Biomedical subjects

Hubert Astner

Publications and source records attributed to Hubert Astner.

11 recordsLinked to original sources

PubMed2006

Proteomic analysis of rat cortical neurons after fluoxetine treatment.

The known neurochemical effect of most currently available antidepressants is the enhancement of the synaptic levels of monoamine neurotransmitters. However, the existence of other mechanisms has been suggested to justify the significant delay between the modulation of the monoaminergic system and the clinical effects. In order to investigate the effects of the antidepressant fluoxetine (a prototypical serotonin selective re-uptake inhibitor) and to improve the understanding of its mechanism of action, we performed a proteomic investigation in rat primary cortical neurons exposed sub-chronically to this antidepressant. Cortical neurons were treated for 3 days with 1 microM fluoxetine or vehicle. Protein extracts were processed for 2D gel characterization. Image analysis allowed the identification of six proteins differently expressed by more than 100% and seven proteins differently expressed by more than 50% (P<0.05). Nine proteins were identified by mass spectrometry. Among them, cyclophilin A, 14-3-3 protein z/delta and GRP78 are involved in neuroprotection, in serotonin biosynthesis and in axonal transport, respectively. This study showed that the primary culture of cortical neurons is a suitable system for studying the effects of fluoxetine action and may contribute to improve the understanding of fluoxetine psychotherapeutic action and the mechanisms mediating the long-term effects of this antidepressant treatment.

Analysis of Variance↗

PubMed2005

Proteomic changes in rat serum, polymorphonuclear and mononuclear leukocytes after chronic nicotine administration.

In order to gain information about the effect triggered at the molecular level by nicotine, its neuroimmunomodulatory properties and its impact on the pathogenesis of inflammatory diseases, peripheral blood serum and leukocytes of rat submitted to passive nicotine administration were subjected to proteomic investigation. Serum, polymorphonuclear (PMN) and mononuclear (MN) leukocytes from chronically treated animals and from control animals were analysed by a two-dimensional (2-D) gel electrophoresis/mass spectrometry approach to detect differentially expressed proteins. The nicotine regimen selected is known to have a stimulatory effect on locomotor activity and to produce a sensitisation of the mesolimbic dopamine system mechanism involved in addiction development. After 2-D gel analysis and matching, 36 spots in serum, seven in PMN and five in MN were found to display a statistical difference in their expression and were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry peptide fingerprinting for protein identification. Fifteen different proteins were identified. The results indicate an overall impact of nicotine on proteins involved in a variety of cellular and metabolic pathways, including acute phase response (suggesting the effect on inflammatory cascades and more in general on the immune system), oxidative stress metabolism and assembly and regulation of cytoskeleton. In particular, the observed changes imply a general reduction in the inflammatory response with a concomitant increased unbalance of the oxidative stress metabolism in the periphery and point to a number of potential noninvasive markers for the central nervous system (CNS) and non-CNS mediated activities of nicotine.

Animals↗

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

A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431.

Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin-resistant subline, A431/Pt, were used as a model system. The experimental set-up involved not just a two-way comparison of the control vs. the drug-resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four-way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat-shock proteins HSP60, HSP90, HSC71, heat-shock cognate 71 kDa protein), Ca2+-binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione-S-transferase, GST), anti-apoptotic proteins (such as 14-3-3 switched on in cisplatin-exposed cells) and ion channels (such as VDAC-1, voltage-dependent anion-selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up-regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up-regulated the anti-apoptotic 14-3-3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug-induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.

Antineoplastic Agents↗

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

Proteomic profiling of pancreatic ductal carcinoma cell lines treated with trichostatin-A.

A pancreatic adenocarcinoma cell line (Paca44) was treated with trichostatin-A (TSA), a potent inhibitor of histone deacetylases, in order to evaluate the effect of this drug on protein expression. Master maps of control and treated Paca44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 51 polypeptide chains, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry analysis enabled the identification of 22 of these spots. Among these proteins, of particular interest are the two downregulated proteins nucleophosmin and translationally controlled tumor protein, as well as the upregulated proteins programmed cell death protein 5 (also designated as TFAR19) and stathmin (oncoprotein 18). The modulation of these four proteins is consistent with our observation that TSA is able to inhibit cell growth of Paca44 by causing cell cycle arrest at the G2 phase and apoptotic cell death.

Antineoplastic Agents↗

PubMed2003

Two-dimensional molecular profiling of mantle cell lymphoma.

The present research establishes standard two-dimensional (2-D) maps for control, reactive lymph node and non-Hodgkin's lymphoma (mantle cell lymphoma, MCL). Medium sensitivity, mass spectrometry compatible colloidal Coomassie has revealed a total of ca. 750 spots in each of the maps. Comparison of 2-D maps by statistical packages, such as the PDQuest, established up- and downregulation of a total of ca. 145 spots, with positive variations of up to 10-folds and negative variations of up to 13-folds in both MCL biopsies' protein extracts. Qualitative and quantitative variations in the two lymphoma samples are consistent. More than 20 proteins have been so far identified by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry, with an additional five spots, which gave very good spectra but could not be matched to any of the presently available databases. Some of the spots, such as the 78 kDa glucose-regulated protein precursor and the glutathione S-transferase P, appear to be in common with other tumors, such as lung adenocarcinoma. Others may simply reflect overall changes in cellular metabolism and growth rate that occur during malignancy and thus might turn out to be in common with any cell population receiving any kind of stress. Some (notably T-cell leukemia/lymphoma protein 1A, TCL1, found to be 10-fold overexpressed) appear to be specific of the non-Hodgkin's lymphoma here studied. Western blot and immunohistochemical analyses were applied to obtain further information about stathmin (Op18) and TCL1, respectively.

Biopsy↗

PubMed2003

Proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2'-deoxycytidine.

A pancreatic adenocarcinoma cell line (PaCa44), which contains, among other alterations, a methylated p16 promoter, was treated with a chemoterapeutic agent, 5-aza-2'-deoxycytidine (DAC), in order to evaluate the effect of this drug on cell growth and protein expression. Cell proliferation was strongly inhibited by a 24 h DAC treatment and this inhibition lasted for at least 10 days. Master maps of control and treated PaCa44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 45 polypeptide chains, of which 32 were downregulated and 13 upregulated, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by mass spectrometry analysis enabled the identification of 36 of these spots. Among the major changes in DAC-treated cells: cofilin and profilin 1 are silenced; coactosin, peptidyl-propyl cis-trans isomerase A and cystatin B are decreased by 22, 16- and 15-fold, respectively; stress-70 protein, superoxide dismutase and protein disulfide isomerase A3 are increased by 13-, 11-, and 5-fold, respectively. The significance of some of these major changes is discussed.

Actin Depolymerizing Factors↗

PubMed2002

Advanced glycation end-products/peptides: a preliminary investigation by LC and LC/MS.

An investigation on AGE-peptides, originating by proteolysis of in vitro glycated proteins, was carried out by LC methods with different detection applied to the mixture produced by proteinase K digestion of in vitro glycated human serum albumin (HSA). Classical approaches, like spectroscopic (UV, fluorescence) and mass spectrometric methods (MALDI, LC/ESI/MS), show that the digestion mixture is highly complex. However, there are clearcut differences between the digestion mixtures of glycated and unglycated HSA, in the former case allowing identification of possible glycated peptides belonging to the AGE-peptide class. MS/ MS experiments on selected species seem to be promising as regards structural information.

Chromatography, Liquid↗