Biomedical subjects

B Alvarez

Publications and source records attributed to B Alvarez.

At least 19 recordsLinked to original sources

PubMed2006

Sulfenic acid in human serum albumin.

Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.

Free Radicals↗

PubMed2006

Phenotypic and functional characterization of porcine granulocyte developmental stages using two new markers.

Here, we describe two new surface antigens, named 6D10 and 2B2, whose expression is restricted to porcine granulocytes. 6D10 is only detected in neutrophils and its expression decreases from promyelocytes to mature cells. By contrast, 2B2 antigen is selectively expressed in mature neutrophils, eosinophils and basophils. The expression of these antigens along granulocyte maturation allows the discrimination of several developmental stages of granulocytes based on phenotypic, morphological and functional characteristics previously established. Moreover, these new markers are useful tools to easily characterize the different granulocytes lineages (neutrophils, eosinophils and basophils). By using multiparameter flow cytometric analysis, we have performed a phenotypic and functional characterization of the granulocyte subsets identified by the combination of 6D10 and 2B2 antigens.

Animals↗

PubMed2006

Influence of target organ lesion detection (assessment of microalbuminuria and echocardiogram) in cardiovascular risk stratification and treatment of untreated hypertensive patients.

European guidelines indicate the importance of the evaluation of global cardiovascular risk (CVR) to determine the management of the hypertensive patients (EH). However, in primary care, the diagnostic work-up (PCD) only includes the metabolic risk factors. The aim of this study was to assess the importance of microalbuminuria (MA) and echocardiogram (ECHO) in the process of risk stratification, and the number of patients to be treated with drugs at diagnosis. In total, 155 nontreated EH were included in the study. Blood pressure, a lipid profile and plasma glucose (LG) were determined after an overnight fast. MA was evaluated with dipstick MICRALTEST, and in those patients with two positive results, it was measured again in two 24-h urine samples and was considered positive (MA+) if the average was >30 mg/24 h. Left ventricular mass index was calculated and values>125 g/m2 were considered as LV hypertrophy (LVH+). When the patients were stratified according to PCD, 22 had to be treated with drugs. When MA, ECHO and both tests used together were added to the risk evaluation, the number of patients to be treated were 42, 51 and 64, respectively (P<0.001 vs PCD). It is mainly in patients who have moderate cardiovascular risk that risk changes, whereas risk hardly changes in those having low and very high risk. In conclusion, in EH with moderate risk, measurement of MA, due to its easy availability and low cost, seems to be a cost effective screening test to avoid the underestimation of the CVR.

Albuminuria↗

PubMed2006

A mutation in Flavobacterium psychrophilum tlpB inhibits gliding motility and induces biofilm formation.

Flavobacterium psychrophilum is a psychrotrophic, fish-pathogenic bacterium belonging to the Cytophaga-Flavobacterium-Bacteroides group. Tn4351-induced mutants deficient in gliding motility, growth on iron-depleted media, and extracellular proteolytic activity were isolated. Some of these mutants were affected in only one of these characteristics, whereas others had defects in two or more. FP523, a mutant deficient in all of these properties, was studied further. FP523 had a Tn4351 insertion in tlpB (thiol oxidoreductase-like protein gene), which encodes a 41.4-kDa protein whose sequence does not exhibit high levels of similar to the sequences of proteins having known functions. TlpB has two domains; the N-terminal domains has five transmembrane regions, whereas the C-terminal domains has the Cys-X-X-Cys motif and other conserved motifs characteristic of thiol:disulfide oxidoreductases. Quantitative analysis of the thiol groups of periplasmic proteins revealed that TlpB is required for reduction of these groups. The tlpB gene is part of the fpt (F. psychrophilum thiol oxidoreductase) operon that contains two other genes, tlpA and tpiA, which encode a thiol:disulfide oxidoreductase and a triosephosphate isomerase, respectively. FP523 exhibited enhanced biofilm formation and decreased virulence and cytotoxicity. Complementation with the tlpB loci restored the wild-type phenotype. Gliding motility and biofilm formation appear to be antagonistic properties, which are both affected by TlpB.

Amino Acid Sequence↗

PubMed2005

Differential expression of chemokine receptors and CD95 in porcine CD4(+) T cell subsets.

Among other differences, naïve and memory T cells show distinct migratory patterns and susceptibility to CD95-mediated cell death. We have recently characterised in the pig two subsets of CD4(+) T cells, based on the expression of the 2E3 marker, that display phenotypic and functional features of naïve (CD4(+)2E3(+)) and effector/memory (CD4(+)2E3(-)) T cells. In this study, we have analysed the expression of several chemokine receptors, as well as the distribution of CD95 antigen (APO-1/Fas) in these CD4(+) T cell subsets. CD4(+)2E3(-) T cells express high levels of CXCR3 and CCR4 transcripts but not of CCR7. On the contrary, CCR7 is clearly detected in CD4(+)2E3(+) T cells, whereas CXCR3 and CCR4 are negative or present at trace levels. These subsets also differ in the expression of CD95 antigen, being CD95 positive cells significantly more abundant in the CD4(+)2E3(-) cell subset. These findings, although based on a small number of animals, fit well with those reported for naïve and memory CD4(+) T cells in humans.

Animals↗

PubMed2005

The STRATEGY project: decision tools to aid sustainable restoration and long-term management of contaminated agricultural ecosystems.

The STRATEGY project (Sustainable Restoration and Long-Term Management of Contaminated Rural, Urban and Industrial Ecosystems) aimed to provide a holistic decision framework for the selection of optimal restoration strategies for the long-term sustainable management of contaminated areas in Western Europe. A critical evaluation was carried out of countermeasures and waste disposal options, from which compendia of state-of-the-art restoration methods were compiled. A decision support system capable of optimising spatially varying restoration strategies, that considered the level of averted dose, costs (including those of waste disposal) and environmental side effects was developed. Appropriate methods of estimating indirect costs associated with side effects and of communicating with stakeholders were identified. The importance of stakeholder consultation at a local level and of ensuring that any response is site and scenario specific were emphasised. A value matrix approach was suggested as a method of addressing social and ethical issues within the decision-making process, and was designed to be compatible with both the countermeasure compendia and the decision support system. The applicability and usefulness of STRATEGY outputs for food production systems in the medium to long term is assessed.

Agriculture↗

PubMed2004

Analysis of functional heterogeneity of porcine memory CD4+ T cells.

Previous studies have identified swine helper memory T cells as CD4+CD8alpha+SLADR+. We have recently described a new porcine surface antigen (2E3) selectively expressed on CD4+ T cells that allows to divide these cells into naive (2E3+) and effector/memory (2E3-). However, although the majority of CD4+2E3- cells are CD8alpha+SLADR+, a minor proportion do not express SLADR and/or CD8alpha. Here, we have analyzed the functional capacity of these CD4+2E3- subsets to proliferate to a recall antigen. Both SLADR- and CD8alpha- cells proliferated in response to lysozyme, but at lower levels compared to the whole population CD4+2E3-. Besides, after activation with PMA plus ionomycin, CD4+2E3-SLADR- T cells produced IFNgamma and TNFalpha, although they did also in lower proportion than the whole CD4+2E3- population. Most of the IFNgamma-TNFalpha+, IFNgamma+TNFalpha+, IFNgamma+TNFalpha- cells were CD8alpha+ and CD45RA-, while IFNgamma-TNFalpha- cells showed a less differentiate phenotype.

Animals↗

PubMed2004

2E3, a new marker that selectively identifies porcine CD4+ naive T cells.

We describe a novel antigen recognized by mAb 2E3 selectively expressed in the periphery by a subset of porcine CD4+ T cells. Both, CD4+CD8alpha- and CD4+CD8alphalow T cell subpopulations express this antigen. CD4+2E3+ T cells show phenotypical and functional characteristics of nai;ve cells. The majority of them are CD29low, CD45RAhigh, CD49dlow, CD11alow, CD18low, and SLA-II-. After mitogen activation CD4+2E3+ T cells express high levels of IL-2 mRNA, but only traces of IFN-gamma or IL-4 mRNA. Indeed a minor percentage of cells stained positive for IFN-gamma when assessed by flow cytometry. Moreover, CD4+2E3+ T cells did not proliferate in response to the recall antigen lysozyme, although they did efficiently to the mitogen ConA. By contrast, CD4+2E3- T cells show phenotypical and functional characteristics of primed cells. They express markers associated to a memory phenotype, respond to the recall antigen lysozyme, and produce high amounts of IFN-gamma and IL-4.

Animals↗

PubMed2004

Phosphoinositide 3-kinase and Forkhead, a switch for cell division.

Cell cycle progression is a tightly controlled process. To initiate cell division, mitogens trigger a number of early signals that promote the G(0)-G(1) transition by inducing cell growth and the activation of G(1) cyclins. Activation of cyclin E/cdk2 (cyclin-dependent kinase 2) at the end of G(1) is then required to trigger DNA synthesis (S phase entry). Among the early signals induced by mitogens, activation of PI3K (phosphoinositide 3-kinase) appears essential to induce cell cycle entry, as it regulates cell growth signalling pathways, which in turn determine the rate of cell cycle progression. Another mechanisms by which PI3K and its downstream effector protein kinase B regulate cell cycle entry is by inactivation of the FOXO (Forkhead Box, subgroup O) transcription factors, which induce expression of quiescence genes such as those encoding p27(kip), p130 and cyclin G2. PI3K/FOXO then work as a complementary switch: when PI3K is active, FOXO transcription factors are inactive. The switch is turned on and off at different phases of the cell cycle, thus regulating cell cycle progression.

Animals↗

PubMed2004

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.

Animals↗

PubMed2003

Peroxynitrite reactivity with amino acids and proteins.

Peroxynitrite, the product of the fast reaction between nitric oxide ((*)NO) and superoxide O(2)(*-) radicals, is an oxidizing and nitrating agent which is able to traverse biological membranes. The reaction of peroxynitrite with proteins occurs through three possible pathways. First, peroxynitrite reacts directly with cysteine, methionine and tryptophan residues. Second, peroxynitrite reacts fast with transition metal centers and selenium-containing amino acids. Third, secondary free radicals arising from peroxynitrite homolysis such as hydroxyl and nitrogen dioxide, and the carbonate radical formed in the presence of carbon dioxide, react with protein moieties too. Nitration of tyrosine residues is being recognized as a marker of the contribution of nitric oxide to oxidative damage. Peroxynitrite-dependent tyrosine nitration is likely to occur through the initial reaction of peroxynitrite with carbon dioxide or metal centers leading to secondary nitrating species. The preferential protein targets of peroxynitrite and the role of proteins in peroxynitrite detoxifying pathways are discussed.

Amino Acids↗

PubMed2003

Purification and properties of a new psychrophilic metalloprotease (Fpp2) in the fish pathogen Flavobacterium psychrophilum.

To go further into the characterization of the proteolysis exocellular system of the salmonid pathogen Flavobacterium psychrophilum, the purification and characterization of a novel protease designated Fpp2 (F. psychrophilum protease 2) was undertaken. A protease (Fpp2) hydrolyzing azocasein was purified. The Fpp2 can be defined as a metalloprotease, it had an estimated molecular mass of 62 kDa with calcium playing an important role in the thermostability of the enzyme. Proteolytic activity was optimal at pH 6.0-7.0 and 24 degrees C and activation energy for the hydrolysis of azocasein was determined to be 5.4 kcal mol(-1), being inactive at temperatures above 42 degrees C. All these results are characteristic of 'cold adapted enzymes'. Fpp2 proved to be a broad range hydrolytic enzyme because in optimal conditions it was able to hydrolyze matrix and muscular proteins. It can be concluded that the Fpp1, a previously characterized 55 kDa metalloprotease, and the Fpp2 protease were produced under different physiological conditions and were immunologically as well as biochemically different.

Calcium↗

PubMed2003

Analysis of 35 priority semivolatile compounds in water by stir bar sorptive extraction-thermal desorption-gas chromatography-mass spectrometry. I. Method optimisation.

A multiresidue method for the determination of 35 organic micropollutants (pesticides and polycyclic aromatic hydrocarbons) in water has been optimised using stir bar sorptive extraction (SBSE) and thermal desorption coupled to capillary gas chromatography-mass spectrometry (GC-MS). In the present work, the different parameters affecting the extraction of the analytes from the water samples to the PDMS-coated stir bars and optimisation of conditions affecting thermal desorption are investigated. The optimised conditions consist of a 100-ml water sample with 20% NaCl addition extracted with 20 mm length x 0.5 mm film thickness stir bars at 900 rpm during 14 h at ambient temperature. Desorption is carried out at 280 degrees C during 6 min under a helium flow of 75 ml/min in the splitless mode while maintaining a cryofocusing temperature of 20 degrees C in the programmed-temperature vaporisation (PTV) injector of the GC-MS system. Finally, the PTV injector is ramped to a temperature of 280 degrees C and the analytes are separated in the GC and detected by MS using full scan mode (m/z 60-400). Under the described conditions, the good repeatability, high analyte recoveries and robustness, make SBSE a powerful tool for routine quality control analysis of the selected semivolatile compounds in water samples.

Adsorption↗

PubMed2003

A new epitope on swine CD5 molecule detected by monoclonal antibody 5F12/9.

This paper describes the production and characterization of a monoclonal antibody (MAb), 5F12/9, that recognizes a new epitope on porcine CD5. Conformation of its CD5 specificity was obtained by means of sequential immunoprecipitation and Western blot experiments in combination with anti-CD5 MAb 1H6/8, whereas cross-blocking experiments with both MAbs showed that they reacted with different epitopes.

Animals↗

PubMed2002

Associations between empirical antimicrobial therapy at the hospital and mortality in patients with severe community-acquired pneumonia.

INTRODUCTION: The aim of the study was to examine different antibiotic choices and their relation to outcomes. METHODS: We reviewed patients with severe community-acquired pneumonia (SCAP) from two multicenter studies. Empirical antimicrobial regimens were classified as: macrolides alone (group M); macrolides plus betalactams (group MB); macrolides plus betalactam/betalactamase inhibitor (group MBI); every regimen including aminoglycosides (group A); non-pseudomonal third-generation cephalosporins alone (group C); another betalactam alone (first- and second-generation cephalosporins, or betalactam/betalactamase inhibitor) (group B); fluoroquinolones (group F); and other regimens (group Misc). RESULTS: Initial distribution of regimens was: group MB: 261 patients; group A: 65 patients; group C: 31 patients; group B: 23 patients; group M: 18 patients; group MBI: 13 patients; group F: 11 patients; group Misc: 38 patients. The lowest overall mortality was associated with initial treatment with a macrolide plus other agent (or alone). No deaths were documented among the 13 patients receiving amoxicillin/clavulanate plus a macrolide. The excess mortality for initial treatment with group A was significantly higher (14.2%; CI 95% 27.3-1.1) than the overall mortality rate between patients receiving a macrolide plus other agents. No significant differences were documented when mortality was adjusted for intubated patients. CONCLUSION: Clinicians select the empirical antibiotic regimen after classifying patients according to likely pathogens and prognosis. The inclusion of a macrolide as part of the initial therapeutic regimen for SCAP appears to be as safe and effective as alternative options. Addition of a macrolide agent to a betalactam/betalactamase inhibitor or using a macrolide alone was a marker for less severe disease.

Adolescent↗

PubMed2002

TNF-alpha is involved in activating DNA fragmentation in skeletal muscle.

Intraperitoneal administration of 100 microg kg(-1) (body weight) of tumour necrosis factor-alpha to rats for 8 consecutive days resulted in a significant decrease in protein content, which was concomitant with a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumour necrosis factor-alpha-treated animals as compared with the non-treated controls. Analysis of muscle DNA fragmentation clearly showed enhanced laddering in the skeletal muscle of tumour necrosis factor-alpha-treated animals, suggesting an apoptotic phenomenon. In a different set of experiments, mice bearing a cachexia-inducing tumour (the Lewis lung carcinoma) showed an increase in muscle DNA fragmentation (9.8-fold) as compared with their non-tumour-bearing control counterparts as previously described. When gene-deficient mice for tumour necrosis factor-alpha receptor protein I were inoculated with Lewis lung carcinoma, they were also affected by DNA fragmentation; however the increase was only 2.1-fold. These results suggest that tumour necrosis factor-alpha partly mediates DNA fragmentation during experimental cancer-associated cachexia.

Animals↗

PubMed2001

Forkhead transcription factors contribute to execution of the mitotic programme in mammals.

Cell cycle progression is a process that is tightly controlled by internal and external signals. Environmental cues, such as those provided by growth factors, activate early signals that promote cell cycle entry. Cells that have progressed past the restriction point become independent of growth factors, and cell cycle progression is then controlled endogenously. The phosphatidylinositol 3OH kinase (PI(3)K)/protein kinase B (PKB) pathway must be activated in G1 to inactivate forkhead transcription factors (FKH-TFs) and allow cell cycle entry. Here we show that subsequent attenuation of the PI(3)K/PKB pathway is required to allow transcriptional activation of FKH-TF in G2. FKH-TF activity in G2 controls mammalian cell cycle termination, as interference with FKH transcriptional activation by disrupting PI(3)K/PKB downregulation, or by expressing a transcriptionally inactive FKH mutant, induces cell accumulation in G2/M, defective cytokinesis, and delayed transition from M to G1 of the cell cycle. We demonstrate that FKH-TFs regulate expression of mitotic genes such as cyclin B and polo-like kinase (Plk). Our results support the important role of forkhead in the control of mammalian cell cycle completion, and suggest that efficient execution of the mitotic programme depends on downregulation of PI(3)K/PKB and consequent induction of FKH transcriptional activity.

3T3 Cells↗

PubMed2001

Nitration and inactivation of tyrosine hydroxylase by peroxynitrite.

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7659-7663). Recent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(423) to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) x 10(3) m(-1) s(-1) at pH 7.4 and 25 degrees C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.

Base Sequence↗