Peroxisome proliferator-activated receptors (PPARs) and related transcription factors in differentiating astrocyte cultures

Peroxisome proliferator-activated receptors (PPARs), retinoid X receptors (RXRs), CCAAT/enhancer binding proteins (C/EBPs) and -catenin are transcription factors involved in cell differentiation. The aim of this work was to investigate the occurrence and variations of these proteins during astrocyte differentiation. Primary cultures of mouse cortical astrocytes were characterized using nestin, A2B5 and glial fibrillary acidic protein (GFAP) as differentiation markers, during a period of 21 days in vitro (DIV). Glycogen and triglyceride accumulation were also studied. At 3 DIV the cultures were mainly constituted by neural progenitor cells, as assessed by their immunofluorescent pattern. At this time PPARs and -catenin were localized to the cytoplasm. Interestingly, some cells contained Oil Red O-positive lipid droplets. Between 7 and 21 DIV, nestin decreased, while GFAP increased, indicating ongoing astroglial differentiation. -catenin, predominantly nuclear at 7 DIV, later localized to membranes. Redistribution of all three PPAR isotypes from the cytoplasm to the nucleus was observed starting from 7 DIV. Between 7 and 14 DIV, C/EBP, PPAR, RXR and glycogen content increased. Between 14 and 21 DIV, PPAR/ decreased, while PPAR, C/EBP and and lipid droplet-containing cells increased. At 21 DIV both A2B5/GFAP and A2B5/GFAP cells were predominantly observed, indicating differentiation toward type-1 and type-2 astrocytes, although the presence of GFAP cells demonstrates the persistence of neural precursors in the culture even at this time point. In conclusion, our results, reporting modifications of PPARs, RXRs, C/EBPs and -catenin during culture time, strongly suggest the involvement of these transcription factors in astrocyte differentiation. Specifically, -catenin translocation from the nucleus to plasma membrane, together with PPAR/ decrease and C/EBP increase, could be related to decreased proliferation at confluence, while PPAR and and all C/EBPs could participate in differentiation processes, such as glycogenesis and lipidogenesis

Liposome-entrapped tyrosinase: a tool to investigate the regulation of the Raper-Mason pathway

The effect of the entrapment of mushroom tyrosinase (EC 1.14.18.1) within liposomes on the enzyme activity and Km vs. L-3,4-dihydroxyphenylalanine is reported in the present work; the effect of cholesterol insertion within liposome membranes on the enzyme activity has also been studied. The oxidation rates of various monophenols and diphenols by free and liposome-integrated mushroom tyrosinase were measured and the oxidation latencies vs. different substrates investigated. The different substrates are apparently oxidized according to the properties of the substituents as electron donors or acceptors; the Km values vs. L-3,4-dihydroxyphenylalanine calculated on measuring O2 consumption are higher than those calculated on measuring the dopachrome production rates. It is interesting that natural substrates of tyrosinase are oxidized according to a negative catalysis by the liposome-entrapped enzyme; this point is discussed in relation to the well known cytotoxicity of some intermediates of the Raper-Mason pathway

The cell death phenomenon during Tuber ectomycorrhiza morphogenesis

Cell death phenomenon was investigated during the formation and establishment of Tuber ectomycorrhiza (ECM) with host trees, both in vitro and in pot culture using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) reaction, Transmission electron microscopy (TEM) and Enzyme-linked immuno sorbent assay (ELISA). Tuber borchii mycelium and plantlets of Tilia plathyphyllos were used for in vitro ECM synthesis, whereas T. melanosporum, T. aestivum and T. borchii spores and seedlings of Corylus avellana, Quercus pubescens and T. plathyphyllos were employed in pot cultures. Non-mycorrhizal roots showed TUNEL-positive nuclei at the level of cap cells and tracheary elements as a result of physiological root morphogenesis. In contrast, during the pre-symbiotic phase and the following ECM developmental stages, progressive accumulation of tannin/polyphenol deposits developed in epidermal and cortical cells, leading to the cell death but without TUNEL positivity. After this necrosis, a further unexpected autophagic cell death was observed in apparently healthy mycorrhizae, first affecting mycoclena and then the Hartig net hyphae. This series of cell death phenomena involving both root cells and fungal ectomycorrhizal hyphae points to the existence of a genetic orchestration between the two symbiotic partners during ECM morphogenesis and deserves further investigation to elucidate the underlying molecular mechanisms and signaling pathways

Isolation and characterization of some mycelia inhabiting Tuber ascomata

Tuber spp. are ectomycorrhizal ascomycetes that produce subterranean ascomata known as truffles. Truffles can be regarded as complex microhabitats hosting bacteria and yeasts. In this paper we show that guest filamentous fungi are also associated to truffle ascomata, regardless of the Tuber spp., and report the morpho-molecular characterization of seven truffle-hosted mycelia isolated from healthy and intact Tuber ascomata. Some of these isolates were shown to be related to the fungal endophytes of plants. Interestingly, the trufflehosted mycelia grew stuck to the hyphal wall of their partner when co-cultivated with the Tuber borchii mycelium, but notwhenco-cultivated with the test species Agaricus macrosporus. The present data suggest that guest filamentous fungi can be added to the list of truffle-interacting microorganisms

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion