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The symbiotic fungus Tuber melanosporum Vittad. (Perigord black truffle) belongs to the Ascomycota and forms mutualistic symbiosis with tree and shrub roots. This truffle has a high value in a global market and is cultivated in many countries of both hemispheres. The publication of the T. melanosporum genome has given researchers unique opportunities to learn more about the biology of the fungus. Real-time quantitative PCR (qRT-PCR) is a definitive technique for quantitating differences in transcriptional gene expression levels between samples. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes must show stable expression under given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in T. melanosporum development. In this study, we present a morphological and microscopical classification of the developmental stages of T. melanosporum fruit body, and investigate the expression levels of 12 candidate reference genes (18S rRNA; 5.8S rRNA; Elongation factor 1-alpha; Elongation factor 1-beta; α-tubulin; 60S ribosomal protein L29; β-tubulin; 40S ribosomal protein S1; 40S ribosomal protein S3; Glucose-6-phosphate dehydrogenase; β-actin; Ubiquitin-conjugating enzyme). To evaluate the suitability of these genes as endogenous controls, five software-based approaches and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. We demonstrate here that the 18S rRNA gene shows the most stable expression during T. melanosporum development and that a set of three genes, 18S rRNA, Elongation factor 1-alpha and 40S ribosomal protein S3, is the most suitable to normalize qRT-PCR data from all the analyzed developmental stages; conversely, 18S rRNA, Glucose-6-phosphate dehydrogenase and Elongation factor 1-alpha are the most suitable genes for fruiting body developmental stages.

Tyrosinase is an enzyme having two copper atoms at the reactive site occurring in prokaryotic and eukaryotic organisms. In animals tyrosinase is responsible for pigmentation, in plants for protection of injured tissues or, as in fungi, to harden cell walls. Some of us have previously shown that tyrosinase is involved in truffle development and differentiation. Here we present the purification, the molecular properties and the reversible inhibition of Tuber melanosporum tyrosinase by dimethyl-sulfide and bis[methylthio]methane, the main flavour compounds of black and whitish truffles. The MWr is 39 000. L -3,4-dihydroxyphenylalanine and L -tyrosine stain corresponding bands as expected for a true tyrosinase. Phenylthiourea, diethyldithiocarbamate and mimosine inhibit L -tyrosine and L -3,4-dihydroxyphenylalanine oxidation.

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.

Ribosome-inactivating proteins are enzymes of plant origin which de-adenilate the major ribosomal RNA, making it unable to bind the elongation factor and thus arresting protein synthesis. Recently the N-glycosidase activity of these enzymes has been extended also to deoxyribonucleotides substrates. In the present study we report the successful entrapment of the type 1 ribosome-inactivating protein saporin, covalently labelled with fluorescein isothiocyanate (FITC) into L-alpha lecitin/cholesterol liposomes and describe its delivery to human melanoma cells in vitro. The fluorescein reacted toxin maintained its enzymatic activity, although to a reduced extent; its interaction with liposomes resulted in the entry of the protein through the lipid bilayers. The resulting vesicles are carriers that can deliver the toxin inside cells; as a consequence the cytotoxic effects of the encapsulated enzyme were evident at a concentration two order of magnitude lower than that of the native one. In particular the nuclear damage, as revealed by micronuclei formation, was evident within 44 hr. The intracellular dynamics of the enzyme, as analyzed by confocal microscopy, point to an endocytic pathway of vesicles entry.

The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD(+)-oxidoreductase) activities of truffles of the genus Tuber (Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K(m) and V(max) values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing pI values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti-T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues.

Truffles live in an environment poor in oxygen, and are a good model for investigation of enzymatic adaptation to microaerobic conditions. We studied the antioxidant and glutathione dependent enzymatic endowment of Tuber truffles. Superoxide dismutase, catalase, glutathione peroxidase Se dependent, glutathione reductase, glyoxalase 1 and glyoxalase 2 are expressed and correlate with the microaerobic metabolism, growth rate and mycorrhizal symbiosis of truffles. A very low or undetectable glutathione S-transferase activity was found. For comparison the same enzyme activities were investigated in Agaricus bisporus which is epigeous and non-mycorrhizal.

The present study describes the liposome-mediated delivery of the type 1 ribosome-inactivating protein luffin to human melanoma cells in vitro. Luffin from Luffa cylindrica seeds has been successfully incorporated into lecithin/cholesterol and lecithin/cholesterol/dicetylphosphate negatively charged liposomes. The exposure of melanoma cells to the two types of liposomes resulted in the inhibition of protein synthesis and cell growth; apoptotic cell death was verified by means of TUNEL reaction and quantitation of cytosolic oligonucleosome-bound DNA. The toxicity of encapsulated luffin varied with the lipid composition of the vesicles; the strongest effect was observed with lecithin/cholesterol liposomes. These results identify liposome-incorporated luffin as a possible alternative to immunotoxins for the treatment of human melanoma in situ.

1. 1. ATP inhibits NAD(P) + -dependent malic enzyme activity by competing with the essential activators Mn 2+ and Mg 2+ . 2. 2. The kinetics fit an equation of co-operative kind with K i of 26 μM and K A of 11.3 μM for ATP/Mn 2+ competition; with K i , of 1.1 mM and K A of 0.96 mM for ATP/Mg 2+ competition. 3. 3. In the absence of the inhibitor, the co-operativity index increases from 1.77 to > 4 in the presence of ATP, in the case of ATP/Mn 2+ competition, while it increases from 1.88 to > 9 for ATP/Mg 2+ competition.