Biomedical subjects

Natascia Campostrini

Publications and source records attributed to Natascia Campostrini.

13 recordsLinked to original sources

PubMed2006

Proteomic analysis of anti-angiogenic effects by a combined treatment with vinblastine and rapamycin in an endothelial cell line.

Angiogenesis controls the new blood supply routes into the tumor mass via the host endothelial cells (ECs). In this study, the EA.hy926 endothelial cell line has been treated with vinblastine (VBL) and rapamycin (RAP), both separately and in combination at low doses. Recently, we demonstrated the synergistic antiangiogenic effects of a combination of VBL and RAP at very low doses in vitro and in vivo. Herein, we confirm the ability of this combined treatment to statistically inhibit the proliferation of ECs, in a synergistic manner, by inducing apoptosis. The aim of this study was to substantiate these findings at the protein level. Differential proteomic analysis was performed on untreated control cells, treated with VBL, incubated with RAP, or subjected to a drug combination. Differentially expressed 113 polypeptide chains were visualized and 65 were identified via MALDI-TOF analysis. Some of the regulated proteins are involved in the processes of angiogenesis, proliferation, migration, and apoptosis. The down-modulation of ATP synthase, annexin A2, heat shock p70, glucose-6-phosphate dehydrogenase, vasodilator-stimulated phosphoprotein, proteasome 26S, tryptophanyl-tRNA synthetase, and stathmin/OP18, as well as the up-modulation of carbonyl reductase, Rho-GDI, and histone H1.0 correlates with the synergistic antiangiogenic activity of VBL and RAP.

Angiogenesis Inhibitors↗

PubMed2005

Proteome analysis in the clinical chemistry laboratory: myth or reality?

BACKGROUND: We review here modern aspects of proteomic analysis, as displayed via orthogonal mass/charge analysis (isoelectric focusing in the first dimension, followed by sodium dodecyl sulphate electrophoresis in polyacrylamide gels, SDS-PAGE, at right angles, in the second dimension). METHODS: This technique is capable of displaying a few thousand polypeptide chains, characterized by a single pI and M(r) value as coordinates, and recognized via elution, digestion and mass spectrometry analysis. Although, up to the present, this technique has been used mostly for advanced research, with no immediate applications in the clinical chemistry laboratory, there are hints that such applications will soon become a reality. RESULTS AND CONCLUSIONS: In the field of cancer research, it is here shown that stathmin (Op18) becomes heavily phosphorylated in cancerous mantle cell lymphomas and that the progression of the disease can be followed by the progression of phosphorylation of Op18 and by the appearance of additional phosphorylated spots. Also chemoresistance of different tumors has been evaluated via 2D-PAGE through quantitative, differential proteomics: among up- and down-regulated proteins in a human cervix squamous cell carcinoma cell line (A431), rendered resistant to cisplatin, one particular protein was found to appear in large quantities by de novo synthesis: 14-3-3, a protein known to impart resistance to apoptosis to cells. In the field of brain disorders, we could set up an easy test for detecting pathological prions in sporadic Creutzfeldt-Jakob disease (sCJD), by simply searching for those pathological forms in the olfactory mucosa (up to this finding, diagnosis could only be confirmed post-mortem). We are currently working on a test for differentiating sCJD from all the other degenerative dementias. Upon 2D mapping of cerebrospinal fluid (CSF) and immunoblot analysis, we could identify a major spot (pI 4.8, M(r) 30 kDa) followed by some two-three minor spots (pIs 5.0-6.0, same M(r) value) of the same 14-3-3 anti-apoptotic protein involved in chemoresistance. By this test, sCJD could be differentiated from all the other degenerative dementias, which are 14-3-3 negative (in sCJD, the rapid and massive brain cell damage releases large quantities of 14-3-3 in the cerebrospinal fluid). Another protein that appears very promising as a marker for sCJD is cystatin C, that is strongly up-regulated in this pathology. Human sera should also be mined for discovery of many more markers for disease. Up to the present, no one could be found, but this was due to the presence of several major proteins, obscuring all rare ones. Via several immuno-subtraction steps, followed by ion exchange and size exclusion chromatography, one can now detect proteins and peptides present in sera at levels below 10 ng/mL, highlighting the road to discovery of novel markers of disease. Another technique that could revolutionize biomarker discovery in biological fluids consists in the use of combinatorial beads to reduce the dynamic range. They consist in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of amino acids, resulting in millions different structures. When these beads are impregnated with complex proteomes (e.g., human sera, CSF, urines) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility of evidencing low-abundance species.

Animals↗

PubMed2005

Two-dimensional mapping as a tool for classification of green coffee bean species.

Two species of the genus Coffea, Coffea arabica (Colombia) and Coffea canephora (Indiano Robusta) were analysed by two-dimensional (2-D) maps in order to obtain fingerprints of the expressed polypeptide chains and to determine which ones would characterize the two species. Green beans were milled under liquid nitrogen. A dry powder was produced by three different extraction protocols aimed at eliminating interfering substances (polyphenols). A reduced powder was produced by two successive extractions performed in acetone. Trichloroacetic acid (TCA; 10% w/v) and beta-mercaptoethanol (0.07% v/v) in acetone were used for the first extraction (a) and 10% w/v TCA in acetone was used for the second extraction (b). Proteins were then solubilized in a solution (40 microL per 1 mg powder) containing 7 M urea, 2 M thiourea, 3% w/v 3-(3-cholamidopropyldimethyl-amino)-1-propanesulfate, 1% v/v carrier ampholytes, 40 mM Tris, 5 mM tributylphosphine and 10 mM acrylamide as alkylating agent. Following incubation at room temperature for 1 hour and centrifugation (7000 rpm for 20 minutes), the supernatant was used for 2-D electrophoresis. The proteins were revealed by Sypro Ruby staining. Master maps of the five replicas of each species were compared by PDQuest analysis. The results of this differential proteome analysis were: sixteen proteins were expressed solely in C. canephora (var. Indiano Robusta) and five proteins were only found in C. arabica (var. Colombia). Another eight proteins were up-regulated in C. canephora (var. Indiano Robusta) in comparison to C. arabica (var. Colombia) and one was down-regulated in the same comparison. A number of these polypeptide chains were further characterized by mass spectrometry in the matrix-assisted laser desorption/ionisation-time of flight mode. Additionally, considering the low number of protein sequences of Coffea present in the databases we also investigated some spots with a more powerful tool, reversed phase-high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry, thus obtaining an internal peptide sequence. The general properties of the identified proteins are presented and discussed.

Chromatography, High Pressure Liquid↗

PubMed2005

Numerical approaches for quantitative analysis of two-dimensional maps: a review of commercial software and home-made systems.

The present review attempts to cover a number of methods that have appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this review to conventional two-dimensional (2-D) map analysis which couples orthogonally a charge-based step (isoelectric focusing) to a size-based separation step (sodium dodecyl sulfate-electrophoresis). The first and oldest method applied to 2-D map data reduction is based on statistical analysis performed on sets of gels via powerful software packages, such as Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately running a number of replicas for control and treated samples. The two sets of data are then merged and compared via a number of software packages which we describe. In addition to commercially-available systems, a number of home made approaches for 2-D map comparison have been recently described and are also reviewed. They are based on fuzzyfication of the digitized 2-D gel image coupled to linear discriminant analysis, three-way principal component analysis or a combination of principal component analysis and soft-independent modeling of class analogy. These statistical tools appear to perform well in differential proteomic studies.

Cluster Analysis↗

PubMed2005

Spot overlapping in two-dimensional maps: a serious problem ignored for much too long.

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.

Adrenal Gland Neoplasms↗

PubMed2005

Proteomic approaches for studying chemoresistance in cancer.

The role of various proteins involved in drug resistance in tumor cells is discussed in this review. Two types of studies are covered: those performed in the preproteomics era and those carried out with modern proteomic tools, namely 2D (electrophoretic) maps and 2D chromatography. In the preproteomic studies, one protein had generally been held responsible for a given chemoresistance. However, analysis via proteomic tools may reveal entire sets of proteins that are up- or downregulated (or switched on/off) in chemoresistant tumor cell lines compared with parental tumor lines. Therefore, it appears more realistic to expect that exposure of cells to drugs results in the activation of different mechanisms of resistance. Such investigations have led to the broadly shared opinion that exposure of cells to drugs results in the activation of different mechanisms of resistance, and that a specific drug-resistant phenotype consists of several molecular mechanisms that are simultaneously active. The proteomic papers reviewed clearly support the hypothesis that many metabolic pathways are affected during the resistance process. Although the modulation of expression levels of such proteins is not clear proof of their role in drug resistance per se, at least some of the themes are very likely to be involved in the resistance phenotype, and thus may be potential targets for new drugs. It is hoped that this review will bring new insight in this field and will stimulate novel and deeper searches with proteomic tools (including prefractionation of subcellular organelles, such as nuclei, to bring to the fore low-abundance proteins that might be responsible for the onset of drug resistance).

ATP-Binding Cassette Transporters↗

PubMed2004

Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches.

The present review attempts to cover a number of methods that appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this excursus only to conventional two-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation (sodium dodecyl sulfate (SDS)-electrophoresis). The first and oldest method applied in 2D mapping is based on statistical analysis performed on sets of gels via powerful software packages, such as the Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately-running a number of replicas for control and treated samples, the merging and comparing between these two sets of data being accomplished via the softwares just mentioned. Recent developments permit analyses on a single gel containing mixed samples differentially labelled and resolved by either fluorescence or isotopic means. In one approach, a set of fluorophors, called Cy3 and Cy5, are selected for differentially tagging Lys residues, via a "minimal labelling" protocol. A variant of this, adopts a newer set of fluorophors, also of the Cy3 and Cy5 type, reacting on Cys residues, via a strategy of "saturation labelling". There are at present two methods for quantitative proteomics in a 2D gel format exploiting stable isotopes: one utilizes tagging Cys residues with [2H0]/[2H3]-acrylamide; the other one, also based on a Cys reactive compound, exploits [2H0]/[2H4] 2-vinylpyridine. The latter reagent achieves 100% efficiency coupled to 100% specificity. The advantages and limitations of the various protocols are discussed.

Amino Acid Sequence↗

PubMed2004

Proteomic analysis of an orthotopic neuroblastoma xenograft animal model.

Neuroblastoma is the most common extracranial solid tumour of childhood and comprises up to 50% of malignancies among infants. There is a great need of designing novel therapeutic strategies and proteome analysis is one approach for defining markers useful for tumour diagnosis, as well as molecular targets for novel experimental therapies. We started by comparing healthy adrenal glands (which are the election organs developing primary neuroblastoma, NB, tumours) and adrenal glands carrying primary NB tumours, taken from nude mice. Standard maps of healthy and tumour samples were generated by analysis with the PDQuest software. The comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups, were analysed by MALDI-TOF mass spectrometry and 14 of these spots were identified so far. Among these proteins, of particular interest are the down-regulated proteins adrenodoxin (21-folds), carbonic anhydrase III (eight-folds) and aldose reductase related protein I (eight-folds), as well as the up-regulated protein peptidyl-propyl cis-trans isomerase A (five-folds). Moreover new proteins, which were absent in control samples, were expressed in tumour samples, such as nucleophosmin (NPM) and stathmin (oncoprotein 18).

Animals↗

PubMed2004

Study of proteomic changes associated with healthy and tumoral murine samples in neuroblastoma by principal component analysis and classification methods.

BACKGROUND: The adrenal gland is the election organ forming primary neuroblastoma (NB) tumours, the most common extracranial solid tumours of infancy and childhood. METHODS: Samples of adrenal gland belonging to healthy and diseased nude mouse were analysed by 2D gel-electrophoresis. The resulting 2D-PAGE maps were digitized by PDQuest and investigated by principal component analysis (PCA). RESULTS: The analysis of the loadings of the first principal component (PC) permitted the evaluation of the spots characterising each class of samples. Moreover, the soft-independent model of class analogy (SIMCA) method confirmed the separation of the samples in the two classes and allowed the identification of the modelling and discriminating spots. Very good correlation was found between the data obtained by analysis of 2D maps via the commercial software PDQuest and the present PCA analysis. In both cases, the comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups were analysed by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and 14 of these spots were identified so far.

Animals↗

PubMed2004

Quantitative proteomics: a review of different methodologies.

The present review attempts to cover the vast array of methods which have appeared in the last few years for performing quantitative proteome analysis. These methods are divided into two classes: those applicable to conventional two-dimensional map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation [sodium dodecylsulfate (SDS)-electrophoresis] and those applicable to two-dimensional chromatographic protocols. The first method, although being by and large the most popular approach, can offer differential display of paired samples with relatively few methods, the oldest one being based on statistical analysis performed on sets of gels via powerful software packages, such as the MELANIE, PDQuest, Z3 and Z4000, Phoretix and Progenesis. Recent developments comprise analysis performed on a single gel containing mixed samples differentially labeled, either with fluorophors (Cy3 and Cy5) or with d(0)/d(3) acrylamide. Conversely, chromatographic approaches, which mostly rely on analysis not of intact proteins but of their tryptic digests, offer a panoply of differential labeling protocols, most of which rely on stable isotope tagging. Essentially, all possible reactions have been described, such as those involving Lys, Asp, Glu, Cys residues, as well as a number of methods exploiting differential derivatization of amine and carboxyl groups generated during proteolysis. All such methods are described and evaluated.

Amino Acid Sequence↗

PubMed2003

The proteome: anno Domini 2002.

We present some current definitions related to functional and structural proteomics and the human proteome, and we review the following aspects of proteome analysis: Classical 2-D map analysis (isoelectric focusing (IEF) followed by SDS-PAGE); Quantitative proteomics (isotope-coded affinity tag (ICAT), fluorescent stains) and their use in e.g., tumor analysis and identification of new target proteins for drug development; Electrophoretic pre-fractionation (how to see the hidden proteome!); Multidimensional separations, such as: (a) coupled size-exclusion and reverse-phase (RP)-HPLC; (b) coupled ion-exchange and RP-HPLC; (c) coupled RP-HPLC and RP-HPLC at 25/60 degrees C; (d) coupled RP-HPLC and capillary electrophoresis (CE); (e) metal affinity chromatography coupled with CE; Protein chips. Some general conclusions are drawn on proteome analysis and we end this review by trying to decode the glass ball of the aruspex and answer the question: "Quo vadis, proteome"?

Animals↗

PubMed2002

Comparative two-dimensional mapping of prion protein isoforms in human cerebrospinal fluid and central nervous system.

The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein abundant in neurons. Although its precise function is unknown, PrP(C) represents the substrate for the generation of a conformational pathogenic isoform (PrP(Sc)) in human and animal transmissible spongiform encephalopathies, or prion diseases. By applying novel solubilization cocktails, we analyzed normal human brain and cerebrospinal fluid (CSF) PrP(C) by immunoblot of two-dimensional (2-D) gel electrophoresis preparations, using specific antibodies. Here, we show that PrP(C) from brain and CSF is composed of several charge isomers of differently glycosylated isoforms of the full-length PrP(C) and two N-terminally truncated fragments of 20 and 18 kDa. In the CSF, substantial amounts of the highly glycosylated PrP(C) isoforms and of the unglycosylated 18 kDa fragment are detected. Our study, for the first time, provides a detailed 2-D map of human PrP(C) both in brain and CSF, and establishes an innovative and sensitive method that might help in detecting the CSF pathological PrP(Sc) isoform in vivo. It also shows the incredible microheterogeneity of such isoforms (ca. 60 spots!), as revealed in 2-D mapping, as opposed to 3-4 main zones by mono-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Brain Chemistry↗

PubMedDate not supplied

Formation of truncated proteins and high-molecular-mass aggregates upon soft illumination of photosynthetic proteins.

Different spot profiles were observed in 2D gel electrophoresis of thylakoid membranes performed either under complete darkness or by leaving the sample for a short time to low visible light. In the latter case, a large number of new spots with lower molecular masses, ranging between 15,000 and 25,000 Da, were observed, and high-molecular-mass aggregates, seen as a smearing in the upper part of the gel, appeared in the region around 250 kDa. Identification of protein(s) contained in these new spots by MS/MS revealed that most of them are simply truncated proteins deriving from native ones, fragments, or aggregates. This resulted from the formation of extremely reactive oxygen species (ROS) that can derive by the exposure of chlorophyll binding proteins of photosynthetic apparatus to low-intensity light during laboratory manipulation of sample for electrophoresis runs.

Chromatography, High Pressure Liquid↗