Beatrice Muscatello

Characterization of proteinaceous binders in wall painting samples by microwave-assisted acid hydrolysis and GC-MS determination of amino acids

AbstractAn analytical procedure for the characterization of proteinaceous binders (milk or casein, egg and animal glue) in artistic wall painting samples is described. The method is based on microwave-assisted acid hydrolysis of the sample, and on derivatization of the amino acids freed with a silylating agent and their quantitative determination by gas chromatography/mass spectrometry. The procedure was tested on wall painting reference samples, and showed a typical protein recovery of about 70% at 0·1mg sample size. Fourteen amino acids were determined and some ratios between their concentrations were selected, so that the proteinaceous binders in the sample could be identified. Results on some samples from frescoes from the Monumental Celnetery in Pisa (Italy) showed that the amino acid composition of casein differed significantly from that expected, as a result of degradation processes due to aging and moisture.

Comparative determination of some phytohormones in wild-type and genetically modified plants by gas chromatography–mass spectrometry and high-performance liquid chromatography–tandem mass spectrometry

The analytical performances of two optimized analytical methodologies used for the determination of auxins, cytokinins, and abscisic acid in plant samples were critically compared. Phytohormones were extracted from Nicotiana glauca samples using a modified Bieleski solvent and determined both by gas chromatography-mass spectrometry (GC-MS), after derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Bieleski extract without any further treatment. HPLC-MS/MS gave better results in terms of higher coefficients of determination of the calibration curves, higher and more reproducible recoveries, lower limits of detection, faster sample preparation, and higher sample throughput. Thus, two sets of N. glauca and N. langsdorffii samples, both wild-type and genetically modified by inserting the glucocorticoid receptor (GR) gene encoding for the rat glucocorticoid receptor, were first characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and then analyzed by HPLC-MS/MS. Significant differences in the phytohormone content between the two sample sets were found and are very important in terms of understanding the mechanisms and effects on growth processes and the development of transgenic plants.

Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene

Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plants hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

Metronomic vinorelbine is directly active on Non Small Cell Lung Cancer cells and sensitizes the EGFRL858R/T790M cells to reversible EGFR tyrosine kinase inhibitors

Graphical abstract Figure. No Caption available. ABSTRACT Metronomic vinorelbine (mVNR) has been described primarily as an antiangiogenic therapy, and no direct effects of mVNR on Non Small Cell Lung Cancer (NSCLC) cells has yet been demonstrated. The aims of this study were i) to establish the direct activity of mVNR on NSCLC cells either EGFR wt or EGFRL858R/T790M, and ii) to quantify the synergism of the combination with reversible EGFR tyrosine kinase inhibitors (TKIs), investigating the underlying mechanism of action. Proliferation assays were performed on A‐549 (wt EGFRhigh), H‐292 (EGFR‐wt), H‐358 (EGFR‐wt), H‐1975 (EGFRL858R/T790M) NSCLC cell lines exposed to mVNR, its active metabolite deacetyl‐VNR (D‐VNR), gefitinib and erlotinib for 144 h treatments. The synergism between mVNR and EGFR TKIs was determined by the combination index (CI) in EGFR‐wt and H‐1975 NSCLC cells. Cyclin‐D1 and ABCG2 genes expression and protein levels were measured by RT‐PCR and ELISA assays, as well as the phosphorylation of ERK1/2 and Akt. Intracellular concentrations of EGFR TKIs and VNR were investigated with a mass spectrometry system. mVNR, and its active metabolite D‐VNR, were extremely active on NSCLC cells, in particular on H‐1975 (IC50 = 13.56 ± 2.77 pM), resistant to TKIs. mVNR inhibited the phosphorylation of ERK1/2 and Akt and significantly decreased the expression of both cyclin‐D1 and ABCG2 m‐RNA and protein. The simultaneous combination of VNR and reversible EGFR TKIs showed a strong synergism on EGFR‐wt NSCLC cells and on H‐1975 cells (e.g. CI = 0.501 for 50% of affected cells), increasing the intracellular concentrations of EGFR TKIs (e.g. +50.5% vs. gefitinib alone). In conclusions, mVNR has direct effects on NSCLC cells and sensitizes resistant cells to EGFR TKIs, increasing their intracellular concentrations.