Maria Antonietta Castiglione Morelli

Characterization of Wines by Nuclear Magnetic Resonance: A Work Study on Wines from the Basilicata Region in Italy

We explored the possibility of differentiating Italian wines produced in different regions by means of nuclear magnetic resonance (NMR) techniques. Ten commercial red Aglianico wines were selected from different areas of the Basilicata region in the south of Italy. Some important components of these wines were identified by the assignments of their (1)H and (13)C resonances using one- and two-dimensional homonuclear and heteronuclear NMR experiments. These data were compared with those obtained from 10 Aglianico wines produced in Campania, another southern Italian region. Differences were found among the wines according to their geographical origin and vintage. A fine discrimination of Aglianico wines from Basilicata and Campania was obtained, suggesting that the selected NMR parameters may be a valuable tool for wine authenticity control.

Chemical Profile of White Wines Produced from ‘Greco bianco’ Grape Variety in Different Italian Areas by Nuclear Magnetic Resonance (NMR) and Conventional Physicochemical Analyses

In this study the characterization of white wines produced from the monovarietal Greco bianco grape variety is presented for the first time. A total of 40 commercial wines, from two different southern Italian regions, Calabria and Campania, from the same grape variety and two different vintages, were investigated. The analyses were performed by means of chromatographic methods, conventional analyses, and nuclear magnetic resonance (NMR) spectroscopy. No differentiation was observed according to the year of production but a significant discrimination was achieved using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). In particular, PLS-DA allowed the selection of compounds (total acidity; citric, malic, succinic, and lactic acids; total polyphenol index; glucose and proline/arginine ratio) useful for differentiating the studied wines on the basis of geographical origin.

The nucleotide-binding domain 2 of the human transporter protein MRP6

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) belongs to the ABC transporter family, whose members share many characteristic features including membrane domains and two nucleotide-binding domains (NBD1 and NBD2). These function cooperatively to bind and hydrolyze ATP for the transport of substrates across biological membranes. In this study, MRP6-NBD2 (residues 1252–1503) was expressed in Escherichia coli, purified and structurally and functionally characterized. CD spectra suggested that the protein is folded. Furthermore, NBD2 is shown to be biologically active as it binds ATP and presents ATPase activity although significantly lower compared with isolated NBD1. The mixture of NBD2 and NBD1 exhibited an activity similar to the NBD2 alone, indicating that NBD1 and NBD2 form a heterodimer with the latter limiting ATP hydrolysis. These findings suggest that NBD1 has a higher tendency to form an active homodimer, which is also supported by in silico analysis of energy-minimized dimers of the homology models of the two domains.

The contribution of the citrate pathway to oxidative stress in Down syndrome

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up‐regulation of the citrate pathway and down‐regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl‐transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.

Study of the nucleotide-binding domain 1 of the human transporter protein MRP6.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family. Proteins in this family share many characteristic structural features, including two membrane-spanning domains and two nucleotide-binding domains (NBD1 and NBD2), that function cooperatively but not equally bind and hydrolyze ATP. The MRP6 protein is structurally and functionally poorly characterized. Previously, we showed, by NMR spectroscopy, that a fragment of MRP6-NBD1 presents helical structure and fluorescence experiments demonstrated that peptide binds ATP. These data suggested that the study on selected regions could be a valid approach to define the structure of MRP6 . In the present study, to better characterize MRP6-NBD1, we report data of CD spectroscopy, nucleotide binding and ATP hydrolysis on two different polypeptides, one corresponding to the full-length NBD1 domain (residues from Asp-627 to Leu-851) and the other is a shorter polypeptide (residues from Arg-648 to Thr-805) without some key residues. We report that both polypeptides are highly structured in aqueous buffer and in 20% trifluoroethanol showing considerable helical and β-structure content. The ATP hydrolysis activity is exhibited only by the full-length NBD1 domain. Comparison between our findings and the structurally well characterized MRP1-NBD1 supports the role of H-loop for the ATP hydrolysis and of A-loop in stabilizing the ATP binding.

Identification of a New Splice Variant of the Human ABCC6 Transporter.

ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that encodes a protein (MRP6) involved in active transport of intracellular compounds to the extracellular environment. Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), an autosomal recessive disorder of the connective tissue characterized by progressive calcification of elastic structures in the skin, the eyes, and the cardiovascular system. MRP6 is codified by 31 exons and contains 1503 amino acids. In addition to a full-length transcript of ABCC6, we have identified an alternatively spliced variant of ABCC6 from a cDNA of human liver that lacks exons 19 and 24. The novel isoform was named ABCC6 Δ19Δ24. PCR analysis from cDNA of cell cultures of primary human hepatocites and embryonic kidney confirms the presence of the ABCC6Δ19Δ24 isoform. Western blot analysis of the embryonic kidney cells shows a band corresponding to the molecular weight of the truncated protein.

Interaction of cisplatin with a CCHC zinc finger motif

The interaction between cisplatin and an 18‐residue CCHC zinc finger motif derived from a retroviral nucleocapsid protein (PyrZf18) has been studied using UV–visible, CD and 1H NMR spectroscopies and ESI‐MS spectrometry.

Expression, Purification and Structural Characterization of Up-Regulated Gene 7 Encoded Protein

Up-Regulated Gene 7 (URG7) is a host gene up-regulated in HBV infected hepatocytes that has been suggested to have an anti-apoptotic activity mediated by caspases 3 and 8 and an endoplasmic reticulum localization. Here we report the structural characterization of the encoded protein URG7 by circular dichroism and fluorescence spectroscopy in different solvent media: phosphate buffer and two membrane-mimetic solvents, i.e. 2,2,2-trifluoroethanol (TFE) and SDS micelles. In all solvents URG7 contains substantial amounts of secondary structures. To obtain information about the structural organization and stability of URG7, its thermal denaturation in a membrane environment was studied and intermediate states of thermal unfolding were observed. Furthermore, fluorescence results in SDS micelles could be compatible with different environments for the four tryptophan residues in URG7. Preliminary NMR data indicate that URG7 in TFE solution is quite flexible and not well folded. These data are the first structural information on URG7 and might provide an insight into its structure-function relationships.

Biochemical Characterization and NMR Study of the Region E748-A785 of the Human Protein MRP6/ABCC6

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family which couple ATP hydrolysis with the transport of molecules across biological membranes. MRP6 topology presents three transmembrane domains and two nucleotide-binding domains (NBDs). The protein is structurally and functionally poorly characterized. Mutations in ABCC6 gene cause Pseudoxanthoma elasticum, a recessive genetic disorder affecting the elastic tissues. Most mutations have been found in NBDs that are critical for ATP binding and hydrolysis. With the aim to better characterize MRP6, we have performed a preliminary study on the fragment E748-A785 of MRP6-NBD1, with the wild type sequence and the R765Q mutation found in PXE affected patients. CD and NMR spectroscopy show the presence of helical structures in both peptides. Fluorescence experiments demonstrate that peptides bind ATP. The NMR structure of the mutated peptide is compared with the corresponding region of the MRP6-NBD1 modeled structure using as a template the X-ray structure of MRP1-NBD1. The finding that both wild type and mutated peptide present the same structure and similar affinity for ATP suggests that the onset of PXE symptoms is a consequence of the different type of interactions involving residue 765 R/Q inside the protein.

NMR metabolomics study of follicular fluid in women with cancer resorting to fertility preservation

PurposeThe purpose of this study was to evaluate the possible application of metabolomics to identify follicular fluid changes in cancer patients undergoing fertility preservation. Although metabolomics have been applied already in cancer studies, this is the first application on follicular fluid of cancer patients.MethodsWe selected for the study ten patients with breast cancer and lymphoma who resorted to oocyte cryopreservation to preserve fertility and ten healthy women undergoing in vitro fertilization treatments. Follicular fluid was collected at the time of oocytes retrieval. Metabolomic analysis of follicular fluids was performed by 1H-nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate analysis to interpret the spectral data. Univariate statistical analysis was applied to find correlations between patients’ features and metabolites identified by NMR.ResultsPartial least squares discriminant analysis allowed to discriminate samples from cancer patients and healthy controls. Univariate statistical analysis found significant correlations between patients’ features and metabolites identified by NMR. This finding allowed to identify biomarkers to differentiate both healthy controls from cancer patients and the two different classes of oncological patients.ConclusionThe follicular fluids of cancer patients display significant metabolic alterations in comparison to healthy subjects. NMR-based metabolomics could be a valid prognostic tool for identifying and selecting the best cryopreserved oocytes and improving the outcome prediction in cancer women undergoing in vitro fertilization.