Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.

Background: Salmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested. Methods: Mutants were constructed in S. Gallinarum by P22 transduction from Salmonella Typhimurium (S. Typhimurium) with deletion of the crp gene. The effect was characterized by measuring biochemical properties and by testing of invasion in a chicken loop model and by challenge of six-day-old chickens. Further, birds were immunized with the deleted strain and challenged with the wild type isolate. Results: The crp deletions caused complete attenuation of S. Gallinarum. This was shown by ileal loop experiments not to be due to significantly reduced invasion. Strains with such deletions may have vaccine potential, since oral inoculatoin with S. Gallinarum Δcrp completely protected against challenge with the same dose of wild type S. Gallinarum ten days post immunization. Interestingly, the mutations did not cause the same biochemical and growth changes to the two biotypes of S. Gallinarum. All biochemical effects but not virulence could be complemented by providing an intact crp- gene from S. Typhimurium on the plasmid pSD110. Conclusion: Transduction of a Tn10 disrupted crp gene from S. Typhimurium caused attenuation in S. Gallinarum and mutated strains are possible candidates for live vaccines against fowl typhoid.

Mauro M. Colombo, Scilla Mastrandrea, Antonella Santona, Anamaria Pinto de Andrade, Sergio Uzzau, Salvatore Rubino, Piero Cappuccinelli; Distribution Of The Ac

As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae , which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.