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Andrea Scaloni

Publications and source records attributed to Andrea Scaloni.

At least 55 records · Page 3Linked to original sources
PubMed2004

Proteomic evaluation of core biopsy specimens from breast lesions.

Analysis of tumour samples by a proteomic technology, which combines two-dimensional gel electrophoresis and mass spectrometry analysis, is a promising approach for molecular characterization of cancer. Proteomic analysis of neoplasms is usually performed on surgical material. The possibility to perform proteomic analysis on pre-operative samples might be useful for diagnostic purposes or for determination of tumour sensitivity to therapy. In this study, we report how tissues from core biopsy of breast lesions can be routinely used to obtain accurate protein expression profiles by proteomic analysis. Protein profiles from fibroadenomas were compared to those from ductal infiltrating carcinomas. By using mass spectrometry, identification of proteins overexpressed in carcinomas with respect to fibroadenomas was obtained. Thus, our study provides a methodology to perform proteomic analysis on pre-operative samples of breast lesions.

Biomarkers, Tumor↗
PubMed2004

Proteome analysis of Neisseria meningitidis serogroup A.

Neisseria meningitidis is an encapsulated Gram-negative bacterium responsible for significant morbidity and mortality worldwide. Meningococci are opportunistic pathogens, carried in the nasopharynx of approximately 10% of asymptomatic adults. Occasionally they enter the bloodstream to cause septicaemia and meningitis. Meningococci are classified into serogroups on the basis of polysaccharide capsule diversity, and serogroup A strains have caused major epidemics mainly in the developing world. Here we describe a two-dimensional gel electrophoresis protein map of the serogroup A strain Z4970, a clinical isolate classified as ancestral to several pandemic waves. To our knowledge this is the first systematically annotated proteomic map for N. meningitidis. Total protein samples from bacteria grown on GC-agar were electrophoretically separated and protein species were identified by matrix-assisted laser desorption/ionization time of flight spectrometry. We identified the products of 273 genes, covering several functional classes, including 94 proteins so far considered as hypothetical. We also describe several protein species encoded by genes reported by DNA microarray studies as being regulated in physiological conditions which are relevant to natural meningococcal pathogenicity. Since menA differs from other serogroups by having a fairly stable clonal population structure (i.e. with a low degree of variability), we envisaged comparative mapping as a useful tool for microevolution studies, in conjunction with established genotyping methods. As a proof of principle, we performed a comparative analysis on the B subunit of the meningococcal transferrin receptor, a vaccine candidate encoded by the tbpB gene, and a known marker of population diversity in meningococci. The results show that TbpB spot pattern variation observed in the maps of nine clinical isolates from diverse epidemic spreads, fits previous analyses based on allelic variations of the tbpB gene.

Agar↗
PubMed2004

Inactivation of Helicobacter pylori cagA gene affects motility.

BACKGROUND: The cytotoxin-associated protein CagA is a Helicobacter pylori immunodominant antigen whose gene resides in the cag pathogenicity island. Our purpose was to determine if the disruption or deletion of cagA gene could have an effect on the expression of other proteins at the proteome level. We analyzed two H. pylori strains, 328 and G27 wild-type, bearing the cag pathogenicity island, and their respective isogenic cagA(-) mutants. METHODS: The proteomes of two H. pylori strains (328 and its isogenic mutant SPM328_DeltacagA) were resolved by two-dimensional electrophoresis and the digitalized images obtained were analysed both quantitatively and qualitatively. Peculiar spots of each strain were identified by mass spectrometry or by Western blotting. RESULTS: The comparison between the proteome expression of an H. pylori cagA(+) strain and an isogenic mutant strain where the cagA gene was disrupted showed that, as well as the lack of expression of CagA, both flagellin A and flagellin B expressions were significantly decreased. The cagA(-) isogenic mutant was nonmotile. G27_DeltacagA, in which CagA was inactivated by gene deletion, was nonmotile as well respecting to motile G27 wild-type strain. Moreover, reintroduction of cagA in G27_DeltacagA restored motility. CONCLUSIONS: Our results suggest that CagA could quantitatively influence flaA and flaB transcription or their subsequent translation and/or correct folding.

Antigens, Bacterial↗
PubMed2004

Mechanistic studies on bovine cytosolic 5'-nucleotidase II, an enzyme belonging to the HAD superfamily.

Cytosolic 5'-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme-phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho-enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P-type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5'-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal region of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena.

5'-Nucleotidase↗
PubMed2003

Tetrapandins, a new class of scorpion toxins that specifically inhibit store-operated calcium entry in human embryonic kidney-293 cells.

Venoms from 14 snakes and four scorpions were screened for inhibitory activities toward store-operated Ca2+ entry (SOCE) in human embryonic kidney-293 cells. An inhibitory activity was found in venom from the African scorpion Pandinus imperator. The active agent of this venom was purified by gel filtration and reverse-phase high pressure liquid chromatography methods. Sequence information on the purified fraction, by automatic Edman degradation and mass spectrometry analysis, identified the activity as being contained in two tetrapeptides, which we have named tetrapandins. We demonstrate that synthesized tetrapandins have inhibitory activity for SOCE in human embryonic kidney-293 cells while having no effect on either thapsigargin- or carbachol-stimulated release of Ca2+ stores. These toxins should be extremely useful in future studies to determine downstream events regulated by SOCE as well as to determine whether multiple pathways exist for thapsigargin-stimulated Ca2+ entry.

Animals↗
PubMed2003

Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma.

Ecto-5'-nucleotidase (ecto-5'-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5'-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln(547) and/or Phe(548). Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn(403). Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5'-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

5'-Nucleotidase↗
PubMed2003

Probing the dimeric structure of porcine aminoacylase 1 by mass spectrometric and modeling procedures.

Aminoacylase 1 is a zinc-binding metalloprotease catalyzing the hydrolysis of N(alpha)-acylated l-amino acids; it presents altered expression levels in different renal and small cell lung carcinomas. A description of its redox and oligomerization state was achieved by combined biochemical and mass spectrometric procedures. A topological analysis of the enzyme structural architecture was derived from limited proteolysis and selective chemical modification experiments, using a broad range of proteases and chemical reagents. The analysis of the reaction products by different mass spectrometric techniques identified 26 amino acids as being accessible on the molecular surface, defining polypeptide regions exposed in the structure of the dimeric protein. The nature of the intermolecular contact zone between monomers was investigated by cross-linking reaction and mass mapping experiments. The cross-linked dimer was isolated, and the intermolecular cross-linked peptides were characterized, thus demonstrating the spatial proximity of Lys220 and Lys231 at the dimerization interface. Standard modeling procedures based on automatic alignment on the structure of members of the M20 peptidase family failed to produce a dimeric model consistent with experimental data. Discrepancies were observed mainly at the dimer interface and at loop regions. Therefore, a refined model for this dimeric protease was calculated by selecting the one able to generate a structure fully compatible with experimental findings, among all possible suboptimal sequence alignments. According to this model, each aminoacylase monomer consists of two domains: a globular catalytic subunit (residues 1-188 and 311-399) consisting of a beta-sheet sandwiched between alpha-helices and a second beta-sheet located on the surface, and the dimerization domain (residues 189-310) folding into a beta-sheet flanked on one side by two alpha-helices. These results indicate that reliable approaches such as limited proteolysis, selective chemical modification, and cross-linking coupled to mass spectrometry can be used to test and optimize molecular models of multimeric proteins and highlight problems in automatic model building.

Amidohydrolases↗
PubMed2003

Nuclear localization of Galectin-3 in transformed thyroid cells: a role in transcriptional regulation.

The differential proteomic approach (2D gel analysis coupled to MALDI-MS analysis) of nuclear proteins can provide an extremely useful tool to understand control of cell proliferation and differentiation. In order to identify possible markers of dedifferentiation between normal and cancerous thyroid cells, we used a differential proteomics approach by comparing nuclear extracts from the normal rat thyroid cell line FRTL-5 and the completely undifferentiated Ki-mol cell line, obtained by transformation with the Ki-ras oncogene. Galectin-3 (Gal-3) was identified as highly expressed, in the nuclear compartment, only in the transformed cell line. By using different human cancer cell lines, we showed that Gal-3 is maximally expressed in nuclei of papillary cancer cells. We focused on the functional relationship existing between Gal-3 and the thyroid-specific transcription factor TTF-1, whose expression is maintained in papillary cancer where it can contribute to the proliferating status. By using gel-retardation and transient tranfection assays, we demonstrate that Gal-3 upregulates the TTF-1 transcriptional activity. GST-pulldown experiments demonstrate the occurrence of interaction between Gal-3 and TTF-1 homeodomain. Since several lines of evidence suggest a role for Gal-3 in controlling proliferation and tumor progression in thyroid cancer, the stimulatory activity played by Gal-3 over TTF-1 would account for a possible molecular mechanism through which the galectin controls proliferation in thyroid cells.

Animals↗
PubMed2003

Platelet-derived growth factor induces the beta-gamma-secretase-mediated cleavage of Alzheimer's amyloid precursor protein through a Src-Rac-dependent pathway.

The beta-amyloid peptide (Abeta) present in the senile plaques of Alzheimer's disease derives from the cleavage of a membrane protein, named APP, driven by two enzymes, known as beta- and gamma-secretases. The mechanisms regulating this cleavage are not understood. We have developed an experimental system to identify possible extracellular signals able to trigger the cleavage of an APP-Gal4 fusion protein, which is detected by measuring the expression of the CAT gene transcribed under the control of the Gal4 transcription factor, which is released from the membrane upon the cleavage of APP-Gal4. By using this assay, we purified a protein contained in the C6 cell-conditioned medium, which activates the cleavage of APP-Gal4 and which we demonstrated to be PDGF-BB. The APP-Gal4 processing induced by PDGF is dependent on the gamma-secretase activity, being abolished by an inhibitor of this enzyme, and is the consequence of the activation of a pathway downstream of the PDGF-receptor, which includes the non-receptor tyrosine kinase Src and the small G-protein Rac1. These findings are confirmed by the observation that a constitutively active form of Src increases Abeta generation and that, in cells stably expressing APP, the generation of A is strongly decreased by the Src tyrosine kinase inhibitor PP2.

Alzheimer Disease↗
PubMed2003

Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae.

We report a study on the adaptive response of a wild-type wine Saccharomyces cerevisiae strain, isolated from natural spontaneous grape must, to mild and progressive physiological stresses due to fermentation. We observed by two-dimensional electrophoresis how the yeast proteome changes during glucose exhaustion, before the cell enters its complete stationary phase. On the basis of their identification, the proteins representing the S. cerevisiae proteomic response to fermentation stresses were divided into three classes: repressed proteins, induced proteins and autoproteolysed proteins. In an overall view, the proteome adaptation of S. cerevisiae at the time of glucose exhaustion seems to be directed mainly against the effects of ethanol, causing both hyperosmolarity and oxidative responses. Stress-induced autoproteolysis is directed mainly towards specific isoforms of glycolytic enzymes. Through the use of a wild-type S. cerevisiae strain and PMSF, a specific inhibitor of vacuolar proteinase B, we could also distinguish the specific contributions of the vacuole and the proteasome to the autoproteolytic process.

Cell Division↗
PubMed2003

Purification, cloning, and characterization of XendoU, a novel endoribonuclease involved in processing of intron-encoded small nucleolar RNAs in Xenopus laevis.

Here we report the purification, from Xenopus laevis oocyte nuclear extracts, of a new endoribonuclease, XendoU, that is involved in the processing of the intron-encoded box C/D U16 small nucleolar RNA (snoRNA) from its host pre-mRNA. Such an activity has never been reported before and has several uncommon features that make it quite a novel enzyme: it is poly(U)-specific, it requires Mn(2+) ions, and it produces molecules with 2'-3'-cyclic phosphate termini. Even if XendoU cleaves U-stretches, it displays some preferential cleavage on snoRNA precursor molecules. XendoU also participates in the biosynthesis of another intron-encoded snoRNA, U86, which is contained in the NOP56 gene of Xenopus laevis. A common feature of these snoRNAs is that their production is alternative to that of the mRNA, suggesting an important regulatory role for all the factors involved in the processing reaction.

Amino Acid Sequence↗
PubMed2003

Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines.

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.

Base Sequence↗
PubMed2003

Proteins from bovine tissues and biological fluids: defining a reference electrophoresis map for liver, kidney, muscle, plasma and red blood cells.

A number of high resolution two-dimensional electrophoresis (2-DE) reference maps for bovine tissues and biological fluids have been determined for animals in basal state. Among the 1863 distinct protein features detected in samples of liver, kidney, muscle, plasma and red blood cells, 509 species were identified and associated to 209 different genes. Difficulties in the identification were related to the poorly characterized Bos taurus genome and were solved by a combined matrix-assisted laser desorption/ionisation-mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry approach. The experimental output allowed us to establish a 2-DE database accessible through the World Wide Web network at the URL address (http://www.iabbam.na.cnr.it/Biochem). These reference maps may serve as a tool in future veterinary medical studies aimed at the evaluation of changes in protein repertoire for altered animal physiological conditions and infectious diseases, to the definition of molecular markers for novel diagnostic kits and vaccines, as well as the characterization of protein modifications in bovine materials following technological processes used in the food industry.

Animals↗
PubMed2003

Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.

GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain.

Binding Sites↗
PubMed2003

Proteomic characterization of a wild-type wine strain of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is the optimal eukaryotic model system to study mammalian biological responses. At the same time Saccharomyces cerevisiae is also widely utilized as a biotechnological tool in the food industry. Enological Saccharomyces cerevisiae strains have been so far routinely analyzed for their microbiological aspects. Nevertheless, wine yeasts are gaining an increasing interest in the last years since they strongly affect both the vinification process and the organoleptic properties of the final product wine. The protein repertoire is responsible of such features and, consequently, 2D-PAGE can be an useful tool to evaluate and select optimal wine yeast strains. We present here the first proteomic map of a wild-type wine Saccharomyces cerevisiae strain selected for the guided fermentation of very high quality wines.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2002

Oxidative modification of aldose reductase induced by copper ion. Definition of the metal-protein interaction mechanism.

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys(303) appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys(298)-Cys(303). However, a Cys(80)-Cys(303) disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys(80)-Cys(303) disulfide to the more stable product Cys(298)-Cys(303) is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys(80)-Cys(303) disulfide. However, no evidence of conditions favoring the formation of the Cys(298)-Cys(303) disulfide was observed. Our proposal is that the generation of the Cys(298)-Cys(303) disulfide, either directly or by rearrangement of the Cys(80)-Cys(303) disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.

Aldehyde Reductase↗
PubMed2002

Characterization of heat-induced lactosylation products in caseins by immunoenzymatic and mass spectrometric methodologies.

Nonenzymatic glycosylation of proteins occur during milk thermal treatment through the Maillard reaction. Immunoenzymatic and spectrometric methodologies were used to investigate caseins modification in samples submitted to different processing. As expected, protein-bound carbonyl content in raw and thermal-treated milks was positively correlated with the severity of the treatment. When immunoblotting and ELISA experiments were carried out either on isolated or whole milk proteins, carbonyl accumulation on caseins and macromolecular aggregates produced by thermal processing was evidenced. A comparative electrospray-mass spectrometry (ES-MS) analysis of different milks allowed verifying the extent of Amadori adducts on the more abundant protein components (alphas1- and beta-casein (alphas1- and beta-CN)). A combined matrix-assisted laser desorption ionization (MALDI)-MS/Edman degradation approach on enzymatic digests identified caseins modification sites. In moderately heat-treated milks, we observed that lactosylation occurred specifically at Lys-34 (alphas1-CN) and Lys-107 (beta-CN); whereas, samples subjected to more severe conditions presented modification at Lys-7, Lys-34, Lys-83, Lys-103, Lys-105, Lys-132 and Lys-193 (alphas1-CN) and at Lys-32, Lys-48, Lys-107, Lys-113 and Lys-176 (beta-CN). Differences in secondary structure of these components was assayed by circular dichroism (CD) spectroscopy.

Animals↗