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1.
Article in English | MEDLINE | ID: mdl-35410049

ABSTRACT

The principal aim of the present study was to develop and apply novel ex vivo tests as an alternative to cell cultures able to evaluate the possible effects of emerging and legacy contaminants in Caretta caretta. To this end, we performed ex vivo experiments on non-invasively collected whole-blood and skin-biopsy slices treated with chrysene, MEHP, or PBDE-47. Blood samples were tested by oxidative stress (TAS), immune system (respiratory burst, lysozyme, and complement system), and genotoxicity (ENA assay) biomarkers, and genotoxic and immune system effects were observed. Skin slices were analyzed by applying a 2D-PAGE/MS proteomic approach, and specific contaminant signatures were delineated on the skin proteomic profile. These reflect biochemical effects induced by each treatment and allowed to identify glutathione S-transferase P, peptidyl-prolyl cis-trans isomerase A, mimecan, and protein S100-A6 as potential biomarkers of the health-threatening impact the texted toxicants have on C. caretta. Obtained results confirm the suitability of the ex vivo system and indicate the potential risk the loggerhead sea turtle is undergoing in the natural environment. In conclusion, this work proved the relevance that the applied ex vivo models may have in testing the toxicity of other compounds and mixtures and in biomarker discovery.


Subject(s)
Turtles , Animals , Biomarkers/metabolism , Chrysenes , Diethylhexyl Phthalate/analogs & derivatives , Halogenated Diphenyl Ethers , Proteomics , Turtles/metabolism
2.
J Proteomics ; 170: 28-42, 2018 01 06.
Article in English | MEDLINE | ID: mdl-28970102

ABSTRACT

Cystic Fibrosis (CF) is a recessively inherited disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. CFTR has a pivotal role in the onset of CF, and several proteins are involved in its homeostasis. To study CFTR interactors at protein species level, we used a functional proteomics approach combining 2D-DIGE, mass spectrometry and enrichment analysis. A human bronchial epithelial cell line with cystic fibrosis (CFBE41o-) and the control (16HBE14o-) were used for the comparison. 73 differentially abundant spots were identified and some validated by western-blot. Enrichment analysis highlighted molecular pathways in which ezrin, HSP70, endoplasmin and lamin A/C, in addition to CFTR, were considered central hubs in CFTR homeostasis. These proteins acquire different functions through post-translational modifications, emphasizing the importance of studying the CF proteome at protein species level. Moreover, serpin H1, prelamin A/C, protein-SET and cystatin-B were associated to CF, demonstrating the importance of heat shock response, cross-talk between the cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating in the pathophysiology of the disease. These results open new perspectives for the understanding of the proteostasis network, characteristic of CF pathology, and could provide a springboard for new therapeutic strategies. BIOLOGICAL SIGNIFICANCE: Homeostasis of CFTR is a dynamic process managed by multiple proteostatic pathways. The used gel-based proteomic approach and enrichment analysis pointed out protein species variations among Human Bronchial (16HBE14o-) and Cystic Fibrosis Bronchial Epithelial cell lines (CFBE41o-) and specific molecular mechanisms involved in CF. In particular, we have highlighted HSP70 (HSP7C), HSP90 (endoplasmin), ERM proteins (ezrin), and lamin-A/C as central hubs of the functional analysis. Moreover, for the first time we consider serpin H1, lamin A/C, protein-SET and cystatin-B important player in CF, affecting acute exacerbation, cytoskeleton reorganization, CFTR gating and chronic inflammation in CF. Due to the presence of different spots corresponding to the same protein, we focalize our attention on the idea that a "protein species discourse" is mandatory to well-define functional roles of proteins. Our approach has permitted to pay attention to the molecular mechanisms which regulate pathways directly or indirectly involved with CFTR defects: heat shock response, cross-talk between cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating. Our data could open new perspectives into the understanding of CF, identifying potential targets for drug treatments in order to alleviate Δ508CFTR membrane instability and consequently increase life expectancy for CF patients.


Subject(s)
Cystic Fibrosis/metabolism , Protein Processing, Post-Translational , Proteome/metabolism , Cell Line, Transformed , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Proteome/genetics
3.
Environ Pollut ; 232: 603-614, 2018 Jan.
Article in English | MEDLINE | ID: mdl-28993024

ABSTRACT

Illicit drugs have been identified as emerging aquatic pollutants because of their widespread presence in freshwaters and potential toxicity towards aquatic organisms. Among illicit drug residues, cocaine (COC) and its main metabolites, namely benzoylecgonine (BE) and ecgonine methyl ester (EME), are commonly detected in freshwaters worldwide at concentration that can induce diverse adverse effects to non-target organisms. However, the information of toxicity and mechanisms of action (MoA) of these drugs, mainly of COC metabolites, to aquatic species is still fragmentary and inadequate. Thus, this study was aimed at investigating the toxicity of two concentrations (0.3 and 1.0 µg/L) of COC, BE and EME similar to those found in aquatic ecosystems on zebrafish (Danio rerio) embryos at 96 h post fertilization through a functional proteomics approach. Exposure to COC and both its metabolites significantly altered the protein profile of zebrafish embryos, modulating the expression of diverse proteins belonging to different functional classes, including cytoskeleton, eye constituents, lipid transport, lipid and energy metabolism, and stress response. Expression of vitellogenins and crystallins was modulated by COC and both its main metabolites, while only BE and EME altered proteins related to lipid and energy metabolism, as well as to oxidative stress response. Our data confirmed the potential toxicity of low concentrations of COC, BE and EME, and helped to shed light on their MoA on an aquatic vertebrate during early developmental period.


Subject(s)
Cocaine/toxicity , Embryo, Nonmammalian/drug effects , Illicit Drugs/toxicity , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Cocaine/analogs & derivatives , Cocaine/metabolism , Embryo, Nonmammalian/physiology , Fresh Water , Oxidation-Reduction , Oxidative Stress/drug effects , Zebrafish/metabolism
4.
J Proteomics ; 167: 46-59, 2017 09 07.
Article in English | MEDLINE | ID: mdl-28802583

ABSTRACT

Osteogenesis imperfecta (OI) is a collagen-related disorder associated to dominant, recessive or X-linked transmission, mainly caused by mutations in type I collagen genes or in genes involved in type I collagen metabolism. Among the recessive forms, OI types VII, VIII, and IX are due to mutations in CRTAP, P3H1, and PPIB genes, respectively. They code for the three components of the endoplasmic reticulum complex that catalyzes 3-hydroxylation of type I collagen α1Pro986. Under-hydroxylation of this residue leads to collagen structural abnormalities and results in moderate to lethal OI phenotype, despite the exact molecular mechanisms are still not completely clear. To shed light on these recessive forms, primary fibroblasts from OI patients with mutations in CRTAP (n=3), P3H1 (n=3), PPIB (n=1) genes and from controls (n=4) were investigated by a functional proteomic approach. Cytoskeleton and nucleoskeleton asset, protein fate, and metabolism were delineated as mainly affected. While western blot experiments confirmed altered expression of lamin A/C and cofilin-1, immunofluorescence analysis using antibody against lamin A/C and phalloidin showed an aberrant organization of nucleus and cytoskeleton. This is the first report describing an altered organization of intracellular structural proteins in recessive OI and pointing them as possible novel target for OI treatment. SIGNIFICANCE: OI is a prototype for skeletal dysplasias. It is a highly heterogeneous collagen-related disorder with dominant, recessive and X-linked transmission. There is no definitive cure for this disease, thus a better understanding of the molecular basis of its pathophysiology is expected to contribute in identifying potential targets to develop new treatments. Based on this concept, we performed a functional proteomic study to delineate affected molecular pathways in primary fibroblasts from recessive OI patients, carrying mutations in CRTAP (OI type VII), P3H1 (OI type VIII), and PPIB (OI type IX) genes. Our analyses demonstrated the occurrence of an altered cytoskeleton and, for the first time in OI, of nuclear lamina organization. Hence, cytoskeleton and nucleoskeleton components may be considered as novel drug targets for clinical management of the disease. Finally, according to our analyses, OI emerged to share similar deregulated pathways and molecular aberrances, as previously described, with other rare disorders caused by different genetic defects. Those aberrances may provide common pharmacological targets to support classical clinical approach in treating different diseases.


Subject(s)
Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Genes, Recessive , Nuclear Lamina/metabolism , Nuclear Proteins/metabolism , Osteogenesis Imperfecta/metabolism , Proteomics , Cytoskeletal Proteins/genetics , Cytoskeleton/genetics , Humans , Nuclear Lamina/genetics , Nuclear Proteins/genetics , Osteogenesis Imperfecta/genetics
5.
Mol Cell Proteomics ; 16(7): 1348-1364, 2017 07.
Article in English | MEDLINE | ID: mdl-28483926

ABSTRACT

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides.


Subject(s)
Aggregatibacter actinomycetemcomitans/metabolism , Escherichia coli/genetics , Lipoproteins/metabolism , Neisseria meningitidis/metabolism , Aggregatibacter actinomycetemcomitans/genetics , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/metabolism , Escherichia coli/cytology , Escherichia coli/metabolism , Lipoproteins/genetics , Neisseria meningitidis/genetics , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transformation, Bacterial
6.
Inflamm Res ; 66(3): 269-280, 2017 Mar.
Article in English | MEDLINE | ID: mdl-27900411

ABSTRACT

BACKGROUND: Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT. METHODS: Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated. RESULTS: CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregulation featuring a bulk increase of anti-inflammatory cytokines, predominantly IL-10, could explain the unchanged erythrocyte sedimentation rate and atypical features of inflammation in CDKL5-RTT. Omega-3 PUFAs were able to counterbalance the pro-inflammatory status. CONCLUSION: For the first time, we revealed a subclinical smouldering inflammation pattern in CDKL5-RTT consisting in the coexistence of an atypical APR coupled with a dysregulated cytokine response.


Subject(s)
Acute-Phase Reaction/immunology , Cytokines/immunology , Rett Syndrome/immunology , Spasms, Infantile/immunology , Acute-Phase Reaction/genetics , Acute-Phase Reaction/metabolism , Adolescent , Blood Proteins/immunology , Blood Proteins/metabolism , Child , Child, Preschool , Cytokines/blood , Dietary Supplements , Epileptic Syndromes , Fatty Acids, Omega-3/pharmacology , Female , Humans , Infant , Methyl-CpG-Binding Protein 2/genetics , Protein Serine-Threonine Kinases/genetics , Rett Syndrome/genetics , Rett Syndrome/metabolism , Spasms, Infantile/genetics , Spasms, Infantile/metabolism
7.
Proteomics ; 16(6): 963-72, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26711811

ABSTRACT

The aim of this work was the functional and proteomic analysis of a mutant, W3110 Bgl(+) /10, isolated from a batch culture of an Escherichia coli K-12 strain maintained at room temperature without addition of nutrients for 10 years. When the mutant was evaluated in competition experiments in co-culture with the wild-type, it exhibited the growth advantage in stationary phase (GASP) phenotype. Proteomes of the GASP mutant and its parental strain were compared by using a 2DE coupled with MS approach. Several differentially expressed proteins were detected and many of them were successful identified by mass spectrometry. Identified expression-changing proteins were grouped into three functional categories: metabolism, protein synthesis, chaperone and stress responsive proteins. Among them, the prevalence was ascribable to the "metabolism" group (72%) for the GASP mutant, and to "chaperones and stress responsive proteins" group for the parental strain (48%).


Subject(s)
Escherichia coli K12/metabolism , Escherichia coli K12/physiology , Escherichia coli Proteins/analysis , Proteome/analysis , Proteomics/methods , Electrophoresis, Gel, Two-Dimensional , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Mass Spectrometry , Proteome/chemistry , Proteome/metabolism
8.
Mol Biosyst ; 9(6): 1162-8, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23385635

ABSTRACT

Fabry disease (FD) is an X-linked progressive multisystem disease due to mutations in the gene encoding the lysosomal enzyme α-galactosidase A (α-GalA). The deficiency in α-GalA activity leads to an intra-lysosomal accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3), in various organs and systems. Enzyme replacement therapy is available and alternative therapeutic approaches are being explored. No diagnostic test, other than sequencing of the α-galactosidase A gene, is available, no biomarker has been proven useful to screen for and predict the disease, and underlying mechanisms are still elusive. The aim of this study is to identify FD specific biomarkers and to better understand the pathophysiological changes that occur over time in FD. We compared peripheral blood mononuclear cells (PBMC) from FD patients (n = 8) with control PBMC from healthy individuals (n = 6), by two-dimensional electrophoresis (2DE) and the detected differentially expressed proteins were then subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). In FD patients we identified, among the down-regulated proteins, Calnexin, Rho GDP-dissociation inhibitor 2, Rho GDP-dissociation inhibitor 1, Chloride intracellular channel protein 1; on the other hand γ-enolase, 14-3-3 protein theta, 14-3-3 protein zeta/delta, and galectin-1 were identified as up-regulated proteins. Calnexin and Rho GDP-dissociation inhibitor-1,2 are related to protein folding, signal transduction and cell proliferation. This is the first time that γ-enolase and galectin-1 are described to be up-regulated in Fabry patients. Levels of γ-enolase increase dramatically in cardiovascular accidents and cerebral trauma, whereas galectins are regulators of acute and chronic inflammation. These findings may improve our understanding of the molecular mechanisms underlying the pathology and provide new insight and knowledge for future studies in this field.


Subject(s)
Fabry Disease/metabolism , Leukocytes, Mononuclear/metabolism , Proteome/metabolism , 14-3-3 Proteins/biosynthesis , Adult , Biomarkers , Calnexin/biosynthesis , Cell Proliferation , Chloride Channels/biosynthesis , Down-Regulation , Fabry Disease/diagnosis , Female , Galectin 1/biosynthesis , Gene Expression , Humans , Inflammation , Male , Middle Aged , Phosphopyruvate Hydratase/biosynthesis , Protein Folding , Proteomics , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Up-Regulation , alpha-Galactosidase/genetics , rho Guanine Nucleotide Dissociation Inhibitor alpha/biosynthesis , rho Guanine Nucleotide Dissociation Inhibitor beta/biosynthesis
9.
Proteomics ; 12(11): 1767-80, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22623105

ABSTRACT

The Saccharomyces cerevisiae gene SCO1 has been shown to play an essential role in copper delivery to cytochrome c oxidase. Biochemical studies demonstrated specific transfer of copper from Cox17p to Sco1p, and physical interactions between the Sco1p and Cox2p. Deletion of SCO1 yeast gene results in a respiratory deficient phenotype. This study aims to gain a more detailed insight on the effects of SCO1 deletion on S. cerevisiae metabolism. We compared, using a proteomic approach, the protein pattern of SCO1 null mutant strain and wild-type BY4741 strain grown on fermentable and on nonfermentable carbon sources. The analysis showed that on nonfermentable medium, the SCO1 mutant displayed a protein profile similar to that of actively fermenting yeast cells. Indeed, on 3% glycerol, this mutant displayed an increase of some glycolytic and fermentative enzymes such as glyceraldehyde-3-phosphate dehydrogenase 1, enolase 2, pyruvate decarboxylase 1, and alcohol dehydrogenase 1. These data were supported by immunoblotting and enzyme activity assay. Moreover, the ethanol assay and the oxygen consumption measurement demonstrated a fermentative activity in SCO1 mutant on respiratory medium. Our results suggest that on nonfermentable carbon source, the lack of Sco1p causes a metabolic shift from respiration to fermentation.


Subject(s)
Electron Transport Complex IV/metabolism , Membrane Proteins/genetics , Mitochondrial Proteins/genetics , Proteome/analysis , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Electron Transport Complex IV/biosynthesis , Electron Transport Complex IV/genetics , Fermentation/genetics , Gene Deletion , Glycolysis/genetics , Mitochondria/genetics , Mitochondria/metabolism , Oxygen Consumption/genetics , Proteomics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
10.
Proteomics ; 11(18): 3725-42, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21761561

ABSTRACT

Several solid tumors are characterized by poor prognosis and few effective treatment options, other than palliative chemotherapy in the recurrent/metastatic setting. Epidermal growth factor receptor (EGFR) has been considered an important anticancer target because it is involved in the development and progression of several solid tumors; however, only a subset of patients show a clinically meaningful response to EGFR inhibition, particularly to EGFR tyrosine kinase inhibitors such as gefitinib. We have recently demonstrated synergistic antitumor effect of the histone deacetylase inhibitor vorinostat combined with gefitinib. To further characterize the interaction between these two agents, cellular extracts from Hep-2 cancer cells that were untreated or treated for 24 h with either vorinostat or gefitinib alone or with a vorinostat/gefitinib combination were analyzed using 2-D DIGE. Software analysis using DeCyder was performed, and numerous differentially expressed protein spots were visualized between the four examined settings. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified; some of these were validated by Western blotting. Finally, a pathway analysis of experimental data performed using MetaCore highlighted a relevant relationship between the identified proteins and additional potential effectors. In conclusion, we performed a comprehensive analysis of proteins regulated by vorinostat and gefitinib, alone and in combination, providing a useful insight into their mechanisms of action as well as their synergistic interaction.


Subject(s)
ErbB Receptors/antagonists & inhibitors , Hydroxamic Acids/pharmacology , Quinazolines/pharmacology , Antineoplastic Agents/pharmacology , Blotting, Western , Cell Line, Tumor , Drug Synergism , Gefitinib , Gene Expression Regulation, Neoplastic , Humans , Mass Spectrometry/methods , Peptide Mapping , Protein Isoforms/chemistry , Proteomics , Software , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Two-Dimensional Difference Gel Electrophoresis , Vorinostat
11.
Clin Chem Lab Med ; 47(11): 1373-9, 2009.
Article in English | MEDLINE | ID: mdl-19778290

ABSTRACT

BACKGROUND: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease. METHODS: Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody. RESULTS: SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated M(r) of 12.5-29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments. CONCLUSIONS: These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood.


Subject(s)
Kidney Failure, Chronic/blood , Kidney Neoplasms/blood , Serum Albumin/analysis , Adult , Aged , Aged, 80 and over , Electrophoresis, Polyacrylamide Gel , Humans , Kidney Function Tests , Middle Aged , Pilot Projects , Renal Dialysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
12.
J Proteome Res ; 8(7): 3377-89, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19408963

ABSTRACT

Freshly ejaculated sperm acquire the fertilizing potential by a continuing process that occurs during sperm transport through the female genital tract, and it is physiologically not complete until the spermatozoon reaches the oocyte. The process termed capacitation can be mimicked in vitro by using appropriate capacitation media. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. This work deals with a proteomic approach to the analysis of protein profile variations in human normospermic samples as a consequence of three hours in vitro capacitation. 2DE gels were produced per freshly ejaculated sperm and per capacitated sperm and several quantitative and qualitative significant variations were found. Among the MS obtained identifications, proteins with a significant decrease after capacitation were found to be involved in protein fate, metabolism, and flagellar organization; on the contrary, increasing proteins were found to be related to cellular stress. Interestingly, the detected flagellar organization proteins decreased during capacitation whereas their corresponding fragments increased. A swim-up selected and three-hour capacitated sperm subpopulation has also been resolved by 2DE, and its synthetic gel has been analyzed for the variations observed in the entire sperm population. An immunofluorescence analysis of this sperm typology was carried out with antiactin and antitubulin antibodies.


Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Mass Spectrometry/methods , Proteins/chemistry , Sperm Capacitation/physiology , Spermatozoa/pathology , Apoptosis , Female , Fertilization , Humans , Male , Microscopy, Fluorescence/methods , Proteomics/methods , Silver Staining , Spectrometry, Fluorescence/methods , Spermatozoa/metabolism
13.
J Proteome Res ; 8(2): 860-9, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19105609

ABSTRACT

Inhabitants of Metsovo, NW Greece, have been exposed to an asbestos whitewash, resulting in malignant pleural mesothelioma (MPM) and pleural calcifications (PCs). Interestingly, those with PCs (PC(+)) are less prone to MPM. They also have lymphocytic alveolitis, and differences in bronchoalveolar lavage (BAL) proteins, compared with those without pleural calcifications (PC(-)). This may mean a different response to the fiber leading to different susceptibility to neoplasia. To further evaluate this, a proteomic analysis of BAL proteins was performed. Proteomic analysis (2D-electrophoresis/Mass Spectrometry) of BAL in Metsovites nonoccupationally exposed to asbestos revealed increased albumin fragments, alpha1-antitrypsin, S100-A9 and HSP27, suggesting ongoing inflammation. In those without pleural calcifications, increased expression of acid ceramidase, glutathione-S-transferase and presence of calcyphosin, all involved in cell cycle regulation and death as well as in the detoxification of mutagenic and toxic agents, lend further support to our thesis of possible "protection against neoplasia" in Metsovites with pleural calcifications.


Subject(s)
Asbestos/adverse effects , Bronchoalveolar Lavage Fluid/chemistry , Mesothelioma , Pleural Diseases/etiology , Proteins/analysis , Proteome/analysis , Cluster Analysis , Greece , Humans , Mesothelioma/chemistry , Mesothelioma/etiology , Molecular Sequence Data
14.
Glycobiology ; 19(4): 337-43, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19023103

ABSTRACT

In this paper, we demonstrate the existence and localization of fucosyl-containing O-glycoforms of nucleolin in cultured bovine endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody raised against gp273, a glycoprotein ligand for the sperm-egg interaction in the mollusc bivalve Unio elongatulus. The function and immunological properties of gp273 mainly depend on clustered Lewis-like, fucose-containing O-glycans. Here an anti-gp273 antibody was used to evaluate whether glycoepitopes similar to those of gp273 are part of potential ligands of selectins in endothelial cells. We found that anti-gp273 strongly and exclusively interacted with a 110 kDa protein in CVEC and A431 tumor cells. After partial purification, mass spectrometry identified the protein as nucleolin. This was confirmed by comparing anti-gp273 and anti-nucleolin antibody immunoblotting after nucleolin depletion. We confirmed that anti-gp273 binding to nuclear and extranuclear nucleolin was against a fucose-containing O-glycoepitope by immunoblot analysis of the protein after chemically removing O-glycans and by lectin-blot analysis of control and nucleolin-depleted samples. Using anti-gp273 IgG, we detected nucleolin on the plasma membrane and cytoplasm. O-Glycosylation may regulate the plethora of functions in which nucleolin is involved.


Subject(s)
Epitopes/chemistry , Fucose/chemistry , Glycoproteins/chemistry , Immunoglobulin G/chemistry , Phosphoproteins/chemistry , RNA-Binding Proteins/chemistry , Animals , Cattle , Cell Line, Tumor , Epitopes/immunology , Epitopes/metabolism , Fucose/immunology , Fucose/metabolism , Glycoproteins/immunology , Glycoproteins/metabolism , Glycosylation , Humans , Immunoglobulin G/immunology , Phosphoproteins/immunology , Phosphoproteins/metabolism , RNA-Binding Proteins/immunology , RNA-Binding Proteins/metabolism , Unio , Nucleolin
15.
J Proteomics ; 72(5): 853-64, 2009 Jul 21.
Article in English | MEDLINE | ID: mdl-19111954

ABSTRACT

Milk fat globules (MFGs) are secretory vesicles assembled and secreted by mammary epithelial cells during lactation. They consist of fat globules surrounded by a lipid bilayer membrane which is derived from the apical membrane of the lactating cells. MFGs contain, besides lipids, proteins from the apical plasma membrane and from the cytoplasmatic material. Their peculiar vesicle nature makes them a suitable and easily available source of biological material in monitoring the physiopathological state of the mammary gland. Unfortunately, the conspicuous lipidic component of MFGs consistently limits protein extraction and purification for MFG proteomic investigations. This work deals with the development of a suitable procedure for protein extraction from the cow MFGs in order to qualitatively and quantitatively improve 2-D electropherograms of the MFG. MFGs were purified from raw milk by centrifugation and then delipidated/precipitated. The resulting protein pellets were solubilised using four different 2-D SDS PAGE compatible lysis buffers. Applied methodological procedures for protein extraction and evaluation of the resulting 2-D protein-pattern are presented and discussed. Using these procedures a reference 2-D map of cow milk fat globules is also reported. The majority of the obtained identifications was represented by proteins involved in lipid synthesis or in fat globule secretion.


Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Glycolipids/chemistry , Glycoproteins/chemistry , Milk/metabolism , Proteomics/methods , Animals , Cattle , Cell Membrane/metabolism , Detergents/pharmacology , Electrophoresis, Polyacrylamide Gel , Lipid Droplets , Lipids/chemistry , Proteins/chemistry , Proteome , Silver Staining/methods
16.
Mol Cell Proteomics ; 7(12): 2337-49, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18676363

ABSTRACT

The presence of autoantibodies in multiple sclerosis (MuS) is well known, but their target antigens have not been clearly identified. In the present study, IgG autoreactivity to neural antigens of normal human white matter separated by bidimensional electrophoresis was assessed in serum and cerebrospinal fluid of 18 MuS and 20 control patients. Broad IgG autoreactivity was detected by two-dimensional immunoblotting in all cases to neural antigens, most of which were identified by mass spectrometry. The comparative analysis of MuS and non-MuS reactive spots showed that a restricted number of neural protein isoforms were specifically recognized by MuS IgG. Almost all MuS patients had cerebrospinal fluid IgG directed to isoforms of one of the oligodendroglial molecules, transketolase, 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I, collapsin response mediator protein 2, and tubulin beta 4. Interestingly 50% of MuS IgG recognized transketolase, which was mostly localized on oligodendrocytes in human white matter from normal and MuS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2, and actin-interacting protein 1) was prevalent in secondary progressive MuS patients. Among the proteins recognized by serum IgG, almost all MuS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, whereas 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I was the oligodendroglial antigen most frequently recognized (44%) by MuS seric IgG. Our immunomics approach shed new light on the autoimmune repertoire present in MuS patients revealing novel oligodendroglial and/or neuronal putative autoantigens with potential important pathogenic and diagnostic implications.


Subject(s)
2',3'-Cyclic-Nucleotide Phosphodiesterases/immunology , Autoantibodies/immunology , Immunoglobulin G/immunology , Multiple Sclerosis/enzymology , Multiple Sclerosis/immunology , Transketolase/immunology , Adult , Aged , Case-Control Studies , Demography , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/cerebrospinal fluid , Isoenzymes/immunology , Male , Middle Aged , Multiple Sclerosis/cerebrospinal fluid , Multiple Sclerosis/pathology , Protein Transport
17.
Eur J Heart Fail ; 10(8): 749-57, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18579441

ABSTRACT

BACKGROUND: In chronic heart failure (CHF), peripheral blood mononuclear cells (PBMC) might undergo structural and/or functional alterations as a consequence of the development and progression of the disease. AIMS: This study was aimed at: (1) assessing the proteome profile of PBMC from Controls and CHF subjects, (2) identifying differentially-expressed proteins in healthy subjects and patients, and (3) analysing the expression of these proteins in patients after heart transplantation. METHODS AND RESULTS: Proteome changes were assessed in PBMC from 8 healthy and 11 end-stage CHF (6 Ischaemic Heart Failure [IHF], 5 Dilated CardioMyopathy [DCM]) subjects by gel electrophoresis, PD-Quest analysis and mass spectrometry. Eighteen proteins were differentially expressed in Controls and CHF patients. However, among CHF patients, these proteins were equally expressed in IHF and DCM subjects. Eleven proteins were found to belong to 4 functional classes (3 cytoskeletal, 4 cell-cycle progression, 2 stress response and DNA repair, 2 energetic metabolism proteins). Changes in three of the differentially-expressed proteins were also confirmed by Western blot and were reversed after heart transplantation. CONCLUSION: Results demonstrate an altered protein expression profile in PBMC of CHF patients compared to Controls, thus providing a basis for further diagnostic and prognostic tests for CHF.


Subject(s)
Blood Proteins/analysis , Heart Failure/blood , Leukocytes, Mononuclear/chemistry , Blotting, Western , Electrophoresis, Gel, Two-Dimensional , Heart Failure/diagnosis , Heart Transplantation , Humans , Prognosis
18.
J Neuroimmunol ; 196(1-2): 153-8, 2008 May 30.
Article in English | MEDLINE | ID: mdl-18407358

ABSTRACT

To identify the target of IgG autoimmune response in Hashimoto's encephalopathy (HE), we studied the binding of IgG present in serum and cerebro-spinal fluid (CSF) from six patients with HE and 15 controls to human central nervous system (CNS) white matter antigens by 2D-PAGE and immunoblotting and by immunohistochemistry. We found that CSF IgG from HE patients specifically recognized 3 spots, which were identified as dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). DDAHI was present in two isoforms recognized respectively by five and four HE patients; immunohistochemistry with anti-DDAHI antiserum depicted endothelial cells in normal human CNS. AKRIAI was recognized by three HE CSF and this enzyme was widely distributed on neurons and endothelia by immunohistochemistry. IgG from HE CSF immunostained both neuronal and endothelial cells in mouse CNS. The presence of these autoantibodies selectively in the CSF of HE patients may have important diagnostic and pathogenetic implications, since the autoimmune response to these enzymes may lead to vascular and/or neuronal damage, two major mechanisms involved in the pathogenesis of HE.


Subject(s)
Autoantigens/immunology , Hashimoto Disease/immunology , Immunoglobulin G/cerebrospinal fluid , Proteomics/methods , Aged , Aldehyde Reductase/metabolism , Amidohydrolases/metabolism , Autoantigens/cerebrospinal fluid , Blood Vessels/metabolism , Electrophoresis, Gel, Two-Dimensional/methods , Female , Hashimoto Disease/cerebrospinal fluid , Hashimoto Disease/pathology , Humans , Male , Mass Spectrometry/methods , Middle Aged
19.
Proteomics ; 8(2): 402-12, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18203263

ABSTRACT

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short- (1 injection) and long-term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short-term and one long-term MPE treatment. One group received EV alone. Two test groups received EV with either a short-term or long-term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2-DE/MALDI-TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV-induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.


Subject(s)
Blood Proteins/analysis , Mucuna/chemistry , Plant Extracts/chemistry , Proteomics , Viper Venoms/antagonists & inhibitors , Animals , Electrophoresis, Gel, Two-Dimensional , Male , Mice , Seeds/chemistry , Viperidae
20.
Proteomics ; 7(11): 1877-91, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17520686

ABSTRACT

This study used proteomic and transcriptomic techniques to understand the molecular basis of the phenotypic variability in the bone disorder osteogenesis imperfecta (OI). Calvarial bone mRNA expression was evaluated by microarray, real-time, and comparative RT-PCR and the bone proteome profile was analyzed by 2-DE, MS, and immunoblotting in the OI murine model BrtlIV, which has either a moderate or a lethal OI outcome. Differential expression analysis showed significant changes for eight proteins. The expression of the ER stress-related protein Gadd153 was increased in lethal mice, whereas expression of the chaperone alphaB crystallin was increased in nonlethal mice, suggesting that the intracellular machinery is involved in the modulation of the OI phenotype. Furthermore, in lethal BrtlIV, the increased expression of the cartilaginous proteins Prelp, Bmp6, and Bmp7 and the lower expression of the bone matrix proteins matrilin 4, microfibril-associated glycoprotein 2, and thrombospondin 3 revealed that both a delay in skeletal development and an alteration in extracellular matrix composition influence OI outcomes. Differentially expressed proteins identified in this model offer a starting point for elucidating the molecular basis of phenotypic variability, a characteristic common to many genetic disorders. The first reference 2-DE map for murine calvarial tissue is also reported.


Subject(s)
Collagen Type I/metabolism , Osteogenesis Imperfecta/metabolism , Transcription Factor CHOP/metabolism , Amino Acid Substitution , Animals , Blotting, Western , Bone and Bones/metabolism , Collagen Type I/genetics , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Genes, Lethal , Genetic Markers , Genetic Variation , Mice , Microarray Analysis , Osteogenesis Imperfecta/genetics , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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