Biomedical subjects

M Villalba

Publications and source records attributed to M Villalba.

At least 55 records · Page 3Linked to original sources

PubMed1998

Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy.

A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen.

Allergens↗

PubMed1998

A possible role for PKC delta in cerebellar granule cells apoptosis.

Cerebellar granule cells (CGC) undergo massive DNA fragmentation, an apoptotic marker, in 8-day-old rat cerebellum. In vitro, they survive in the presence of depolarizing concentrations of KCl. Bisindolylmaleimide, a specific PKC inhibitor, blocks CGC apoptosis in vitro. Here I show that PKC delta, which has been involved in apoptosis in different cell lines, is constitutively cleaved in CGC, suggesting that its catalytic subunit is active per se. Moreover, KCl deprivation induces cyclin D1 expression and accumulation in nuclei. This process is blocked by bisindolylmaleimide. A model is proposed where, in the absence of survival signals, activated PKC delta induces cyclin D1 expression and accumulation in the nucleus, which subsequently, would lead to an aborted cell cycle and apoptosis.

Animals↗

PubMed1998

Bisindolylmaleimide prevents cerebellar granule cells apoptosis: a possible role for PKC.

Cerebellar granule cells (CGC) are amongst the most abundant neuronal cells in the mammalian central nervous system. In vitro, they undergo apoptosis when deprived of depolarizing concentrations of KCl, providing an interesting system to study neuronal apoptosis. Despite its significance, the intracellular mechanisms that follow an apoptotic insult are poorly understood. Bisindolylmaleimide, a specific protein kinase C (PKC) inhibitor, blocks cell death and apoptosis with an optimal concentration of 10 microM. Bisindolylmaleimide also blocks apoptosis up to 7 h after KCl deprivation, suggesting that it blocks a secondary process. Ro-31-8220, another PKC inhibitor, also blocks apoptosis, but it is less effective, probably due to its lack of specificity. A possible PKC role in increasing the transcription or activating protein(s) involved in neuronal apoptosis is proposed.

Animals↗

PubMed1998

Protection by pyruvate and malate against glutamate-mediated neurotoxicity.

Pyruvate and malate (P/M) increase the contribution of mitochondria to neuronal calcium homeostasis. We have now found that cortical neuronal cultures utilize pyruvate preferentially over glucose. The supply of pyruvate and malate protects hippocampal and cortical neurons against delayed cell death occurring 24 h after glutamate exposure. High [Ca2+]i levels attained during and after glutamate exposure were reduced when neurons were incubated in the presence of P/M. At the single cell level, this was reflected in a decrease in the number of neurons that respond to glutamate with high rises in [Ca2+]i. The results suggest that the ability to prevent large increases in [Ca2+]i may underlie the beneficial effects of pyruvate and malate during glutamate excitotoxicity.

Animals↗

PubMed1998

Insulin-like growth factor-I modulation of cerebellar cell populations is developmentally stage-dependent and mediated by specific intracellular pathways.

Although development of transgenic animals overexpressing insulin-like growth factor-I has allowed the establishment of a role of this trophic factor in brain growth, detailed knowledge of the action of insulin-like growth factor-I on different brain areas is still lacking. We now provide evidence for a pleiotrophic role of this growth factor on cerebellar development. Insulin-like growth factor-I produced by cerebellar cultures is a survival factor for Purkinje cells and a mitogen/differentiation factor for cerebellar glioblasts. Trophic effects of insulin-like growth factor-I were observed only during specific developmental stages. In addition, insulin-like growth factor-I increased intracellular Ca2+ levels in Purkinje cells and c-Fos in dividing glioblasts. Survival-promoting effects of insulin-like growth factor-I on Purkinje cells required activation of protein kinase C, while glioblast division induced by insulin-like growth factor-I depended on phosphatidylinosytol 3-kinase activation. We conclude that insulin-like growth factor-I is a paracrine/autocrine pleiotrophic factor for both glia and neurons in the cerebellum. Its effects are mediated by distinct intracellular signals and appear to be specific to the developmental stage of the target cell. Since development of the different cell populations that compose a specific brain territory is not synchronized, the pleiotrophic action of growth factors such as insulin-like growth factor-I may be essential to ontogenetic processes underlying normal brain growth.

Animals↗

PubMed1998

Olive-pollen profilin. Molecular and immunologic properties.

Olive-pollen profilin has been isolated and characterized as a significant allergen. Its molecular properties, such as a molecular mass of 15 kDa; amino-acid composition; and secondary repetitive structure percentages of 15% alpha-helix, 33% beta-strand, 20% beta-turn, and 32% random coil, have been determined. Its allergenic capability, a recognition frequency estimated at 24% of olive-hypersensitive patients, and high cross-reactivity with all the pollen used have been found. The presence of conformation epitopes in the olive profilin, as well as a high structural and immunologic similarity to other pollen sources such as birch and ash, can be established from these studies.

Amino Acids↗

PubMed1997

Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen.

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH2-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH2-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X3-Cys-X3-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.

Allergens↗

PubMed1997

Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain.

The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation and apoptosis. The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders. We report here the cloning of a novel zinc finger protein which regulates apoptosis and cell cycle arrest and was accordingly named Zac1. In vitro Zac1 inhibited proliferation of tumor cells, as evidenced by measuring colony formation, growth rate and cloning in soft agar. In vivo Zac1 abrogated tumor formation in nude mice. The antiproliferative activity of Zac1 was due to induction of extensive apoptosis and of G1 arrest, which proceeded independently of retinoblastoma protein and of regulation of p21(WAF1/Cip1), p27Kip1, p57Kip2 and p16INK4a expression. Zac1-mediated apoptosis was unrelated to cell cycle phase and G1 arrest was independent of apoptosis, indicating separate control of apoptosis and cell cycle arrest. Zac1 is thus the first gene besides p53 which concurrently induces apoptosis and cell cycle arrest.

Amino Acid Sequence↗

PubMed1997

Concomitant induction of apoptosis and necrosis in cerebellar granule cells following serum and potassium withdrawal.

Serum and potassium withdrawal-induced cell death of cerebellar granule cells is one of the most popular models used for studying neuronal apoptosis in vitro, and it is generally assumed that compounds preventing cell death in this model prevent apoptosis. In the present study we demonstrate that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. Each of these death mechanisms could be blocked by activation of different pathways, such as raising cAMP production or stimulation of the IGF-1 receptor. Studies on serum and potassium withdrawal-induced cell death of cerebellar granule cells should therefore carefully assess whether a given compound is preventing apoptosis or necrosis using appropriate techniques.

Androstadienes↗

PubMed1997

A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines.

The tetracycline-dependent expression system has gained increasing popularity for the expression of any gene of interest. Careful choice of the expression vector has been suggested to exploit the full potential of this system. A novel tetracycline-sensitive expression vector based on a modified mouse mammary tumor virus promoter achieved considerably improved regulatory properties in a series of cell lines tested under transient and stable conditions. Therefore, the applicability of the tetracycline-dependent expression system can be largely enhanced by careful adaptation of the expression vector to the host cell line.

Animals↗

PubMed1997

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway.

Pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions, including cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP-38 on apoptosis induced by potassium deprivation. We demonstrated that PACAP-38 increased survival of cerebellar neurons in a dose-dependent manner by decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP-38 induced activation of the extracellular signal-regulated kinase (ERK)-type of mitogen-activated protein (MAP) kinase through a cAMP-dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the antiapoptotic effect of PACAP-38, suggesting that MAP kinase pathway activation is necessary for PACAP-38 action.

Animals↗

PubMed1997

Detection, isolation and complete amino acid sequence of an aeroallergenic protein from rapeseed flour.

BACKGROUND: Seed proteins have been found to cause hypersensitivity by ingestion or inhalation. Rapeseed flour was responsible for allergic symptoms in a patient, who develops into allergy to mustard spice. OBJECTIVE: To determine the presence of allergenic proteins in rapeseed flour, and analyse the structure of the main component and its crossreactivity with the mustard allergen. METHODS: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequent immunoblotting with a serum from a rapeseed allergic patient were performed to detect IgE-binding proteins. Proteolytic digestions and high performance liquid chromatography were used to obtain the peptides from the allergenic BnIII napin from rapeseed flour. Automatic Edman degradations were carried out to determine their amino acid sequences, which were compared with other sequences in nucleotide and amino acid sequence databases. Crossreactivity assays were carried out by ELISA inhibition using sera from a rapeseed allergic patient and from patients allergic to mustard. RESULTS: The 2S albumins of rapeseed were recognized by the serum from a patient allergic to this seed. The most abundant isoform of the allergenic napins, BnIII, was used for structural and immunological analysis. The protein consists of two different chains of 9.5 and 4.5 kDa. Their complete amino acid sequences were determined. The protein exhibited structural relationships with other napin-like storage proteins from seeds. IgE and IgG crossreactivity between rapeseed and mustard allergens was also demonstrated. Considering the structural and immunological data, certain polypeptide regions are suggested to be involved in the allergenicity of these proteins. CONCLUSIONS: Rapeseed contains 2S storage proteins which may cause allergy in hypersensitive individuals. These proteins exhibit great sequence similarity with 2S albumins from different seeds. Crossreactivity between mustard and rapeseed flours can be explained by sequence homology.

2S Albumins, Plant↗

PubMed1997

Nonoperative management of adult blunt splenic trauma: a 15-year experience.

From January 1989 to December 1993, 40 consecutive adult patients with ruptured spleen from blunt trauma were examined. Fourteen patients (35%) were taken to the operating room initially because of hemodynamic instability and generalized peritoneal signs. Twenty-six patients (65%) were hemodynamically stabilized at admission and treated by nonoperative management, which included strict bed rest, intensive care unit monitoring, frequent physical examinations, and serial hematocrits. Four patients failed nonsurgical management and required a splenectomy, three because of clinical deterioration within 1 to 3 days of admission; the fourth patient had recurrent bleeding 7 days after injury. The patients in the operative group had a greater severity of injury with a mean injury severity score of 26.6, four deaths, and mean transfusion requirements of 3.7 to 4.0 units of blood, compared to a mean injury severity score of 14.6, one late death from cardiac causes, and average blood requirement of 0.4 to 0.7 units. Splenic injury grading averaged 3.2 in the surgical group (grade 1, one patient; grade 2, four patients; grade 3, eight patients; grade 4, no patients; and grade 5, one patient) and differed significantly from that of the nonoperative group (mean = 2.4; grade 1, 12 patients; grade 2, seven patients; grade 3, six patients; grade 4, two patients; and grade 5, no patients). Recent ultrasound analysis of select grades I to IV has shown excellent resolution or repair of these injuries. This report extends our series from 1978 to 1993 and includes 144 adult patients sustaining blunt splenic ruptures. Seventy-nine (55%) of these patients were treated nonsurgically. Seven patients (of 80) failed nonoperative management and required interval laparotomy, representing a 91 per cent success rate. Follow-up on more than 90 per cent of the patients has shown no sequelae from their splenic injuries. We conclude that adult patients with splenic injuries from blunt trauma who are hemodynamically stable and are without abdominal findings requiring celiotomy can be safely managed by a nonoperative approach.

Adolescent↗

PubMed1996

Ole e 3, an olive-tree allergen, belongs to a widespread family of pollen proteins.

An allergen has been isolated from a saline extract of olive tree (Olea europaea) pollen. The protein consists of a single polypeptide chain of 9.2-kDa, as determined by mass spectrometry. It contains neither tryptophan nor tyrosine residues, and displays an acidic isoelectric point. The secondary structure of the protein, estimated from the analysis of the circular-dichroism spectrum in the peptide-bond region, is composed of 52% alpha-helix, 10% beta-strand, 29% beta-turn and 9% non-regular conformation. The N-terminal end of the protein is blocked. Amino-acid-sequence data have been obtained from peptides produced by CNBr treatment of the native allergen. A partial sequence of 36 amino acids has thus been elucidated. The protein exhibits sequence similarity with pollen allergens from Brassica species and contains a Ca(2+)-binding motif. The isolated protein displays IgE-binding activity against sera of patients allergic to olive-tree pollen. It has been named Ole e 3, according to the recommendations of the IUIS Nomenclature Committee. IgG ELISA inhibition assays with polyclonal antibodies specific for Ole e 3 reveal the presence of proteins similar to Ole e 3 in the pollen from non-related plant species, which may explain allergic cross-reactivity processes.

Allergens↗

PubMed1996

Expression in Escherichia coli of Sin a 1, the major allergen from mustard.

Sin a 1, the major yellow mustard allergen, is a seed storage protein that belongs to the 2S albumin family. It is composed of two disulfide-bonded polypeptide chains. The cloning of this allergen has been carried out by means of the polymerase chain reaction using non-degenerate oligonucleotides encoding the N-terminal and C-terminal regions of the mature protein as primers. Five genomic nucleotide sequences have been analyzed, encoding both mature polypeptide chains linked by the internal processed fragment. The sequence data show the existence of microheterogeneities at ten positions, demonstrating the polymorphism exhibited by the natural protein. One of the genomic clones was expressed in Escherichia coli by fusion to glutathione S-transferase from Schistosoma japonicum. The resulting chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B matrix, and digested with thrombin to release the recombinant allergen. The recombinant Sin a 1 is recognized by rabbit polyclonal and mouse monoclonal antisera raised against natural Sin a 1, as well as by the IgE of mustard-sensitive human sera. In addition, recombinant Sin a 1 possesses a high resistance to trypsin digestion, like the native mustard allergen.

Allergens↗

PubMed1996

Mouse microglial cells express a plasma membrane pore gated by extracellular ATP.

We have investigated responses to extracellular ATP (ATPe) in the microglial cell lines N9 and N13 and in freshly isolated mouse microglial cells. Upon stimulation with this nucleotide, N9 and N13 cells underwent an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i), a sustained depolarization of the plasma membrane, and an uptake of extracellular markers such as ethidium bromide and lucifer yellow; increases in plasma membrane permeability were paralleled by striking morphologic changes. ATPe, as well as other nucleotides, activated a spiking Ca2+ release from intracellular stores; however, only ATPe was also able to cause a massive transmembrane Ca2+ influx. The ATP analogue 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) triggered a sustained Ca2+ influx accompanied by little release from stores. The ATP derivative oxidized ATP (oATP) strongly inhibited Ca2+ influx, minimally affecting Ca2+ release. From ATPe-sensitive microglial cell lines, we selected several ATPe-resistant clones that showed complete lack of ATPe-mediated plasma membrane permeability changes, although they retained the Ca2+ mobilization response from intracellular stores. ATPe-dependent plasma membrane permeability changes were also greatly reduced in growth-arrested microglial cells. Finally, ATPe triggered IL-1 beta release from wild-type but not ATPe-resistant microglial cells. These results show that microglial cells express at least two purinergic receptor subtypes, metabotropic (P2Y) and ionotropic (P2Z), and that the latter is modulated during cell cycle and coupled to IL-1 beta release.

Adenosine Triphosphate↗

PubMed1996

Bedside tracheostomy in the intensive care unit.

OBJECTIVE: To prove that tracheostomy performed at the bedside in the intensive care unit is a safe, cost-effective procedure. DESIGN: Retrospective review of all adult patients undergoing elective bedside tracheostomy in the intensive care unit between January 1983 and December 1988. Two hundred four patients were identified. SETTING: A private 1200-bed tertiary care center with a 120-bed critical care facility. MAIN OUTCOME MEASURES: Major and minor perioperative complications, cost savings, and comparison of risk between bedside tracheostomy and that performed in the operating room. RESULTS: There were six major complications (2.9%): one death due to tube obstruction, two bleeding episodes requiring reoperation, one tube entrapment requiring operative removal, one nonfatal respiratory arrest, and one bilateral pneumothorax; and seven minor complications (3.4%): five episodes of minor bleeding, one tube dislodgement in a tracheostomy with a well-developed tract, and one episode of mucus plugging. One late complication (tracheal stenosis) was identified. CONCLUSIONS: Bedside tracheostomy in the intensive care unit can be performed with morbidity and mortality rates comparable to operative tracheostomy. In addition, it provides a significant cost savings for the patient.

Cost-Benefit Analysis↗

PubMed1996

Cytosolic and mitochondrial calcium in synaptosomes during aging.

Synaptosomal [Ca2+]i levels increase during aging, particularly in the old rat hippocampus, both under basal conditions and after high K depolarization. This is probably the result of age-dependent modifications in calcium buffering and extrusion systems rather than due to increased calcium influx, since calcium uptake through synaptosomal voltage gated calcium channels decreases in old animals. The calcium binding capacity of the cytosolic compartment (i.e, that excluded from mitochondria and endoplasmic reticulum) of synaptosomes was markedly reduced in old rats. Calcium compartmentation in synaptosomal mitochondria, is also reduced during aging, and this is associated with a decrease in activity of the mitochondrial calcium uniporter. Taken together, these modifications point towards a clear deterioration of the cell calcium homeostatic mechanisms towards increased [Ca2+]i in old age, specially under conditions of high calcium loads, a situation that may exacerbate neuronal vulnerability to excitotoxicity.

Aging↗