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1.
Exp Cell Res ; 316(12): 1977-84, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20399774

ABSTRACT

Micro-vesicles can be released by different cell types and operate as 'safe containers' mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway. In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases.


Subject(s)
DNA, Mitochondrial/metabolism , Myoblasts, Skeletal/metabolism , Signal Transduction , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Insulin-Like Growth Factor Binding Protein 5/genetics , Insulin-Like Growth Factor Binding Protein 5/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Mice , Microscopy, Electron, Transmission , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Eur J Histochem ; 53(4): e31, 2009 Dec 29.
Article in English | MEDLINE | ID: mdl-22073363

ABSTRACT

Skeletal muscle cell differentiation is a multistage process extensively studied over the years. Even if great improvements have been achieved in defining biological process underlying myogenesis, many molecular mechanisms need still to be clarified.To further highlight this process, we studied cells at undifferentiated, intermediate and highly differentiated stages, and we analyzed, for each condition, morphological and proteomic changes. We also identified the proteins that showed statistical significant changes by a ESI-Q-TOF mass spectrometer. This work provides further evidence of the involvement of particular proteins in skeletal muscle development. Furthermore, the high level of expression of many heat shock proteins, suggests a relationship between differentiation and cellular stress. Intriguingly, the discovery of myogenesis-correlated proteins, known to play a role in apoptosis, suggests a link between differentiation and this type of cell death.


Subject(s)
Cell Differentiation/physiology , Heat-Shock Proteins/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Proteomics/methods , Animals , Cell Line , Electrophoresis, Gel, Two-Dimensional , Mass Spectrometry , Mice , Microscopy, Electron, Transmission , Muscle Development/physiology , Myoblasts/physiology
3.
Mol Genet Genomics ; 269(6): 807-16, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12898221

ABSTRACT

The nitrate assimilation pathway represents a useful model system in which to study the contribution of a mycorrhizal fungus to the nitrogen nutrition of its host plant. In the present work we cloned and characterized the nitrate reductase gene (tbnr1) from Tuber borchii. The coding region of tbnr1 is 2,787 nt in length, and it encodes a protein of 929 amino acids. Biochemical and Northern-blot analyses revealed that nitrate assimilation in T. borchii is an inducible system that responds mainly to nitrate. Furthermore, we cloned a nitrate reductase cDNA (tpnr1) from Tilia platyphyllos to set up a quantitative real-time PCR assay that would allow us to determine the fungal contribution to nitrate assimilation in ectomycorrhizal tissue. Using this approach we demonstrated that the level of tbnr1 expression in ectomycorhizae is eight times higher than in free-living mycelia, whereas tpnr1 transcription was found to be down-regulated after the establishment of the symbiosis. Enzymatic assays showed that NADPH-dependent nitrite formation markedly increases in ectomycorrhizae. These findings imply that the fungal partner plays a fundamental role in nitrate assimilation by ectomycorrhizae. Amino acid determination by HPLC revealed higher levels of glutamate, glutamine and asparagine in symbiotic tissues compared with mycelial controls, thus suggesting that these amino acids may represent the compounds that serve to transfer nitrogen to the host plant.


Subject(s)
Ascomycota/genetics , Mycorrhizae/metabolism , Nitrate Reductases/genetics , Plant Roots/microbiology , Symbiosis/genetics , Amino Acid Sequence , Ascomycota/growth & development , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , DNA, Fungal/genetics , DNA, Plant/genetics , Gene Expression Regulation, Fungal , Gene Expression Regulation, Plant , Gene Library , Genes, Fungal , Molecular Sequence Data , Mycorrhizae/genetics , NAD/metabolism , NADH, NADPH Oxidoreductases/metabolism , NADP/metabolism , Nitrate Reductase , Nitrate Reductases/metabolism , Nitrates/metabolism , Plant Roots/metabolism , Polymerase Chain Reaction , Sequence Homology, Amino Acid
4.
Mol Cell Biochem ; 209(1-2): 145-53, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10942212

ABSTRACT

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pKa and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues. Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.


Subject(s)
Dehydroascorbic Acid/pharmacology , Hexokinase/antagonists & inhibitors , Amino Acids/analysis , Anaerobiosis , Hexokinase/chemistry , Humans , Kinetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry
5.
FEMS Microbiol Lett ; 188(2): 119-24, 2000 Jul 15.
Article in English | MEDLINE | ID: mdl-10913693

ABSTRACT

Very little information is available to date about the complex truffle life cycle which involves the succession of three developmental phases. In order to gain more knowledge about ectomycorrhizal formation and fruit body development an ectomycorrhizal model system was used to study fungal biomass and plant and fungal transcript levels. They were evaluated in ectomycorrhizal development using the ergosterol assay and the internal transcribed spacer-5.8S ribosomal DNA from Tilia platyphyllos and Tuber borchii as molecular probes respectively. The results obtained from different approaches revealed a decrease in fungal biomass, transcript and protein levels during ectomycorrhizal development.


Subject(s)
Ascomycota/growth & development , Ascomycota/genetics , Plant Roots/genetics , Plant Roots/microbiology , Biomass , DNA Primers , DNA, Fungal/genetics , DNA, Plant/genetics , Electrophoresis, Gel, Two-Dimensional , Ergosterol/analysis , Fungal Proteins/analysis , Gene Expression Profiling , Molecular Sequence Data , Nucleic Acid Hybridization , Plant Proteins/analysis , RNA, Fungal/genetics , RNA, Ribosomal/genetics , RNA, Ribosomal, 5.8S/analysis , Symbiosis
6.
Electrophoresis ; 21(17): 3710-6, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11271490

ABSTRACT

This paper reports the first results in the proteome analysis of Tuber borchii Vittad. mycelium, an ectomycorrhizal fungus poorly defined genetically, but known for its generation of edible fruit bodies known as white truffles. Employing isoelectric focusing on immobilized pH gradients, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we obtained an electropherogram presenting over 800 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Different reducing agents were tested in the sample preparation buffers, and the standard lysis buffer plus 2% w/v polyvinylpolypyrrolidone allowed the best solubilization and resolution of the proteins. The T. borchii proteins separated in micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and visualized by Coomassie staining. Twenty-three proteins were excised and analyzed by the combination of amino acid and N-terminal analysis. One protein was identified by matching its amino acid composition, estimated isoelectric point and molecular mass against the SWISS-PROT and EMBL databases. Four spots were successfully tagged by Edman microsequencing but no homologous sequences were found in databases.


Subject(s)
Ascomycota/chemistry , Fungal Proteins/analysis , Amino Acid Sequence , Amino Acids/analysis , Ascomycota/growth & development , Dithioerythritol , Electrophoresis, Gel, Two-Dimensional/methods , Expressed Sequence Tags , Fungal Proteins/genetics , Molecular Sequence Data , Phosphines
8.
Fungal Genet Biol ; 25(2): 87-99, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9974220

ABSTRACT

This paper reports the purification and localization of a Tuber borchii Vittad, fruitbody protein (TBF-1) and the cloning of the encoding gene. TBF-1 is detectable by SDS-PAGE analyses only in this white truffle species and presents a molecular mass of 11,994 Da. TBF-1 was purified by one-step Reversed-Phase HPLC and its complete amino acid sequence was determined after digestion with trypsin and N-Asp endoproteinase. Polyclonal antibodies were produced and tested in immunofluorescence and immunogold experiments, providing information about the protein localization. It was detected mostly on the hyphal walls, where it was colocalized with beta-1,3-glucans and chitin. The sporal wall was not labeled. The encoding gene (tbf-1) was cloned using several techniques involving PCR. The coding region consists of a 360-bp open reading frame interrupted by an intron, with another intron following the stop codon. A putative signal peptide of 12 amino acids was found at the N-terminal. Northern blot analysis revealed that tbf-1 is highly expressed in unripe and ripe fruitbodies and was not detectable in culture mycelium or ectomycorrhizal roots.


Subject(s)
Ascomycota/genetics , Fungal Proteins/genetics , Amino Acid Sequence , Ascomycota/physiology , Base Sequence , Cell Wall/metabolism , Fungal Proteins/biosynthesis , Fungal Proteins/chemistry , Genes, Fungal , Introns , Molecular Sequence Data , Open Reading Frames
9.
Electrophoresis ; 16(4): 625-9, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7588535

ABSTRACT

High performance capillary electrophoresis (HPCE) was applied to the separation of protein and peptide mixtures with molecular masses ranging from 1300 to 96000 Da using a new bonded hydrophilic phase capillary, CElect-P150. This coated capillary reduces the interaction between proteins and silanol groups in capillary walls, allowing a complete recovery of the proteins and peptides of interest. HPCE was also used for the analysis of a complex mixture of tryptic fragments and to monitor the process of enzymatic digestion. Moreover, using a CElect-P150 capillary, highly reproducible analysis was possible without preconditioning the capillary with acid or basic solutions before each new analysis.


Subject(s)
Electrophoresis/methods , Proteins/analysis , Animals , Cattle , Cytochrome c Group/chemistry , Cytochrome c Group/metabolism , Electrophoresis/instrumentation , Molecular Weight , Peptides/analysis , Reproducibility of Results , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism , Trypsin
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