolic compounds. Numerous trichomes were observed at the lower epidermis of 10-day-treated laminae. They were rich in phenolics and characterised by Mn concretions at their base. As they are associated with a high concentration of the metal in culture water and sediments, trichomes may constitute a morphological differentiation for the secretion of Mn-chelating molecules into the culture water, as a probable "avoidance" mechanism. Finally, monitoring of the photosynthetic apparatus showed that photosynthetic function was not impaired, though differences in development occurred.

Modulations of the thylakoid system in snow xanthophycean alga cultured in the dark for two months: comparison between microspectrofluorimetric responses and morphological aspects.

The response of the plastid was studied, with a special emphasis on thylakoid structure and function, in a snow filamentous xanthophycean alga (Xanthonema sp.) incubated in darkness for two months. Microspectrofluorimetric analyses were performed on single living cells to study the variations in the assembly of the chlorophyll-protein complexes of photosystem II, in comparison with cells grown in light. In parallel, changes in micro- and submicroscopic plastid morphology and in photosynthetic pigment content were monitored. Throughout the experiment, the lamellar architecture of thylakoids in the alga was relatively well preserved, whereas photosystem II underwent disassembly and degradation triggered by prolonged darkness. Conversely, the light-harvesting complex of photosystem II proved to be relatively stable for long periods in darkness. Moreover, a role of the peripheral antennae in determining thylakoid arrangement in xanthophycean algae is implied. Although the responses observed in Xanthonema sp. can be considered in terms of acclimation to darkness, the progressive destabilisation of the light-harvesting complex of photosystem II testifies to incipient ageing of the cells after 35 days.

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess.

Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10 mM MnSO4. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72 h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96 h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion-tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.

Specific intra-tissue responses to manganese in the floating lamina of Trapa natans L.

Plant tolerance to heavy metals requires morpho-physiological mechanisms that are still poorly understood, especially in hydrophytes. This study focuses on the young floating lamina of the rhyzophyte Trapa natans exposed for 10 d to 130 microM Mn. The lamina has the ability to bioaccumulate Mn (> 3000 microg g(-1)). X-ray microanalysis of Mn cellular distribution revealed accumulation in the upper epidermis, in the first palisade layer, and in the idioblasts of the spongy tissue, which were shown with electron microscopy to contain osmiophilic vacuolar deposits, also observed to a minor extent in the control leaves. On the basis of biochemical and histochemical tests, these deposits were attributed to phenolic compounds that were probably able to chelate Mn. Net photosynthesis, photosynthetic pigments, room temperature microspectrofluorimetric analyses, and ultrastructural studies of plastids were performed to evaluate the status of the photosynthetic apparatus. A greater development of thylakoid membranes was observed in plastids of the second palisade and spongy tissue, which, however, did not accumulate Mn. Only the spongy tissue experienced inadequate assembly of PS II, but this did not significantly influence the photosynthetic yield of the whole lamina. It was concluded that T. natans can optimise productivity in the presence of Mn by means of specific intra-tissue responses within the framework of the floating lamina.

Carotenoid and ultrastructure variations in plastids of Arum italicum Miller fruit during maturation and ripening.

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.

Functional conservation of calreticulin in Euglena gracilis.

Calreticulin is the major high capacity, low affinity Ca2+ binding protein localized within the endoplasmic reticulum. It functions as a reservoir for triggered release of Ca2+ by the endoplasmic reticulum and is thus integral to eukaryotic signal transduction pathways involving Ca2+ as a second messenger. The early branching photosynthetic protist Euglena gracilis is shown to possess calreticulin as its major high capacity Ca2+ binding protein. The protein was purified, microsequenced and cloned. Like its homologues from higher eukaryotes, calreticulin from Euglena possesses a short signal peptide for endoplasmic reticulum import and the C-terminal retention signal KDEL, indicating that these components of the eukaryotic protein routing apparatus were functional in their present form prior to divergence of the euglenozoan lineage. A gene phylogeny for calreticulin and calnexin sequences in the context of eukaryotic homologues indicates i) that these Ca2+ binding endoplasmic reticulum proteins descend from a gene duplication that occurred in the earliest stages of eukaryotic evolution and furthermore ii) that Euglenozoa express the calreticulin protein of the kinetoplastid (trypanosomes and their relatives) lineage, rather than that of the eukaryotic chlorophyte which gave rise to Euglena's plastids. Evidence for conservation of endoplasmic reticulum routing and Ca2+ binding function of calreticulin from Euglena traces the functional history of Ca2+ second messenger signal transduction pathways deep into eukaryotic evolution.

Multiple transcription start sites of the carrot dihydrofolate reductase-thymidylate synthase gene, and sub-cellular localization of the bifunctional protein.

The analysis of clones obtained by rapid amplification of the 5' end and by primer extension of the mRNA for carrot bifunctional dihydrofolate reductase-thymidylate synthase showed transcripts of differing lengths that belonged to two sub-populations. The longer transcripts were found to contain a translation start site 147 nt upstream of, and in frame with, the one which is present in the shorter transcripts. The ORF that begins at this ATG codes for a protein of 64714 Da, which is much larger than mature DHFR-TS subunit. The N-terminus region of this polypeptide shows features typical of plant transit peptides. Immunogold labelling studies and immunorecognition of the plastid-containing sub-cellular fraction suggested a plastidial localisation of the bifunctional protein. Although plant cells were shown to contain folate pools in plastids, in mitochondria and in the cytosol, few enzymes of the folate pathway have been associated with any sub-cellular compartment. Thus, this is the first indication for the presence of an enzyme of the folate biosynthetic pathway in plastids. The longer transcripts revealed the presence of a TC microsatellite at the 5'-untranslated end.

Morphogenetic effects induced by spermine and ruthenium red in yeasts.

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.

Stimulation of the autophagic activity in blastospores of Candida albicans exposed in vitro to fluconazole.

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.

Interaction of monensin on dimorphic transition and hyphal growth in Candida albicans.

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.

5-Azacytidine accelerates yeast-mycelium conversion in Candida albicans.

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.

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.

Hemodynamic effects of felodipine in congestive heart failure.

The hemodynamic effects of increasing dosages of felodipine, a new calcium antagonist with selective vasodilator properties, were studied in 13 patients with chronic cardiac failure. A Swan-Ganz thermodilution catheter was positioned in the pulmonary artery and hemodynamic parameters were monitored from 9 am to 6 pm for five days. On the first and the fifth day patients received placebo (P) and on the second, third, and fourth day patients received felodipine 5, 10, and 20 mg, respectively. Symptom-limited exercise tests with a bicycle ergometer were performed on both days of P and on the fourth day. A marked reduction of systemic vascular resistance (SVR) and a significant increase of cardiac index without increments of heart rate (HR) were observed after felodipine at rest. A dose response effect could be demonstrated. During exercise a significant increment of cardiac index and decrease of pulmonary wedge pressure was observed after felodipine. Felodipine showed a potent vasodilator action on systemic circulation with significant changes on both stroke volume and filling pressures at rest and during exercise without side effects.

[Vertebral fracture caused by electric cardioversion]. / Frattura vertebrale da cardioversione elettrica.

A case of vertebral fracture following DC shock for ventricular fibrillation is reported. After a short review of the complications of this procedure the case is described and compared with two other cases known in literature. We conclude that, although this complication is uncommon, it must be kept in mind owing to its potential severity.

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.

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.