Elena Barbieri

Creatine as an antioxidant

Creatine monohydrate (Cr), the most diffuse supplement in the sports industry, is receiving greater attention because of its beneficial effects in a wide number of human degenerative diseases and conditions. These effects can be barely explained on the basis of the sole ergogenic role of the Cr/CrP system. Indeed, a wide number of research articles indicate that Cr is capable of exerting multiple, non-energy related, effects on diverse and relevant cellular targets. Among these effects, the antioxidant activity of Cr emerges as an additional mechanism which is likely to play a supportive role in the Cr-cytoprotection paradigm.

Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

ABSTRACT Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to theCytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genusTuber.

Occurrence and expression of virulence-related properties of Vibrio species isolated from widely consumed seafood products

In this study, widely consumed fresh seafood products were examined for the presence of Vibrio spp. Thirteen percent of the samples examined were found to be contaminated with halophilic vibrios belonging to the species V. alginolyticus (81.48%), V. parahaemolyticus (14.8%) and V. cholerae non 0:1 (3.7%). A greater isolation frequency (18.9%) was found for mussels. Significant adhesiveness and strong cytotoxicity factors were revealed in a significant number of the Vibrio spp. isolated. These results confirm that the presence of Vibrio spp. in seafood products is common, and suggest that routine examination of such products for these pathogenic agents would be advisable.

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste‐water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae, 46 (22%) as Aer. sobria, 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii. Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP‐types) based on their biochemical phenotypes. Several different PhP‐types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP‐types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico‐chemical conditions. The production of toxins capable of causing cytoskeletal‐dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.

Shewanella pealeana sp. nov., a member of the microbial community associated with the accessory nidamental gland of the squid Loligo pealei.

A new, mesophillic, facultatively anaerobic, psychrotolerant bacterium, strain ANG-SQ1T (T = type strain), was isolated from a microbial community colonizing the accessory nidamental gland of the squid Loligo pealei. It was selected from the community on the basis of its ability to reduce elemental sulfur. The cells are motile, Gram-negative rods (2.0-3.0 microns long, 0.4-0.6 micron wide). ANG-SQ1T grows optimally over the temperature range of 25-30 degrees C and a pH range of 6.5-7.5 degrees C in media containing 0.5 M NaCl. 16S rRNA sequence analysis revealed that this organism belongs to the gamma-3 subclass of the Proteobacteria. The closest relative of ANG-SQ1T is Shewanella gelidimarina, with a 16S rRNA sequence similarity of 97.0%. Growth occurs with glucose, lactate, acetate, pyruvate, glutamate, citrate, succinate, Casamino acids, yeast extract or peptone as sole energy source under aerobic conditions. The isolate grows anaerobically by the reduction of iron, manganese, nitrate, fumarate, trimethylamine-N-oxide, thiosulfate or elemental sulfur as terminal electron acceptor with lactate. Growth of ANG-SQ1T was enhanced by the addition of choline chloride to growth media lacking Casamino acids. The addition of leucine or valine also enhanced growth in minimal growth media supplemented with choline. The results of both phenotypic and genetic characterization indicate that ANG-SQ1T is a Shewanella species. Thus it is proposed that this new isolate be assigned to the genus Shewanella and that it should be named Shewanella pealeana sp. nov., in recognition of its association with L. pealei.

Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts

It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor‐specific regulation is available and data on their post‐transcriptional and post‐translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post‐transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage‐dependent localization suggestive of a post‐translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half‐life regulation to modulate the differentiation stage‐dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo‐cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms. J. Cell. Biochem. 108: 1302–1317, 2009.

Creatine supplementation prevents the inhibition of myogenic differentiation in oxidatively injured C2C12 murine myoblasts.

Creatine (Cr), one of the most popular nutritional supplements among athletes, has been recently shown to prevent the cytotoxicity caused by different oxidative stressors in various mammalian cell lines, including C2C12 myoblasts, via a direct antioxidant activity. Here, the effect of Cr on the differentiating capacity of C2C12 cells exposed to H(2)O(2) has been investigated. Differentiation into myotubes was monitored using morphological, ultrastructural, and molecular techniques. Treatment with H(2)O(2) (1 h) not only caused a significant (30%) loss of cell viability, but also abrogated the myogenic ability of surviving C2C12. Cr-supplementation (24 h prior to H(2)O(2) treatment) was found to prevent these effects. Interestingly, H(2)O(2)-challenged cells preconditioned with the established antioxidants trolox or N-acetyl-cysteine, although cytoprotected, did not display the same differentiating ability characterizing oxidatively-injured, Cr-supplemented cells. Besides acting as an antioxidant, Cr increased the level of muscle regulatory factors and IGF1 (an effect partly refractory to oxidative stress), the cellular availability of phosphocreatine and seemed to exert some mitochondrially-targeted protective activity. It is concluded that Cr preserves the myogenic ability of oxidatively injured C2C12 via a pleiotropic mechanism involving not only its antioxidant capacity, but also the contribution to cell energy charge and effects at the transcriptional level which common bona fide antioxidants lack.

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum.

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.

Geographical traceability of Italian white truffle (Tuber magnatum Pico) by the analysis of volatile organic compounds

Results are presented that were obtained on the geographic traceability of the white truffle Tuber magnatum Pico. Solid-phase microextraction coupled to gas chromatography/mass spectrometry (SPME-GC/MS) was employed to characterize the volatile profile of T. magnatum white truffle produced in seven geographical areas of Italy. The main components of the volatile fraction were identified using SPME-GC/MS. Significant differences in the proportion of volatile constituents from truffles of different geographical areas were detected. The results suggest that, besides genetic factors, environmental conditions influence the formation of volatile organic compounds. The mass spectra of the volatile fraction of the samples were used as fingerprints to characterize the geographical origin. Next, stepwise factorial discriminant analysis afforded a limited number of characteristic fragment ions that allowed a geographical classification of the truffles studied.

Effect of storage on biochemical and microbiological parameters of edible truffle species

The effects of different storage treatments on the most common edible truffle species, such as Tuber magnatum and Tuber borchii (white truffles), Tuber melanosporum and Tuber aestivum (black truffles), were analysed. Biochemical and microbiological profiles were monitored, in order to evaluate possible alterations during truffle preservation. After harvesting, some fresh samples were kept at 4°C for 30days, other samples were frozen at -20°C for one month, thawed and preserved at 4°C; the remainder were autoclaved. The biochemical parameters studied were sugar and protein content, the activity of some enzymes involved in the central metabolism of the fungi and the electrophoretic pattern of soluble proteins. Total mesophilic bacteria were also counted. The results obtained showed that the storage at 4°C is the treatment that best preserves the biochemical and microbiological characteristics of fresh truffles. Black truffles were more resistant to biochemical spoilage than the white ones, while T. magnatum was the most resistant to microbial spoilage.