Biomedical subjects

M Villalba

Publications and source records attributed to M Villalba.

At least 73 records · Page 4Linked to original sources

PubMed1996

Cross-reactivity between the major allergen from olive pollen and unrelated glycoproteins: evidence of an epitope in the glycan moiety of the allergen.

Ole e 1, the major allergen from olive pollen, is a glycoprotein containing a single Asn-linked glycan moiety. Rabbit antiserum against this protein has been obtained; and its immunologic cross-reactivities in Western blotting with ascorbate oxidase, horseradish peroxidase, bromelain, ovalbumin, and honeybee venom phospholipase A2 have been studied. Ascorbate oxidase, peroxidase, and bromelain are recognized by the Ole e 1 antiserum. When these three proteins are deglycosylated by periodate treatment, such an immunologic reaction does not occur. The relative affinities of these proteins have been analyzed by direct and inhibition ELISA experiments. A commercially available antibody against horseradish peroxidase has also been considered in these studies. This antibody reacts with Ole e 1 but not with the periodate-deglycosylated allergen. Horseradish peroxidase, bromelain, and ascorbate oxidase are recognized by the IgE of sera from patients who are hypersensitive to olive tree pollen. This binding is also abolished by periodate treatment. The results are interpreted in terms of the presence of an epitope in the carbohydrate moiety of Ole e 1, which would contain a xylose involved in recognition by both IgE and IgG antibodies.

Allergens↗

PubMed1996

Modulation of presynaptic calcium homeostasis by nitric oxide.

In the present work, we have adapted established microfluorimetric techniques based on the calcium indicator Fura-2, for the study of synaptosomal calcium homeostasis regulation in an immobilized synaptosomal preparation from the rat hippocampus. With this tool, we have addressed the actions of two proposed interneuronal messengers, nitric oxide (NO) and arachidonic acid (AA). NO donors (sodium nitroprusside, SNP and hydroxylamine, HX) and AA induced an increase in depolarization-induced calcium transients (both in magnitude and duration). However, resting calcium levels were not modified by NO, whereas AA application resulted in an steady increase in Ca. The effects of SNP were blocked when EGTA was present between depolarizations, suggesting that a minimum level of internal calcium load is required for NO effects. The effects of NO on Cai transients are persistent up to 90 min after drug application, and could be involved in some of the forms of synaptic plasticity where NO plays a role.

Animals↗

PubMed1996

Spermatic cord hematoma: case report and literature review.

Spermatic cord hematoma is a rare diagnosis. The etiology may be idiopathic, traumatic, secondary to anticoagulation therapy, or as an extension of a retroperitoneal hemorrhage. It has been misdiagnosed as an incarcerated inguinal hernia, a testicular torsion, or a tumor. A review of the English literature is presented. An additional case is presented here as a complication of anticoagulation therapy after aortic valve replacement. Risk factors for a spermatic cord hematoma may warrant an ultrasound examination.

Anticoagulants↗

PubMed1996

Isolation, cDNA cloning and expression of Lig v 1, the major allergen from privet pollen.

BACKGROUND: An olive allergen-like protein has been detected in privet pollen. This protein could be involved in the allergenic cross-reactivity described for privet and olive tree pollen extracts. OBJECTIVE: Isolation and characterization of natural Lig v 1. Cloning and expression of its cDNA in order to assess its structural similarity with the olive allergen. METHODS: Current chromatographic methods were used to isolate the privet counterpart of Ole e 1. A pool of sera from subjects allergic to olive tree pollen was used to immunodetect the protein in the elution profiles. Ole e 1-specific polyclonal antibody and allergic sera were used in immunoblotting assays of the isolated protein. Polymerase chain reaction amplification of the first strand cDNA synthesized from the privet pollen total RNA was carried out to prepare a full-length fragment encoding Lig v 1. After nucleotide sequencing, expression of one clone was performed in Escherichia coli, under the form of a fusion protein with glutathione S-transferase. The IgE binding capability of the recombinant protein was also analysed. RESULTS: The major allergen from privet pollen. Lig v 1, was purified to homogeneity by two gel filtration chromatographies and one reverse-phase high-performance liquid chromatography. Its amino acid composition and N-terminal amino acid sequence were determined. Two different clones encoding Lig v 1 were sequenced. Strong sequence similarity between Lig v 1 and Ole e 1 was observed, the identity being 85 and 96%. One of the sequenced clones was expressed and the recombinant product exhibited IgG and IgE binding activities against both anti-Ole e 1 polyclonal antibodies and olive-allergic sera. CONCLUSION: Privet pollen contains a protein structurally and immunologically related to the major allergen of olive pollen. The similarity exhibited by these proteins could explain the cross-reactivity observed between the two pollen extracts. Since these allergens are highly polymorphic, the expression of an immunologically active recombinant Lig v 1 will permit the preparation of well defined molecules for both research and clinical purposes.

Allergens↗

PubMed1995

Ionic regulation of endonuclease activity in PC12 cells.

We have investigated the Ca2+ dependency of DNA degradation into nucleosome-sized fragments in intact chromaffin-like PC12 cells and PC12 nuclear fractions. In intact cells we were unable to trigger DNA fragmentation by inducing either transient or sustained elevations of cytoplasmic Ca2+ ([Ca2+]i) with the Ca2+ ionophore ionomycin. On the contrary, DNA fragmentation was induced in intact cells by the intracellular Zn2+ chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). To characterize further PC12 cell endonuclease activity, we then investigated digestion by purified PC12 cell fractions of exogenously added plasmids. In nuclear fractions two endonuclease activities were identified: an acidic (pH 5.0) endonuclease activity that was fully Ca2+- and Mg(2+)-independent; and a neutral (pH 7.6) endonuclease activity that was Ca(2+)-independent but Mg(2+)-dependent. Both endonuclease activities were inhibited by Zn2+. Nuclear membrane permeabilization greatly enhanced plasmid digestion at pH 7.6, but not at pH 5.0. This suggests that neutral endonuclease was located in a membrane-bound compartment, whereas acidic endonuclease was freely accessible to the substrate even in the presence of an intact nuclear membrane. In intact nuclei, digestion of genomic DNA could not be triggered by increasing the bivalent cation composition of the medium. On the contrary, in hypotonic medium we observed a large spontaneous nucleolytic DNA degradation that was increased by Zn2+ chelation. However, an acidic pH shift was a potent stimulus for DNA fragmentation in isotonic as well as hypotonic medium.

Animals↗

PubMed1995

Activation of microglial cells by beta-amyloid protein and interferon-gamma.

Alzheimer's disease is the most common cause of progressive intellectual failure. The lesions that develop, called senile plaques, are extracellular deposits principally composed of insoluble aggregates of beta-amyloid protein (A beta), infiltrated by reactive microglia and astrocytes. Although A beta, and a portion of it, the fragment 25-35 (A beta (25-35)), have been shown to exert a direct toxic effect on neurons, the role of microglia in such neuronal injury remains unclear. Here we report a synergistic effect between A beta and interferon-gamma (IFN-gamma) in triggering the production of reactive nitrogen intermediates and tumour-necrosis factor-alpha (TNF-alpha) from microglia. Furthermore, using co-culture experiments, we show that activation of microglia with IFN-gamma and A beta leads to neuronal cell injury in vitro. These findings suggest that A beta and IFN-gamma activate microglia to produce reactive nitrogen intermediates and TNF-alpha, and this may have a role in the pathogenesis of neuronal degeneration observed in ageing and Alzheimer's disease.

Aging↗

PubMed1995

Colorectal complications following cardiac surgery. Six-year experience.

PURPOSE: This study was undertaken to assess colorectal complications following cardiopulmonary bypass surgery. METHOD: This is a retrospective review of 5,801 patients who underwent 5,801 cardiopulmonary bypass procedures from 1985 to 1991. Patients were evaluated for type of bypass procedure, postoperative colorectal complications, age, sex, bypass time, aortic cross-clamp time, elective vs. emergency cases, uses of intra-aortic balloon pump, perioperative hypotension, and use of vasopressors. Statistical analysis was performed using chi-squared analysis and Student's t-test. RESULTS: Nineteen of the 5,801 patients developed 19 colorectal complications, a prevalence of 0.3 percent for the initial hospital stay following bypass surgery. Mortality in those with complications was 37 percent (7/19). Of the 19 complications, 9 (47 percent) followed coronary artery bypass grafting, whereas 10 (53 percent) followed valve replacement or combined coronary artery bypass grafting with other cardiac procedures. Five (26 percent) of the complications followed emergency cases, whereas 14 (74 percent) followed elective cases. Average age of those with complications was 69.8 years, compared with 63.2 years for those without complications. Average aortic cross-clamp time for those with complications was 71 +/- 25 minutes; pump time was 106 +/- 34 minutes. That was significantly higher than in those without complications. Nine (47 percent) patients with complications required vasopressors during the perioperative period, whereas eight (42 percent) suffered prolonged hypotension (systolic blood pressure, < 90 mmHg). CONCLUSIONS: It appears that increased age, valve replacement, or combined cardiac procedures, emergency procedures, and prolonged aortic cross-clamp and bypass pump times are risk factors for development of colorectal complications. Hypoperfusion, as suggested by prolonged pump times, clamp times, and emergency procedures may be a possible cause for development of colorectal complications.

Aged↗

PubMed1994

Food mustard allergen interaction with phospholipid vesicles.

Sin a I, the major allergen from mustard seeds, interacts with acid phospholipid vesicles. The protein binds to dimyristoylglycerophosphoglycerol vesicles with an apparent dissociation constant of approximately 2.4 microM, the number of phospholipid molecules affected by one protein molecule being approximately 20. Sin a I promotes an increase in the light scattering of a vesicle suspension. This process becomes saturated at approximately a lipid/protein molar ratio of 20:1. Sin a I also modifies the thermotropic behaviour of the negatively charged vesicles, which has been studied by measuring the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene incorporated into the hydrophobic core of the bilayer. Sin a I also promotes lipid mixing between vesicles. This mixing has been analyzed by measuring the variation of the fluorescence energy transfer between N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylglycerophosphoe thanolamine (donor) and N-(lissamine rhodamine B sulphonyl)-PtdEtn (acceptor) incorporated into dimyristoylglycerophosphoglycerol vesicles. This effect is also corroborated by observing a single thermotropic transition in a mixture of independent dipalmitoylglycerophosphoglycerol and dimyristoylglycerophosphoglycerol vesicles when Sin a I is added to the lipid suspension. The allergen promotes release of aqueous contents of PtdGro vesicles, as determined by an aminonaphthalenetrisulfonic acid/p-xylylenebis(pyridinium)bromide dequenching assay. This study shows that the allergen Sin a I is able to interact with membrane lipids. This interaction is discussed in terms of its potential involvement in the allergenicity of this protein.

Allergens↗

PubMed1994

Cloning and expression of Ole e I, the major allergen from olive tree pollen. Polymorphism analysis and tissue specificity.

Ole e I, the major allergen from the olive tree (Olea europaea), is one of the main causes of allergy in Mediterranean countries and some areas of North America. The cloning and sequencing of several cDNAs coding for the olive allergen have been achieved. cDNA has been synthesized from total pollen RNA and amplified by using the polymerase chain reaction. The nucleotide sequence data demonstrate the existence of microheterogeneities in at least 37 positions out of the 145 amino acids of Ole e I, thus explaining the high degree of polymorphism exhibited by the natural protein. One of the sequenced cDNAs encoding a full-length isoform was inserted into the plasmid vector pGEX-2T and overexpressed. The recombinant Ole e I has been produced in Escherichia coli as a fusion protein with glutathione S-transferase of Schistosoma japonicum. This chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B column and digested with thrombin to release the recombinant allergen. Both the fusion protein and the recombinant Ole e I were recognized in Western blot analysis by rabbit polyclonal and mouse monoclonal antisera raised against native Ole e I as well as by the IgE of olive pollen-sensitive human sera. This indicates that the recombinant production of individual isoforms may be useful for the improvement of reagents to be used in diagnosis and therapy of IgE-mediated disorders. In addition, Ole e I mRNA has been observed to be pollen-specific as shown in a Northern blot analysis.

Allergens↗

PubMed1994

Isolation and characterization of an olive allergen-like protein from lilac pollen. Sequence analysis of three cDNA encoding protein isoforms.

An olive allergen-like protein has been isolated from lilac (Syringa vulgaris) pollen extract. The protein can be considered as an allergen since is recognized by IgE from olive hypersensitive human sera, and has been called Syr v I (IUIS nomenclature). This protein consists of a glycosylated polypeptide of 20 kDa, which has an amino acid composition, spectroscopic properties, and an N-terminal sequence similar to the major allergen from olive pollen, Ole e I. The lilac allergen is recognized by rabbit polyclonal antisera raised against olive allergen as well as by an Ole e I-specific monoclonal antibody. Using a polymerase chain reaction strategy, based on the similarities observed between these olive and lilac proteins, three cDNA clones encoding Syr v I have been isolated and sequenced. These clones code for a polymorphic protein of 145 residues with a derived molecular mass of about 16,400Da, which contains a potential N-glycosylation site. Comparison of the deduced amino acid sequences of these Syr v I isoforms to each other revealed identities of 90-97%. Moreover, these sequences showed a high degree of similarity (85.5-89.6% identity) with Ole e I. The structural and immunological characterization of Syr v I justify the cross-reactions observed between olive and lilac pollen extracts. The molecular cloning of Syr v I is relevant for the epitope mapping in Oleaceae allergens, and may contribute to an improvement in the design of reagents for diagnosis and therapy of IgE-dependent allergic reactions.

Allergens↗

PubMed1994

The role of pyruvate in neuronal calcium homeostasis. Effects on intracellular calcium pools.

It has long been known that pyruvate is essential for survival of prenatal neurons in culture. To understand the role of exogenous pyruvate in neuronal calcium homeostasis, we have investigated the effects of pyruvate (plus malate) addition to dissociated adult rat hippocampal and cerebral cortex cells and cultured CNS neurons having an unrestricted glucose supply. We found that pyruvate (plus malate) increased the respiration rate while ATP levels were unchanged. At the same time, cytosolic free calcium concentrations, [Ca2+]i, decreased while total 45Ca2+ and 40Ca2+ accumulation increased. The extra Ca2+ accumulated by the cells is attributable to an increase in the size of the intracellular calcium pools. Two such pools were identified on the basis of their sensitivity to specific drugs. The first pool was mobilized by thapsigargin plus tert-butyl hydroquinone and caffeine while the second pool was discharged by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxphenylhydrazone (FCCP) (plus oligomycin). The two pools represented about 15-20% and 15-30%, respectively, of the rapidly exchangeable 45Ca2+ pools in cerebral cortex cells. In cultured hippocampal neurons, the collapse of the mitochondrial membrane potential (as induced by uncouplers (FCCP) or respiratory chain inhibitors (antimycin) caused a large increase in [Ca2+]i which varied in size and shape among cells and was reduced by external Ca2+ chelation. The latter condition also resulted in a partial discharge of FCCP-releasable 45Ca2+. The effects of FCCP did not result simply from ATP depletion since incubation in glucose-free medium and sequential additions of 2 mM deoxyglucose and 10 microM oligomycin, conditions that led to a dramatic reduction in cellular ATP levels, did not abolish the FCCP-induced [Ca2+]i rise. Taken together, the results indicate that mitochondria harbor a significant proportion of cellular Ca2+. The sensitivity of the mitochondrial pool size to pyruvate (plus malate) questions previous hypotheses concerning a kinetic limitation for Ca2+ accumulation in mitochondria in resting neurons.

Acetylcholine↗

PubMed1994

The activity of synaptosomal calcium channels is inversely correlated with working memory performance in memory impaired, aged rats.

Aged, memory-impaired rats do not learn an 8-arm radial maze task but differ in their performance along testing. The aim of this study was to determine whether any of the systems that govern calcium homeostasis in synaptosomes may be related to that difference in performance. A negative correlation between initial (5 s) K(+)-stimulated 45Ca2+ uptake and the behavioral scores from the last testing sessions was obtained K(+)-stimulated 45Ca2+ uptake showed also a negative correlation with an improvement score that evaluates the progress made by the rat along testing. The results support the notion that calcium inflow through synaptosomal voltage gated calcium channels in old rats is inversely correlated with their behavior. This may explain the beneficial effects of organic calcium channel blockers on behavioral performance in aged animals.

Aging↗

PubMed1994

Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety.

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.

Allergens↗

PubMed1994

Ole e I: epitope mapping, cross-reactivity with other Oleaceae pollens and ultrastructural localization.

Ole e I is the major allergen derived from olive tree pollen (Olea europaea) and it is composed of two polypeptides with molecular weights (MWs) of 18 and 20 kD. A panel of six monoclonal antibodies (mAbs) has been prepared and used to map antigenic determinants on this molecule. Four epitope determinants have been identified on Ole e I. Using the purified mAbs produced against Ole e I, we have analyzed the common epitope determinants in olive (O. europaea) and different Oleaceae pollens: ash (Fraxinus excelsior); privet (Ligustrum vulgare); lilac (Syringa vulgaris), and forsythia (Forsythia suspensa). ELISA showed three reactivity groups depending on the recognition of monoclonal antibodies: (1) olive and ash; (2) olive, ash, privet and lilac; and (3) olive, ash, privet, lilac and forsythia. Immunoblotting studies on Oleaceae pollen extracts with these mAbs showed a very similar cross-reactivity pattern. The 18- and 20-kD MW proteins were present in each pollen, except in the case of forsythia. In this case the reactivity pattern was associated with 50- to 55-kD protein bands. This band was recognized by a pool of sera from olive-allergic patients. Finally, ultrastructural localization of Ole e I antigen was performed on the mature olive pollen grain. Ole e I was located in association with dilated endoplasmic reticulum cisternae. Pollen grain walls, nuclei and cytoplasmic organelles were totally devoid of the allergen.

Allergens↗

PubMed1993

The amino acid sequence of Ole e I, the major allergen from olive tree (Olea europaea) pollen.

The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which have been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.

Allergens↗

PubMed1993

Characterization of a new oriental-mustard (Brassica juncea) allergen, Bra j IE: detection of an allergenic epitope.

Bra j IE, a major allergen from oriental-mustard (Brassica juncea) seeds, has been isolated and characterized. Its primary structure has been elucidated. This protein is composed of two chains (37 and 92 amino acids) linked by disulphide bridges. The amino acid sequence obtained is closely related to that previously determined for Sin a I, an allergen isolated from yellow mustard (Sinapis alba). A common epitope has been detected in the large chain of both Bra j IE and Sin a I by means of electroblotting and immunodetection with 2B3, which is a monoclonal antibody raised against the yellow-mustard allergen. A histidine residue of the large chain of both mustard allergens has been found to be essential for the recognition by 2B3 antibody. A synthetic multiantigenic peptide containing this His was recognized by 2B3 as well as by sera of mustard-hypersensitive individuals. Therefore this antigenic determinant must be involved in the allergenicity of these proteins.

2S Albumins, Plant↗

PubMed1993

Sequence analysis and antigenic cross-reactivity of a venom allergen, antigen 5, from hornets, wasps, and yellow jackets.

Ag 5 is a major allergen of vespid venom. The complete amino acid sequences of Ag 5 from two species each of hornets (Dolichovespula arenaria and maculata), wasps (Polistes annularis and exclamans), and yellow jackets (Vespula maculifrons and vulgaris) have been determined by amino acid sequencing and/or cDNA cloning. The sequence data reveal several conserved and variable regions for vespid Ag 5. The conserved regions show greater sequence similarity than do the less conserved regions to a family of proteins from human, mouse, and rat testis and to a class of pathogenesis-related proteins from tobacco and tomato leaves. Tests in the murine system for both T and B cell responses demonstrate a high degree of cross-reactivity of hornet Ag 5 with wasp or yellow jacket Ag 5 and a low degree of cross-reactivity of yellow jacket Ag 5 with wasp Ag 5. This pattern of cross-reactivity is in the same order as their sequence similarity.

Allergens↗

PubMed1993

Cloning and expression of the major allergen from yellow mustard seeds, Sin a I.

The cloning and bacterial expression of the major allergen from yellow mustard (Sin a I) is reported. Cloning has been carried out by means of the polymerase chain reaction using non-degenerate oligo primers encoding the N- and C-terminal regions of the mature protein. Two nucleotide sequences (SA2S1 and SA2S2) have been found and analysed. The observed polymorphism suggests the existence of multiple isoforms for Sin a I allergen. SA2S2 has been expressed as a fusion protein, linked to the choline-binding domain of the Streptococcus pneumoniae murein hydrolase. The resulted fusion protein is recognized by Sin-a-I-specific antibodies.

Allergens↗