MALDI-ToF Ms Analysis Of Plants Treated with Acybenzolar-S Methyl.

Acybenzolar-S-methyl (ASM) is a synthetic inducer and a functional analogue of salicylic acid, involved in systemic acquired resistance (SAR). In our study, we assessed the expression of proteins, in Solanum lycopersicum. leaves treated with ASM. Tomato leaves were gound into a fine powder with liquid nitrogen and stored at -70°C. For protein extraction, the powder was washed twice with ethanol and deionized water, after centrifugation the final pellets were dried in speed-vac and dissolved in 1% (w/v) SDS, 20% (v/v) glycerol, 40 mM DTT, 50 mM Tris-HCl, pH 7.5. Protein content was determined according to the method of Bradford. Aliquots of extracts from untreated and ASM-treated samples were precipitated with chloroform/methanol and the protein pellets were dried and analyzed by 2-D electrophoresis. IEF (Isoelectrophocusing) steps were performed on IPG dry-strips of 13 cm in non-linear pH gradient of 3-11 (GE-Heathcare), proteins were separated in the 2nd dimension (SDS-PAGE) in a 13% (w/v) polyacrylamide gel. Gels were coomassie stained and analyzed by the ImageMaster 2D Elite software (Amersham Biosciences). Three gels were produced for untreated and ASM-treated samples, respectively. Protein spots present on at least two gels were used for comparison of the obtained spots. Image analysis of 2-DE gels revealed that 116±8 and 124±11 spots were extracted from control and ASM-treated samples, respectively. Proteins were considered differentially expressed when spot intensities (normalized volume) varied of at least a two-fold differences (ANOVA; P < 0.05) in up or down regulation respect to the untreated sample (control). Protein spots that significantly changed in response to the ASM-treatment with respect to the control, were successfully identified by MALDI-ToF analysis. Twentyeight spots identified as corresponding to 17 different proteins were classified in three groups. on the basis of their function, Of these, the majority (18 spots) were involved in defense mechanisms/ stress responses, seven spots in photosynthetic metabolism and three spots as proteins associated to energy metabolism

The adaptor protein Rai/ShcC promotes astrocyte-dependent inflammation during experimental autoimmune encephalomyelitis

Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model.We found that, unexpectedly, EAE was less severe in Rai2/2 mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai+/+ mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis

Development of novel cyclic peptides as pro-apoptotic agents

Our recent finding that paclitaxel behaves as a peptidomimetic of the endogenous protein Nur77 inspired the design of two peptides (PEP1 and PEP2) reproducing the effects of paclitaxel on Bcl-2 and tubulin, proving the peptidomimetic nature of paclitaxel. Starting from these peptide-hits, we herein describe the synthesis and the biological investigation of linear and cyclic peptides structurally related to PEP2. While linear peptides (2a,b, 3a,b, 4, 6a-f) were found inactive in cell-based assays, biological analysis revealed a pro-apoptotic effect for most of the cyclic peptides (5a-g). Cellular permeability of 5a (and also of 2a,b) on HL60 cells was assessed through confocal microscopy analysis. Further cellular studies on a panel of leukemic cell lines (HL60, Jurkat, MEC, EBVB) and solid tumor cell lines (breast cancer MCF-7 cells, human melanoma A375 and 501Mel cells, and murine melanoma B16F1 cells) confirmed the pro-apoptotic effect of the cyclic peptides. Cell cycle analysis revealed that treatment with 5a, 5c, 5d or 5f resulted in an increase in the number of cells in the sub-G0/G1 peak. Direct interaction with tubulin (turbidimetric assay) and with microtubules (immunostaining experiments) was assessed in vitro for the most promising compounds