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Biomedical subjects

E Varela

Publications and source records attributed to E Varela.

At least 19 recordsLinked to original sources
PubMed2005

HUVECs from newborns with a strong family history of myocardial infarction overexpress adhesion molecules and react abnormally to stimulating agents.

Atherosclerosis is a complex disease involved in major fatal events such as myocardial infarction and stroke. It is the result of interactions between metabolic, dietetic and environmental risk factors acting on a genetic background that could result in endothelial susceptibility. Our aim was to determine the patterns of expression of adhesion molecules and whether phosphatidylserine is translocated to the cell surface of human umbilical vein endothelial cells (HUVECs) isolated from healthy newborns born to parents with a strong family history of myocardial infarction under TNF-alpha or oxLDL stimulated conditions. Compared to control HUVECs, experimental cords showed: (a) a four-fold increase in VCAM-1 expression under basal conditions, which showed no change after stimulation with the pro-atherogenic factors; (b) a two-fold increase in basal P-selectin expression that reached a 10-fold increase with any of the pro-atherogenic factors; (c) a basal ICAM-1 expression similar to P-selectin that was not modified by the pro-atherogenic molecules; (d) a similar PECAM-1 expression. Unexpectedly, phospathidylserine expression in experimental cord HUVECs was significantly increased (211 817 versus 3354 TFU) but was not associated to apoptotic death as the percentage of dead cells induced by TNF-alpha treatment was very low (0.55 versus 9.87% in control HUVECs). The latter result was corroborated by TUNEL staining. T cell adherence to HUVECs was highly up-regulated in the genetically predisposed samples. The analysis of nonpooled HUVECs, from newborns to family predisposed myocardial-infarction individuals, might represent a useful strategy to identify phenotypical and functional alterations, and hopefully, to take early preventive actions.

Case-Control Studies↗
PubMed2002

Characterisation of nanoparticulate systems by hydrodynamic chromatography.

Particle size and particle size distribution can have a fundamental effect on the physical properties of colloidal dispersions. For many systems the measurement of average particle size is not sufficient, the presence of different size populations will have a strong influence on properties and could be related to the production process. Hydrodynamic chromatography (HDC) provides a method for the separation of polymers in solution or particles in suspension based on their size. In a packed column, the separation takes place in the inter-particle channels and the elution order is from large to small, analogous to gel permeation chromatography. The dynamic range of packed column HDC is from molecular size up to particles of greater than 1 microm. New instrumentation which can be used to determine the particle size distribution of a range of colloidal dispersions by packed column HDC is described. Data to support accuracy and precision of average particle size determination is presented as well as a number of case studies to illustrate the applicability of the technique to samples with polydisperse or multi-modal particle size distributions.

Chromatography↗
PubMed2001

Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: purification and characterization of the recombinant enzyme.

Aryl-alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin biodegradation by some white-rot fungi. The enzyme catalyzes the extracellular oxidation of aromatic alcohols to the corresponding aldehydes. The electron acceptor is molecular oxygen yielding H(2)O(2) as the product. Herein we describe, for the first time, the expression of AAO from Pleurotus eryngii in the ascomycete Aspergillus nidulans. The activity of the recombinant enzyme in A. nidulans cultures is much higher than found in the extracellular fluid of P. eryngii. The recombinant enzyme showed the same molecular mass, pI and catalytic properties as that of the mature protein secreted by P. eryngii. The enzymic properties are also similar to those reported from other Pleurotus and Bjerkandera species.

Alcohol Oxidoreductases↗
PubMed2001

Determination of xanthine oxidoreductase forms: influence of reaction conditions.

Xanthine oxidoreductase (XOR) has been implicated in ischaemia-reperfusion injury, and increases in this enzyme have been found in plasma of patients with different illnesses. The catalytic concentrations of the XOR forms found in plasma, using various reaction conditions, greatly differ in the related literature. We studied the effect of the assay conditions on the xanthine oxidation rate catalysed by the XOR forms. Our results demonstrate inhibition of XOR by the reaction products and a time-dependent decrease in the reaction rates of XOR forms. Substrate consumption and inhibition by the products did not account for this decrease. Determination at 60 min incubation leads to catalytic concentrations up to 80% lower for the XOR forms than those obtained at 10 min. We conclude that elimination of the reaction products (NADH, H(2)O(2) and O(2)) from the reaction mixture, and short incubation times, are necessary for accurate measurement of the XOR activities.

Animals↗
PubMed2000

Southern blot screening for lignin peroxidase and aryl-alcohol oxidase genes in 30 fungal species.

Screening to detect genes encoding lignin peroxidase (LiP) and aryl-alcohol oxidase (AAO) has been carried out with 30 fungal strain using DNA probes from genes lpo of Phanerochaete chrysosporium (encoding LiP isoenzyme H8) and aao of Pleurotus eryngii. Evidence for the presence of genes closely related to lpo was found in Bjerkandera adusta, Fomes fomentarius, Ganoderma applanatum, Ganoderma australe, Lentinula degener, Peniophora gigantea, P. chrysosporium, Phanerochaete flavido-alba and Trametes tersicolor, whereas the gene aao was detected in Pleurotus species and B. adusta. The presence of both genes was only detected in B. adusta. These results suggest that different enzymatic system, formed by enzymes encoded by different genes, are responsible for lignin degradation by white-rot fungi.

Alcohol Oxidoreductases↗
PubMed2000

Aryl-alcohol oxidase protein sequence: a comparison with glucose oxidase and other FAD oxidoreductases.

Aryl-alcohol oxidase (AAO), an FAD-dependent enzyme involved in lignin degradation, has been cloned from Pleurotus eryngii. The AAO protein is composed of 593 amino acids, 27 of which form a signal peptide. It shows 33% sequence identity with glucose oxidase from Aspergillus niger and lower homology with other oxidoreductases. The predicted secondary structures of both enzymes are very similar. For AAO, it is predicted to contain 13 putative alpha-helices and two major beta-sheets, each of the putative beta-sheets formed by six beta-strands. The ADP binding site and the signature-2 consensus sequence of the glucose-methanol-choline (GMC) oxidoreductases were also present. Moreover, residues potentially involved in catalysis and substrate binding were identified in the vicinity of the flavin ring. They include two histidines (H502 and H546) and several aromatic residues (Y78, Y92 and F501), as reported in other FAD oxidoreductases.

Alcohol Oxidoreductases↗
PubMed2000

Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius.

Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulmonarius has been purified and characterized. The enzyme was produced in glucose-peptone medium and isolated in a sole chromatographic step using Sephacryl S-200. The purified enzyme is an extracellular glycoprotein with 14% N-carbohydrate content and an estimated molecular mass of 70.5 kDa and pI of 3.95. The kinetic studies showed the highest enzyme affinity against p-anisyl alcohol, with constants similar to those of Pleurotus eryngii and Bjerkandera adusta AAO but different from the intracellular AAO described in Phanerochaete chrysosporium, which present the highest activity on m-anisyl alcohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and sequenced. The translation of this sequence consisted of 593 amino acids including a signal peptide of 27 amino acids. The comparison with other alcohol oxidases, 35% amino acid identity with glucose oxidase, showed highly conserved amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried out for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of its catalytic site and general structure.

Alcohol Oxidoreductases↗
PubMed2000

Diagnostic accuracy of pleural fluid polymorphonuclear elastase in the differentiation between pyogenic bacterial infectious and non-infectious pleural effusions.

BACKGROUND AND OBJECTIVES: To establish the diagnostic accuracy of the markers of neutrophil activity (elastase and lysozyme) determined in pleural fluid, for differentiating between pyogenic bacterial infectious and non-infectious pleural effusions. PATIENTS AND METHODS: At our tertiary referral teaching hospital, 160 patients over 14 years with pleural effusion (PE), classified as pyogenic bacterial infectious (41 parapneumonic complicated, 32 parapneumonic non-complicated) and non-infectious (32 neoplasm and 55 undiagnosed pleural exudates) were examined in a prospective study. Polymorphonuclear elastase (PMN-E) was determined by an immunoactivation method and lysozyme by a turbidimetric method. Receiver operating characteristic (ROC) curves were used to evaluate diagnostic accuracy. RESULTS: Pleural fluid PMN-E was the biochemical marker that best differentiated between pyogenic bacterial infectious and non-infectious PE. The ROC area under the curve (AUC) for PMN-E was 0.8276. A PMN-E value over 230 microg/l diagnosed infectious PE with a specificity of 0.81 and a sensitivity of 0.74. The ROC AUC for proteins plus lactate dehydrogenase was 0. 7430. Differences between the two ROC curves were significant (p = 0. 032). After excluding purulent parapneumonic complicated PE, the sensitivity of a pleural fluid PMN-E value equal to or greater than 230 microg/l was 0.64 and the specificity 0.81. CONCLUSIONS: Pleural fluid PMN-E was the marker that best differentiated infectious from non-infectious PE, and PMN-E values lower than 230 microg/l suggest non-infectious PE.

Adolescent↗
PubMed1999

Molecular cloning of aryl-alcohol oxidase from the fungus Pleurotus eryngii, an enzyme involved in lignin degradation.

Aryl-alcohol oxidase (AAO), an extracellular enzyme characteristic of fungi from the genus Pleurotus, constitutes a source for H2O2 required in lignin biodegradation. The gene aao has been cloned, sequenced and characterized for the first time in Pleurotus eryngii. Both cDNA and genomic libraries were screened with probes obtained by PCR using as primers oligonucleotides corresponding to the N-terminus and internal sequences of AAO. DNA sequences from positive clones showed a unique open reading frame of 1779 nucleotides interrupted by 12 introns. The conceptual translation of the protein agrees with the partial amino acid sequences obtained from protein sequencing. A search for proteins with related amino-acid sequences revealed that glucose oxidase from Aspergillus niger has 33% identity and 51% similarity. A comparison with other oxidoreductases showed common motifs in both N- and C-terminal regions corresponding, respectively, to the FAD-binding region and the enzyme active site. However, AAO probably has structural differences with other oxidases, as deduced from its unique ability to generate H2O2 from the oxidation of aromatic alcohols.

Alcohol Oxidoreductases↗
PubMed1999

Secretion of IFN-gamma by bovine peripheral blood mononuclear cells stimulated with Mycobacterium bovis protein fractions obtained by isoelectric-focusing.

Due to the complexity and variety of biological effects found in Mycobacterium bovis (M. bovis) proteins analyzed solely on a molecular weight (MW) basis, we approached the purification of M. bovis proteins through their isoelectric point (pI). Twenty M. bovis culture filtrate protein extract (CFPE) isoelectric focused (IEF) protein fractions, confined between pI3 and 10, were isolated. The MW of the major proteins isolated in the various fractions correlated with protein already reported 14-, 18-, 20-, 25-, 31-, 38-, 45-, 64-, 67- and 70 kDa by SDS-PAGE. Since several different pI fractions showed proteins of the same MW we tested the ability of all IEF fractions to stimulate interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) isolated from cattle with well defined M. bovis tuberculosis (TB) infection. In animals with few lesions IFN-gamma inductive IEF fractions were in the acid range. As the number of lesions increased, neutral fractions were also inductive. Some fractions with relatively few proteins induced as much IFN-gamma production as others with abundant proteins. None of the 20 IEF fractions enhanced IFN-gamma production by anergic cells. We conclude that IFN-gamma production in diseased animals is induced mainly by acidic mycobacterial proteins and that the response towards these proteins is enhanced as the disease progresses, what coincides with higher PPD reactivity. However, the IFN-gamma production in anergic status was severely affected. We found that this cytokine production is spontaneous and antigen-independent.

Animals↗
PubMed1999

Interleukin-6, nitric oxide, and the clinical and hemodynamic alterations of patients with liver cirrhosis.

OBJECTIVE: Nitric oxide has been proposed as a mediator of hyperdynamic circulation in cirrhosis. Endotoxin and cytokines induce the synthesis of nitric oxide. The aim of this study was to investigate the relationship between endotoxemia, cytokines, and nitric oxide in patients with cirrhosis, and to correlate these findings with clinical, biochemical, and hemodynamic parameters. METHODS: Clinical, biochemical, and hemodynamic parameters were assessed in 66 patients with cirrhosis and 15 controls. Levels of antidiuretic hormone, plasma renin activity, aldosterone, interferon gamma, interleukin-1, interleukin-6, tumor necrosis factor alpha, endotoxin, and nitrates-nitrites were determined. RESULTS: Mean arterial pressure was lower and interleukin-6, tumor necrosis factor alpha, nitrites-nitrates levels, and endotoxin positivity rates were higher in cirrhotics than in controls (p < 0.005). Mean arterial pressure decreased and interleukin-6 levels increased with worsening of Child score (p < 0.005). Patients with ascites had higher levels of interleukin-6, tumor necrosis factor alpha, and nitrates-nitrites than patients without ascites (p < 0.01). Elevated levels of interleukin-6 were found in patients with encephalopathy grade I, compared with patients without (p < 0.001); this association was independent of the severity of liver disease. In patients with low mean arterial pressure, interleukin-6 levels were higher than in patients with high mean arterial pressure (p = 0.001), whereas tumor necrosis factor alpha and nitrates-nitrites levels were not different. By multivariate analysis, high interleukin-6 levels showed independent associations with the presence of ascites, encephalopathy, and low mean arterial pressure. Only interleukin-6 levels had significant correlations with Child score, plasma renin activity, serum and urinary sodium, and mean arterial pressure (r > or = 0.4, p < 0.005). CONCLUSIONS: Although the activity of the nitric oxide pathway is increased in patients with cirrhosis and might contribute to the hemodynamic alteration, other factors are involved. Interleukin-6, possibly through nitric oxide-independent mechanisms, also might play a role in the vasodilatation of cirrhosis and the pathogenesis of hepatic encephalopathy.

Aged↗
PubMed1999

Collagen degrading activity associated with Mycobacterium species.

BACKGROUND: The mechanism of Mycobacterium tuberculosis penetration into tissues is poorly understood but it is reasonable to assume that there is a contribution from proteases capable of disrupting the extracellular matrix of the pulmonary epithelium and the blood vessels. A study was undertaken to identify and characterise collagen degrading activity of M tuberculosis. METHODS: Culture filtrate protein extract (CFPE) was obtained from reference mycobacterial strains and mycobacteria isolated from patients with tuberculosis. The collagen degrading activity of CFPE was determined according to the method of Johnson-Wint using 3H-type I collagen. The enzyme was identified by the Birkedal-Hansen and Taylor method and its molecular mass determined by SDS-PAGE and Sephacryl S-300 gel filtration chromatography using an electroelution purified enzyme. RESULTS: CFPE from Mycobacterium tuberculosis strain H37Rv showed collagenolytic activity that was four times higher than that of the avirulent strain H37Ra. The 75 kDa enzyme responsible was divalent cation dependent. Other mycobacterial species and those isolated from patients with tuberculosis also had collagen degrading activity. CONCLUSIONS: Mycobacterium species possess a metalloprotease with collagen degrading activity. The highest enzymatic activity was found in the virulent reference strain H37Rv.

Bacteriological Techniques↗
PubMed1998

Stomatococcus mucilaginosus septicemia in a patient with acute lymphoblastic leukaemia.

A 16-year-old patient with acute lymphoblastic leukaemia which had relapsed for the third time developed clinical signs and symptoms of septicemia during a period of neutropenia. The patient had signs of oral mucositis, and Stomatococcus mucilaginosus was isolated from blood cultures. The patient responded well to antibiotic therapy. The biochemical characteristics and antimicrobial susceptibility patterns of 68 other pharyngeal isolates of Stomatococcus mucilaginosus from immunocompromised patients are presented.

Adolescent↗
PubMed1998

Identification of major glycoconjugates from Mycobacterium bovis culture filtrate by biotin-hydrazide labeling.

To identify Mycobacterium bovis glycoproteins, carbohydrates present in a delipidized M. bovis culture filtrate protein extract were biotin-hydrazide labeled. 11 carbohydrate-containing protein with a molecular weight of 15-, 19-, 25-, 32-, 35-, 39-, 42-, 48-, 52-, 58-, and 62-kDa were detected. The 52- and 32-kDa protein were deglycosylated by endoglycosidase-F.

Bacterial Proteins↗
PubMed1998

Interleukin-8 and markers of neutrophil degranulation in pleural effusions.

In order to know the degree of interleukin-8 (IL-8) production in the pleural space and its relationship to neutrophil activation, IL-8, neutrophil elastase (NE), and myeloperoxidase (MPO) were assessed in blood and pleural fluid (PF) of 219 patients with pleural effusions. Correlations between blood and PF IL-8, NE, and MPO were either absent or weak, except for IL-8 in transudates (r = 0.6745, p < 0.001). PF IL-8, NE, and MPO concentrations in cases of empyema were higher than in cases of effusion of other causes (p < 0.001). No significant differences in inflammatory markers were observed between parapneumonic and tuberculous fluids. IL-8, NE, and MPO levels in malignant, nonspecific, and transudative effusions were lower than in those due to infection, the lowest levels corresponding to transudates. No significant correlation was observed between PF IL-8 and neutrophil count in any group; in contrast, IL-8 was associated with NE and MPO in empyema (r = 0.7545, and r = 0.7283; p < 0.001), tuberculosis (r = 0.4016, p = 0.008 and r = 0.6545, p < 0.001), and nonspecific effusions (r = 0.3748, p = 0.007 and r = 0.3085, p = 0.028). Our results indicate that local production of markers of the nonspecific inflammatory response is high in both chronic and acute pleural infection, and suggest a role for IL-8 in the release of NE and MPO.

Adolescent↗