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ABSTRACT: CELLFOOD (CF) is an innovative nutritional supplement containing 78 ionic/colloidal trace elements and minerals combined with 34 enzymes and 17 amino acids, all suspended in a solution of deuterium sulfate. The aim of this study was to investigate, for the first time, the antioxidant properties of CF in vitro in different model systems. Three pathophysiologically relevant oxidants were chosen to evaluate CF protection against oxidative stress: hydrogen peroxide, peroxyl radicals, and hypochlorous acid. Both biomolecules (GSH and plasmid DNA) and circulating cells (erythrocytes and lymphocytes) were used as targets of oxidation. CF protected, in a dose-dependent manner, both GSH and DNA from oxidation by preserving reduced GSH thiol groups and supercoiled DNA integrity, respectively. At the same time, CF protected erythrocytes from oxidative damage by reducing cell lysis and GSH intracellular depletion after exposure to the oxidant agents. In lymphocytes, CF reduced the intracellular oxidative stress induced by the three oxidants in a dose-dependent manner. The overall in vitro protection of biomolecules and cells against free radical attacks suggests that CF might be a valuable coadjuvant in the prevention and treatment of various physiological and pathological conditions related to oxidative stress, from aging to atherosclerosis, from neurodegeneration to cancer.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 06/2011; 49(9):2292-8. · 2.99 Impact Factor
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ABSTRACT: Lectins, proteins that are able to bind carbohydrate structures, are typically involved in cell recognition mechanisms. We demonstrate here that TBF-1, the main soluble protein in the Tuber borchii Vittad. fruiting body, is a phase-specific lectin that is able selectively to bind the exopolysaccharides produced by ascoma-associated Rhizobium spp. Characterization of TBF-1 was performed using both the protein purified from the truffles and the recombinant protein overexpressed in Escherichia coli. The two proteins exhibit the same hemagglutination activity toward rabbit red blood cells and the same sugar binding specificity. The discovery of lectin activity for TBF-1 led us to propose revising the protein name to 'T. borchii fruiting body lectin 1' with the acronym TBFL-1.
FEMS Microbiology Letters 07/2008; 284(2):197-203. · 2.04 Impact Factor
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ABSTRACT: The TBF-1 is an 11.9-kDa fruiting body specific protein of the Ascomycetes hypogeous fungus Tuber borchii Vittad. found in aqueous extract and the hyphal cell wall. The tbf-1 gene codes a 12-amino acid N-terminal stretch not present in mature protein. This sequence does not match with any homologous signal sequences stored in data banks. To investigate the role of the N-terminus in TBF-1 localization, cDNA was expressed in Saccharomyces cerevisiae under the control of the 3-phosphoglycerate kinase promoter. Like Tuber, yeast also produces and secretes TBF-1 and the foreign protein binds with the cell wall. A signalless mutant protein was constructed; this DeltaTBF-1 was expressed but not exported by yeast. The secretion of TBF-1 was also suppressed using the sec18(ts) yeast mutant strain grown at nonpermissive temperature as host. Thus we demonstrated that the N-terminal 12-amino acid stretch is a noncanonical signal peptide that leads the TBF-1 toward the classical secretory pathway in yeast.
FEMS Microbiology Letters 08/2007; 272(1):114-9. · 2.04 Impact Factor
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ABSTRACT: Here, we report the first evidence of a hexose transporter gene, Tbhxt1, in the ectomycorrhizal ascomycete Tuber borchii Vittadini. The protein encoded by Tbhxt1 functionally complements the hxt-null mutant Saccharomyces cerevisiae EBYVW.4000. TBHXT1 has a strong preference for d-glucose (K(m)=38+/-10 microM) over d-fructose (K(m)=16+/-5mM) and uncoupling experiments indicate that TBHXT1 catalyzes the transport via a proton-symport mechanism. The investigations on the substrate specificity reveal that TBHXT1 also imports d-mannose, and the use of deoxyglucose analogues shows that the hydroxyl groups at C1, C3 and C4 are important for substrate recognition. Tbhxt1 is not regulated by fructose, but it reaches its highest level of expression at 3mM glucose and is repressed by very high glucose concentration. Prolonged carbon starvation condition upregulates Tbhxt1, while its expression remains at basal level in the ectomycorrhizal tissue. The mode of regulation of Tbhxt1 is consistent with its role as a high-affinity d-glucose transporter.
Fungal Genetics and Biology 04/2007; 44(3):187-98. · 3.74 Impact Factor
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ABSTRACT: TBF-1 is a fruitbody-specific protein present in the white truffle species Tuber borchii Vittad. A similar protein has been found only in the closely related species Tuber dryophilum (TDF-1), but not in other truffles. The protein from T. borchii was overexpressed as fusion protein in E. coli and was purified to homogeneity by affinity chromatography. Recombinant protein was used for generating polyclonal antibodies. The antiserum strongly reacted with TBF-1, weakly recognized TDF-1, and did not detect correlate band in the other white truffle species. The high level of expression of this protein in the fruitbody and the specificity of the antibody anti-TBF-1 make it possible to set up a diagnostic tool for detecting these species in natural samples and foodstuffs.
Preparative Biochemistry & Biotechnology 02/2005; 35(2):145-53. · 0.47 Impact Factor
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ABSTRACT: Enolase from Tuber borchii mycelium was purified to electrophoretical homogeneity using an anion-exchange and a gel permeation chromatography. Furthermore, the corresponding gene (eno-1) was cloned and characterized. The purified enzyme showed a higher affinity for 2-PGA (0.26 mM) with respect to PEP; the stability and activity of enolase were dependent of the divalent cation Mg2+. T. borchii eno-1 has an ORF of 1323 bp coding for a putative protein of 440 amino acids and Southern blotting analysis revealed that the gene is present as a single copy in T. borchii. The enzymatic activity and the mRNA expression level evaluated in mycelia grown either in different carbon sources, in pyruvate or during starvation were the same in all the conditions tested, while biochemical and Northern blotting analyses performed with mycelia at different days of growth showed T. borchii eno-1 regulation in response to the growth phase. Finally, Western blotting analysis demonstrated that enolase is localized only in the cytosolic fraction confirming its important role in glycolysis.
Fungal Genetics and Biology 03/2004; 41(2):157-67. · 3.74 Impact Factor
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ABSTRACT: Inhibition by its product, glucose, is a kinetic property of hexokinase type III. In this paper, we report the overexpression in Escherichia coli of human hexokinase type III. The recombinant enzyme was genetically fused with a hexahistidine peptide at the C-terminal end. This modification confers to the product the ability to bind the Ni2+ ion immobilised into agarose by nitrilotriacetic acid (NTA) groups. The purification was performed by one-step column chromatography using ammonium sulphate as stabilising agent. Recombinant hexokinase type III appears as a single band of approximately 100 kDa on a SDS-PAGE gel and shows specific activity of 16 U/mg. Its kinetic parameters are comparable to those of the native enzyme, including the fact that it can be inhibited by glucose. The comparison of these results with the properties of the overexpressed carboxyl-domain led us to suppose that the inhibition site for glucose required the presence of the N-terminal domain.
Preparative Biochemistry & Biotechnology 12/2002; 32(4):393-403. · 0.47 Impact Factor
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ABSTRACT: Polymorphisms of a ribosomal DNA region (ITS) have been analysed using a specific pair of primers, in order to type fruitbodies and ectomycorrhizae of different truffle species. The identification of ectomycorrhizae was obtained by digestion of the PCR products using restriction enzymes. The results show that the strategy used is both suitable and sensitive to characterize the symbiotic fungi from few mycorrhized root tips.
Biotechnology Letters 06/1996; 18(7):821-826. · 1.68 Impact Factor
