The Tuber borchii fruiting body-specific protein TBF-1, a novel lectin which interacts with associated Rhizobium species.

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.

Novel and simple high-performance liquid chromatographic method for determination of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity.

We present here a high-performance liquid chromatographic method for the evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. The automated method was applied to fungal and mouse liver extracts and validated by the addition of mevastatin to the reaction mixture and by several intra- and inter-day assays. This method offers important advantages over those previously reported because no radiolabeled substrates or expensive techniques such as mass spectrometry are required, and the time of analysis is relatively short. Moreover, the method can be successfully applied to different biological samples; hence, it should be very useful in evaluating potential inhibitors of the HMG-CoA enzyme and investigating cholesterol metabolism, cell growth and differentiation processes.

Biochemical characterization and antioxidant and antiproliferative activities of different Ganoderma collections.

The aim of this study was to conduct a molecular and biochemical characterization and to compare the antioxidant and antiproliferative activities of four Ganoderma isolates belonging to Ganoderma lucidum (Gl-4, Gl-5) and Ganoderma resinaceum (F-1, F-2) species. The molecular identification was performed by ITS and IGS sequence analyses and the biochemical characterization by enzymatic and proteomic approaches. The antioxidant activity of the ethanolic extracts was compared by three different methods and their flavonoid contents were also analyzed by high-performance liquid chromatography. The antiproliferative effect on U937 cells was determined by MTT assay. The studied mycelia differ both in the enzymatic activities and protein content. The highest content in total phenol and the highest antioxidant activity for DPPH free radical scavenging and chelating activity on Fe(2+) were observed with the Gl-4 isolate of G. lucidum. The presence of quercetin, rutin, myricetin, and morin as major flavonoids with effective antioxidant activity was detected. The ethanolic extracts from mycelia of G. lucidum isolates possess a substantial antiproliferative activity against U937 cells in contrast to G. resinaceum in which the antiproliferative effects were insignificant. This study provides a comparison between G. lucidum and G. resinaceum mycelial strains, and shows that G. resinaceum could be utilized to obtain several bioactive compounds.

Tuber borchii fruit body: 2-dimensional profile and protein identification.

The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified.

Identification and characterization of the Tuber borchii D-mannitol dehydrogenase which defines a new subfamily within the polyol-specific medium chain dehydrogenases.

A novel NADP(+)-dependent D-mannitol dehydrogenase and the corresponding gene from the plant symbiotic ascomycete fungus Tuber borchii was identified and characterized. The enzyme, called TbMDH, is a homotetramer with two zinc atoms per subunit. It catalyzed both D-fructose reduction and D-mannitol oxidation, although it showed the highest substrate specificity and catalytic efficiency for D-fructose. Co-factor specificity was restricted to NADP(H) and the reaction proceeded via a sequential ordered Bi Bi mechanism. The carbon responsive transcriptional pattern showed that Tbmdh is up-regulated when mycelia are transferred to a culture medium containing D-mannitol or D-fructose. The phylogenetic analysis showed TbMDH to be the first example of a fungal D-mannitol-2-dehydrogenase belonging to the medium-chain dehydrogenase/reductases (MDRs). The enzyme identified a new group of proteins, most of them annotated in databases as hypothetical zinc-dependent dehydrogenases, forming a distinct subfamily among the polyol dehydrogenase family.

Headspace solid-phase microextraction with gas chromatography and mass spectrometry in the investigation of volatile organic compounds in an ectomycorrhizae synthesis system.

Ectomycorrhizae formation represents one of the most significant steps in the truffle life cycle and is determined by a complex molecular signaling between two symbionts. In order to understand the molecular pathway of ectomycorrhiza development, we focused on the signaling interaction between the ectomycorrhizal fungus Tuber borchii Vittad. and the Tilia americana L. plant roots. The medium of a pre-symbiotic (T. americana-T. borchii) in vitro system was analysed by headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry. In total, 73 volatile organic compounds (VOCs) were identified. Twenty-nine of these VOCs were produced only during the interaction phase between the two partners, leading to a hypothesis that these molecules might act as molecular messengers in order to pilot the ectomycorrhizae formation.

Enolase from the ectomycorrhizal fungus Tuber borchii Vittad.: biochemical characterization, molecular cloning, and localization.

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.