Patrizia Cesare

Truffle thio‐flavours reversibly inhibit truffle tyrosinase

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 MW(r) is 39000. 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.

Improved Mitochondrial and Methylglyoxal-Related Metabolisms Support Hyperproliferation Induced by 50 Hz Magnetic Field in Neuroblastoma Cells

Extremely low frequency magnetic fields (ELF‐MF) are common environmental agents that are suspected to promote later stages of tumorigenesis, especially in brain‐derived malignancies. Even though ELF magnetic fields have been previously linked to increased proliferation in neuroblastoma cells, no previous work has studied whether ELF‐MF exposure may change key biomolecular features, such as anti‐glycative defence and energy re‐programming, both of which are currently considered as crucial factors involved in the phenotype and progression of many malignancies. Our study investigated whether the hyperproliferation that is induced in SH‐SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field is supported by an improved defense towards methylglyoxal (MG), which is an endogenous cancer‐static and glycating α‐oxoaldehyde, and by rewiring of energy metabolism. Our findings show that not only the ELF magnetic field interfered with the biology of neuron‐derived malignant cells, by de‐differentiating further the cellular phenotype and by increasing the proliferative activity, but also triggered cytoprotective mechanisms through the enhancement of the defense against MG, along with a more efficient management of metabolic energy, presumably to support the rapid cell outgrowth. Intriguingly, we also revealed that the MF‐induced bioeffects took place after an initial imbalance of the cellular homeostasis, which most likely created a transient unstable milieu. The biochemical pathways and molecular targets revealed in this research could be exploited for future approaches aimed at limiting or suppressing the deleterious effects of ELF magnetic fields. J. Cell. Physiol. 231: 2014–2025, 2016.

Validation of reference genes for quantitative real-time PCR in Périgord black truffle (Tuber melanosporum) developmental stages

The symbiotic fungus Tuber melanosporum Vittad. (Périgord 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.

Antiproliferative effect and apoptotic response in vitro of human melanoma cells to liposomes containing the ribosome-inactivating protein luffin

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.

NUCLEAR DAMAGE INDUCED BY LIPOSOMES CONTAINING FITC-LABELLED SAPORIN ON HUMAN MELANOMA CELLS IN VITRO

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-α 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.

Power frequency magnetic field promotes a more malignant phenotype in neuroblastoma cells via redox-related mechanisms

In accordance with the classification of the International Agency for Research on Cancer, extremely low frequency magnetic fields (ELF-MF) are suspected to promote malignant progression by providing survival advantage to cancer cells through the activation of critical cytoprotective pathways. Among these, the major antioxidative and detoxification defence systems might be targeted by ELF-MF by conferring cells significant resistance against clinically-relevant cytotoxic agents. We investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field was supported by improved defence towards reactive oxygen species (ROS) and xenobiotics, as well as by reduced vulnerability against both H2O2 and anti-tumor ROS-generating drug doxorubicin. ELF-MF induced a proliferative and survival advantage by activating key redox-responsive antioxidative and detoxification cytoprotective pathways that are associated with a more aggressive behavior of neuroblastoma cells. This was coupled with the upregulation of the major sirtuins, as well as with increased signaling activity of the erythroid 2-related nuclear transcription factor 2 (NRF2). Interestingly, we also showed that the exposure to 50 Hz MF as low as 100 µT may still be able to alter behavior and responses of cancer cells to clinically-relevant drugs.

ATP inhibition competes with activating cations in modulating the NAD(P)+-malic enzyme activity in the mitochondrial matrix of Xenopus laevis oocytes

1. ATP inhibits NAD(P)(+)-dependent malic enzyme activity by competing with the essential activators Mn2+ and Mg2+. 2. The kinetics fit an equation of co-operative kind with Ki of 26 microM and KA of 11.3 microM for ATP/Mn2+ competition; with Ki of 1.1 mM and KA of 0.96 mM for ATP/Mg2+ competition. 3. In the absence of the inhibitor, the co-operativity index increases from 1.77 to greater than 4 in the presence of ATP, in the case of ATP/Mn2+ competition, while it increases from 1.88 to greater than 9 for ATP/Mg2+ competition.

Leptin contributes to long-term stabilization of HIF-1α in cancer cells subjected to oxygen limiting conditions

Leptin, a cytokine produced by the adipose tissue in response to food intake, is a key player in the regulation of energy balance and body weight control. Physiological action of leptin in modulating the metabolic adaptation of different peripheral tissues supports the hypothesis that it could also exert a direct effect on cancer cells. In vitro, treatment with leptin up-regulated HIF-1α and stimulated adhesion and invasion of prostate cancer cells cultured in hypoxia. Leptin action was effective in both low and high glycolytic cancer cell lines, and determined the up-regulation of lactate exporter MCT4 and its associated protein CD147. HIF-1α stabilization was oligomycin-independent and was associated with an important modulation of mitochondrial homeostasis. In fact, leptin treatment produced mitochondrial biogenesis, stabilization of mitochondrial membrane potential and increased uncoupled respiration through the up-regulation of UCP2. Furthermore, leptin counteracted the downmodulation of SIRT1 induced by hypoxia, and persistent high levels of SIRT1 were directly involved in HIF-1α stabilization. Leptin can sustain cancer progression in hypoxic environment and when mitochondrial respiration is impaired. Leptin signaling axis, including the new proposed intermediate SIRT1, could represent a new diagnostic and therapeutic target in prostate cancer.

Transcriptional analysis of tyrosinase gene expression during Bufo bufo development

Tyrosinase (EC.1.14.18.1.) is a widespread enzyme, in the phylogenetic scale, that produces melanin, from bacteria to man, by using as substrates monophenols, o-diphenols and molecular oxygen. In this work we have confirmed and demonstrated that during Bufo bufo development tyrosinase activity and gene expression first occur at developmental stages 17–18 (tail bud-muscular response) as detected by a spectrophotometric assay and qRT-PCR. As expected, also during B. bufo development tyrosinase gene is expressed after the late gastrula (stage 12), differently from Rana pipiens development when tyrosinase mRNA appears at the neural plate stage and enzyme activity at stage 20 (gill circulation). We have cloned and sequenced the B. bufo tyrosinase cDNA in order to prepare B. bufo tyrosinase cDNA specific primers (forward and reverse). Tyrosinase mRNA cloning has been performed by using degenerate primers prepared according to the anuran tyrosinase gene sequence coding for the copper binding sites. The expressions of tyrosinase gene and enzymatic activity during B. bufo development support that until the developmental stage 17, embryo melanin is of maternal origin and at this stage can start embryo melanin synthesis. A correlation exists between tyrosinase expression and O2 consumption during B. bufo development.

The challenge for identifying the fungi living inside mushrooms: the case of truffle inhabiting mycelia

Abstract The Tuber ascomata (truffles) are a microhabitat for bacteria, viruses, and fungi (yeasts and filamentous fungi). In this survey, we tried to develop a method that would make it possible to define the mycobiome of the truffle-inhabiting filamentous fungi using culture independent molecular methods. The nested quantitative Real-Time PCR allowed us to demonstrate that each truffle is home to multiple species of filamentous fungi and that their DNA is present within the healthy ascoma at the ratio of 10−6 compared to that of the truffle. Probably due to their insignificant presence, Denaturing Gradient Gel Electrophoresis of the amplification of ITS amplicons showed only those of the host. Based on the results, the possibilities of being able to detect the fungicolous fungi present in very small amounts within a fungal host are discussed.