Biomedical subjects

Antonio Núñez

Publications and source records attributed to Antonio Núñez.

8 recordsLinked to original sources

PubMed2006

Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts.

Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.

Animals↗

PubMed2005

An efficient expression system for the production of functionally active human LKB1.

Human LKB1, also known as STK11, is a tumour-suppression protein that mediates important functions in cellular proliferation and polarization. It might constitute an important target in cancer therapy. In order to produce large amounts of recombinant protein for biochemical and functional studies, a full-length cDNA clone was subcloned and expressed in Escherichia coli and insect cells. Although fusion proteins corresponding to LKB1 with 6xHis, GST and MBP tags could be overexpressed in E. coli, only MBP-LKB1 was recovered in a soluble, but heavily degraded form. Further studies demonstrated that this protein was not functional. Subsequent expression in insect cells of LKB1 with 6xHis and GST tags yielded insoluble products also. However, when chaperones Hsp70 and its cofactors Hsp40 and Hsdj were co-expressed with GST-LKB1, a clear increase in the solubility of the final protein was obtained. Moreover, this soluble, purified recombinant GST-LKB1 demonstrated to be a phosphoprotein, with at least residue Ser325 phosphorylated. The purified protein was functionally active as being able to demonstrate autophosphorylation in the absence of any associated kinase.

AMP-Activated Protein Kinase Kinases↗

PubMed2005

Oxidation of carboxyamidomethyl cysteine may add complexity to protein identification.

Protein identification by interrogation of databases requires a comprehensive compilation of modified amino acids forms. Here, we describe the chemical oxidation of carboxyamidomethyl cysteine to the sulfoxide and sulfone forms, species that may add more complexity to peptide analyses. They can be easily distinguished by tandem mass spectrometry (MS/MS) due to their characteristic pattern of side chain neutral eliminations either from the parent ion or ion series that generate dehydroalanine as detected by MS(3). This finding was supported by the MS(n) spectra recorded for a peptide isolated from a mixture of tryptic peptides and for a derivatized/oxidized synthetic peptide with a different sequence. These modifications and their diagnostic neutral losses should be included in the list of chemical modifications and in algorithms designed for the automatic sequencing of peptides and database searching.

Acetamides↗

PubMed2005

Proteomic expression analysis of colorectal cancer by two-dimensional differential gel electrophoresis.

The identification of specific protein markers for colorectal cancer would provide the basis for early diagnosis and detection, as well as clues for understanding the molecular mechanisms governing cancer progression. In this report, we describe the proteomic analysis of the samples of colorectal cancer corresponding to seven patients. We have used the highly sensitive two-dimensional differential gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) for the identification of proteins differentially expressed in tumoral and neighboring normal mucosa. We have detected differences in abundance of 52 proteins with statistical variance of the tumor versus normal spot volume ratio within the 95th confidence level (Student's t-test; p < 0.05). Forty-one out of 52 analyzed proteins were unambiguously identified by matrix-assisted laser desorption/ionization-time of flight MS coupled with database interrogation as being differentially expressed in colorectal cancer. An ontology analysis of these proteins revealed that they were mainly involved in regulation of transcription (synovial sarcoma X5 protein, metastasis-associated protein 1), cellular reorganization and cytoskeleton (cytokeratins, vimentin, beta actin), cell communication and signal transduction (annexins IV and V, relaxin, APC), and protein synthesis and folding (heat shock protein 60, calreticulin, cathepsin D, RSP4) among others. Preliminary studies demonstrated that the differentially expressed proteins found by 2-D DIGE could be confirmed and validated by immunoblotting and immunohistochemistry analyses in those few cases where antibodies were available. We believe that the incorporation of more samples and new datasets will permit the definition of a collection of proteins with a potential interest as biomarkers for colorectal cancer.

Biopsy↗

PubMed2005

Efficacy of three ELISA measurements of anti-cyclic citrullinated peptide antibodies in the early diagnosis of rheumatoid arthritis.

The objective of the present study was to determine the efficacy of anti-cyclic citrullinated peptide (anti-CCP) antibody detection in the early diagnosis of rheumatoid arthritis (RA), as well as to compare three commercially available enzyme-linked immunosorbent assay (ELISA) kits used to detect such antibodies. We analysed the presence of anti-CCP antibodies in the sera of 78 patients who had been newly referred from primary healthcare centres to the Early Polyarthritis Unit. We also included in the study a group of 50 healthy controls. None of the patients had previously received treatment for the disease. After 1-year follow-up, the diagnosis of RA was confirmed in 53 of these patients. The ELISA kits under study were IMMUNOSCAN RA (Euro-Diagnostica AB), QUANTA Lite CCP IgG ELISA (INOVA Diagnostic) and DIA-STAT Anti-CCP (Axis-Shield Diagnostics); the sensitivity obtained was 52.8%, 58.5% and 52.8%, respectively, with 100% specificity for all three kits. Anti-CCP antibodies detected the presence of RA in 26% of patients without positive rheumatoid factor (RF). The sum of anti-CCP antibodies or the presence of RF gave a sensitivity of up to 67%, with specificity ranging between 94 and 97%. Anti-CCP antibodies show high specificity for the diagnosis of RA. The three ELISAs analysed offer the same degree of diagnostic accuracy.

Adolescent↗

PubMed2004

Proteomic analysis of plasma from patients during an acute coronary syndrome.

OBJECTIVES: The aim of this study was to analyze modifications in the plasma protein map during an acute coronary syndrome (ACS) using proteomics. BACKGROUND: Proteomics is a new technology that allows the detection and identification of several proteins at a given time in a sample. METHODS: Plasma from 19 patients, 11 with acute myocardial infarction (AMI) and 8 with unstable angina (UA), was investigated. The control group included nine age-matched volunteers. RESULTS: In two-dimensional electrophoresis using a pH range of 4 to 7, constant differences were found in at least four different areas within the plasma protein map. In area 1, we identified the presence of seven alpha(1)-antitrypsin (AAT) isoforms in plasma from control subjects. alpha(1)-antitrypsin isoform 1 was undetectable in plasma from UA and AMI patients. The AAT isoforms 5, 6, and 7 were reduced in plasma from AMI patients when compared with UA patients. Three fibrinogen gamma chain isoforms were identified in area 2. Fibrinogen gamma chain isoforms 1 and 2 were increased in AMI patients with respect to UA patients. Five apolipoprotein A-I isoforms were identified in area 3. All of them were reduced in plasma from AMI patients with respect to UA patients. In area 4, the gamma-immunoglobulin heavy chains were detected and were found increased in plasma from ACS patients. CONCLUSIONS: Plasma proteomic analysis makes it possible to develop a map of the protein isoforms that are expressed in plasma during an ACS.

Acute Disease↗

PubMed2004

Prosthetic valve endocarditis due to Listeria monocytogenes. Report of two cases and reviews.

INTRODUCTION: Endocarditis due to Listeria monocytogenes is a rare but serious disease often leading to valve dysfunction and heart failure. Two cases of listerial prosthetic valve endocarditis are reviewed along with 66 cases previously reported. RESULTS: The mean age of patients with listerial endocarditis increased from 47.1 years in the decades from 1955-1984 to 65.5 years from 1985-2000. Chronic debilitating diseases, solid tumours and immunosuppression associated with organ transplantation, hematologic neoplasia or AIDS were found in 41.1% of cases. Listerial endocarditis was a vegetative and destructive process, with dehiscense of the prosthesis and occasionally, abscess formation, fistulization and pericarditis. Treatment with penicillin or ampicillin alone or combined with gentamicin was adequate therapy in most cases. Vancomycin together with gentamicin may be a reasonable alternative therapy. CONCLUSIONS: Despite problems associated with microbial persistence and relapses in other forms of human listeriosis, antimicrobial therapy alone may be a successful treatment for listerial endocarditis, including cases occurring on prosthetic valves. Valve replacement may be reserved for complicated cases with valve dehiscense, cardiac failure or myocardial abscess. Overall mortality was 35.3%, although most patients who died did so before 1985 and since then mortality has been significantly reduced to 12%.

Aged↗

PubMed2003

[Intrahospital mortality after discharge from the ICU (hidden mortality) in patients who required mechanical ventilation].

BACKGROUND AND OBJECTIVE: Our goal was to determine the hidden mortality (HM) in patients who underwent an episode of mechanical ventilation (MV). We also analyzed the factors associated with an increase in the risk of hidden mortality. PATIENTS AND METHOD: Prospective cohort study. Patients admitted to an ICU who required MV and who were monitored until their discharge from hospital. We performed a multivariate study with a logistic regression model including all the variables that were present in a univariate analysis p < 0.20. RESULTS: Forty-one of the 215 patients who were discharged from the ICU died when they were admitted to hospital, which represents a hidden mortality rate of 19% (CI 95% 11%-27%). A mean period of 9 days elapsed between discharge from the ICU and patient's death, with 25% of patients dying within the first two days. Commonest cause of death was respiratory failure (37%). Factors independently associated with an increase in the risk of hidden mortality were (values expressed as adjusted odds ratio (CI 95%): age > 74 years 1.15 (1.01 to 1.26) (p = 0.02); APACHE II > 29 1.14 (1.01 to 1.27) (p = 0.04); reason for MV being coma 1.21 (1.07 to 1.37) (p = 0.002); reason for MV being cardiopulmonary arrest 1.28 (1.18 to 1.68) (p < 0.001); tracheotomy in ICU 1.31 (1.19 to 1.68) (p < 0.001) and stay in the ICU longer than 16 days 1.35 (1.01 to1.70) (p = 0.04). CONCLUSIONS: An important number of patients discharged from the ICU after an episode of MV die in hospital. Risk factors associated with an increased risk of death in hospital identify a group of patients who, after excluding those with non-cardiopulmonary resuscitation orders, would possibly benefit from high surveillance or intermediate care units.

APACHE↗