Biomedical subjects

E Pintado

Publications and source records attributed to E Pintado.

At least 19 recordsLinked to original sources

PubMed2006

Hypomethylation of DNA and resistance to apoptosis in tonsillar hypertrophy in children.

We analyzed the hypomethylation of DNA and the sensitivity to apoptosis of tonsillar cells and peripheral blood lymphocytes (PBL) in twenty children with either recurrent tonsillitis (RT) or tonsillar hypertrophy (TH). We found no significant differences in DNA methylation of PBL obtained from RT and TH groups. Hypomethylation of DNA extracted from tonsillar tissue was higher in TH than in RT and was associated with lower spontaneous and thapsigargin-induced apoptosis. By contrast, RT showed a low level of DNA hypomethylation and was associated with high sensitivity to spontaneous and thapsigargin-induced apoptosis.

Apoptosis↗

PubMed2002

Macrophage inducible nitric oxide synthase gene expression is blocked by a benzothiophene derivative with anti-HIV properties.

Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNF alpha-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharide-elicited production of nitrite (NO(-)(2)) by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO(-)(2) production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFN gamma. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNF alpha, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNF alpha synthesis. Additionally, PD144795 did not block NF-kappa B nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug.

Animals↗

PubMed2001

[Methylation and expression of the FMR1 gene].

OBJECTIVE: In this paper we present a brief review on DNA methylation, the enzymes and proteins involved in the repression complex and the importance of methylation of FMR1 gene in fragile X syndrome. DEVELOPMENT: Methylation status of control region in the genome plays a critical role in the regulation of gene expression. In susceptible genes containing CpG island in the promoter, cytosine methylation favors a repressive chromatin structure that prevents the binding of transcriptional activators to the promoter. The enzyme DNA methyltransferase transfers a methyl group from the S-adenosylmethionine to the 5 carbon of cytosine in the CG sequences. Several proteins have been described that recognize the methyl cytosine and recruit the co-repressor and the histones deacetylases. The lost of the acetyl groups produces the compacting of the chromatin. Fragile X syndrome is due, in the majority of the cases, to the expansion above a threshold of the CGG repeats in the first exon of FMR1 gene. These expansions are concomitant with the methylation of the promoter and the silencing of FMR1. CONCLUSIONS: DNA methylation is a tag that enables different phenotypic expression from an identical nucleotide sequence. Aberrant methylation is the cause of different pathologies including fragile X syndrome. The comprehension of the mechanisms by which methylation induces the silencing of the genes and the study of agents that could revert this process are important for the treatment of these diseases.

DNA Methylation↗

PubMed1999

Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production.

Interleukin (IL)-1beta is a pleiotropic cytokine implicated in a variety of activities, including damage of insulin-producing cells, brain injury, or neuromodulatory responses. Many of these effects are mediated by nitric oxide (NO) produced by the induction of NO synthase (iNOS) expression. We report here that IL-1beta provokes a marked repression of genes, such as fragile X mental retardation 1 (FMR1) and hypoxanthine phosphoribosyltransferase (HPRT), having a CpG island in their promoter region. This effect can be fully prevented by iNOS inhibitors and is dependent on DNA methylation. NO donors also cause FMR1 and HPRT gene silencing. NO-induced methylation of FMR1 CpG island can be reverted by demethylating agents which, in turn, produce the recovery of gene expression. The effects of IL-1beta and NO appear to be exerted through activation of DNA methyltransferase (DNA MeTase). Although exposure of the cells to NO does not increase DNA MeTase gene expression, the activity of the enzyme selectively increases when NO is applied directly on a nuclear protein extract. These findings reveal a previously unknown effect of IL-1beta and NO on gene expression, and demonstrate a novel pathway for gene silencing based on activation of DNA MeTase by NO and acute modification of CpG island methylation.

Animals↗

PubMed1999

Characterization of calcineurin in human neutrophils. Inhibitory effect of hydrogen peroxide on its enzyme activity and on NF-kappaB DNA binding.

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-kappaB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-kappaB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-kappaB inactivation in neutrophils occurs in concert with inhibition of calcineurin.

Calcineurin↗

PubMed1999

Phenylarsine oxide increases intracellular calcium mobility and inhibits Ca(2+)-dependent ATPase activity in thymocytes.

A rise in intracellular Ca(2+) levels has been implicated as a regulatory signal for the initiation of lymphocyte proliferation. In the present study the mechanism underlying the elevation of [Ca(2+)] induced by phenylarsine oxide [PAO] was investigated in thymocytes. This agent inhibits HIV-1 replication and also NF-kappaB-mediated activation. It has been reported that the PAO-induced Ca(2+) elevation results from an enhanced plasma membrane calcium permeability in T cells. Here, we present biochemical evidence that the PAO-induced Ca(2+) increase was independent of external Ca(2+). Consistent with these facts, when [Ca(2+)](i) was depleted by prolonged incubation of the cells in Ca(2+)-free medium, PAO addition did not lead to [Ca(2+)](i) increase. These data indicate the involvement of intracellular organelles of thymocytes as the source of Ca(2+). Moreover, evidence is presented that PAO inhibited Ca(2+)-dependent ATPase activity from thymocytes and sarcoplasmic reticulum from skeletal muscle. This inhibition was dose-dependent, with a IC(50) of about 30 microM for both preparations of enzyme. The ability of PAO to inhibit Ca(2+)-dependent ATPase represents a novel mechanism of action for this drug. Present data suggest that the PAO-dependent [Ca(2+)](i) increase could be mainly the result of inhibition of Ca(2+)-dependent ATPase. In addition, we describe also a Ca(2+)-dependence for PAO effect on tyrosine phosphorylation.

Animals↗

PubMed1998

Assessment of FMR1 expression by reverse transcriptase-polymerase chain reaction of KH domains.

The fragile X syndrome is the most frequent form of inherited mental retardation. This is caused by the transcriptional inactivation of the FMR1 gene. The KH domain is an evolutionarily conserved sequence motif present in many RNA-binding proteins including the fragile X mental retardation gene product. We have studied the expression of the gene in fresh leukocytes derived from patients and normal controls by using a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol that amplifies the region of the FMR1 that contains the KH1 and KH2 domains and that has not been used in previous studies. As expected, normal expression was observed in control subjects and carriers, but FMR1 mRNA was absent in male patients with fragile X syndrome. This method was also proved to be useful for testing the expression of FMR1 in samples from several species and tissues. In all cases we obtained a similar and unique transcript. We suggest that RT-PCR from the KH domains could be the method of choice for studying FMR1 expression.

Female↗

PubMed1997

Highly informative single-stranded conformation polymorphism (SSCP) of short tandem repeats in DNA identification.

The analysis of short tandem repeats (STR) by PCR is a useful technique widely used in DNA identification. Some loci have alleles differing in the number of complete repeat units, whereas others are polymorphic with alleles differing even to the level of a single base. Minor differences in the sequence of STR can confuse the analysis when the PCR product are poorly resolved in denaturing PAGE. The study by SSCP of STR in loci CSF1PO, TPOX, and THO1 and the alleles of locus DIS80 is shown in a representative case of paternity dispute. The analysis of short tandem repeats (STR) by the conformation polymorphisms of single-stranded DNA (SSCP) clearly enhanced the degree of resolution. The high resolution of SSCP of the STR may be the result of the arrangement of very prominent secondary structure and conformation due to the primary repetitive sequence. The method may be of particular interest to distinguish STR with similar PAGE mobility under denaturing conditions and to distinguish tandem repeats of the same size but different sequence.

Alleles↗

PubMed1995

Effect of thimerosal and other sulfhydryl reagents on calcium permeability in thymus lymphocytes.

We have studied the effects of thimerosal, a mercurial compound extensively used as a preservative, as well as other sulfhydryl reagents (e.g. p-hydroxymercurybenzoate, hydrogen peroxide, bromophenacyl bromide, and mercuric chloride) on Ca2+ homeostasis and the redox status of sulfhydryl groups in thymus lymphocytes. They all induced an increase in [Ca2+]i which was blocked with dithiothreitol, suggesting that they act via the oxidation or blockade of sulfhydryl groups. [Ca2+]i increase could be directly related to the effect of the different reagents on cellular protein sulfhydryl content. Experiments with ethidium bromide indicate that the observed rise in [Ca2+]i was not due to a non-specific increase in membrane permeability. Thimerosal differs from the other agents studied in its oxidative properties, which is probably linked to the production of a potent reductor molecule, thiosalicylic acid, which may modulate its oxidative capacity.

Animals↗

PubMed1995

Modulation of phorbol ester-induced respiratory burst by vanadate, genistein, and phenylarsine oxide in mouse macrophages.

The effect of the inhibitors of tyrosine phosphatase (vanadate and phenylarsine oxide) and of an inhibitor of tyrosine kinase (genistein) on O2.- production in mouse peritoneal macrophages was examined. Vanadate and phenylarsine oxide produced a dose-dependent inhibition of phorbol myristate acetate (PMA)-induced O2.- production, whereas genistein potentiated O2.- production triggered by phorbol ester. Vanadate had no effect on the respiratory burst in human neutrophils challenged with fMLP, in agreement with previously published data on human intact neutrophils. It did not alter reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in membrane preparations of mouse peritoneal macrophages. These data suggest that the phosphorylation of protein(s) in tyrosine residues blocked the PMA-dependent respiratory burst in mouse macrophages.

Animals↗

PubMed1995

Instability of the CGG repeat at the FRAXA locus and variable phenotypic expression in a large fragile X pedigree.

Fragile X syndrome is the major cause of inherited mental retardation. The molecular basis for the expression of the fragile X phenotype is the expansion of an unstable CGG repeat element which inhibits transcription of the FMR1 gene. The fragile X syndrome shows great diversity in its phenotype as well as in its cytogenetic and molecular status. We have studied, in a large fragile X family, the correlation between the molecular data and the phenotypic expression of the syndrome. We report two brothers who carry identical unmethylated premutated alleles but present different clinical phenotypes. We also suggest that reductions in allele size from one generation to another may be, as in other diseases, because of triplet amplifications, more common at the FRAXA locus than previously thought.

Alleles↗

PubMed1994

Molecular analysis and test of linkage between the FMR-1 gene and infantile autism in multiplex families.

Approximately 2%-5% of autistic children show cytogenetic evidence of the fragile X syndrome. This report tests whether infantile autism in multiplex autism families arises from an unusual manifestion of the fragile X syndrome. This could arise either by expansion of the (CGG)n trinucleotide repeat in FMR-1 or from a mutation elsewhere in the gene. We studied 35 families that met stringent criteria for multiplex autism. Amplification of the trinucleotide repeat and analysis of methylation status were performed in 79 autistic children and in 31 of their unaffected siblings, by Southern blot analysis. No examples of amplified repeats were seen in the autistic or control children or in their parents or grandparents. We next examined the hypothesis that there was a mutation elsewhere in the FMR-1 gene, by linkage analysis in 32 of these families. We tested four different dominant models and a recessive model. Linkage to FMR-1 could be excluded (lod score between -24 and -62) in all models by using probes DXS548, FRAXAC1, and FRAXAC2 and the CGG repeat itself. Tests for heterogeneity in this sample were negative, and the occurrence of positive lod scores in this data set could be attributed to chance. Analysis of the data by the affected-sib method also did not show evidence for linkage of any marker to autism. These results enable us to reject the hypothesis that multiplex autism arises from expansion of the (CGG)n trinucleotide repeat in FMR-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1991

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes.

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.

Animals↗

PubMed1990

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.

Benzofurans↗

PubMed1989

Calcium release induced by inositol 1,4,5-trisphosphate in thymocyte microsomes. Inhibition by barium and strontium.

The properties of calcium transport in microsomes and the effect of inositol 1,4,5-trisphosphate (IP3) on accumulated calcium were studied in rat thymocytes. Active calcium transport shows an apparent affinity constant for calcium of 0.2 +/- 0.01 microM and a maximal velocity of 2.3 +/- 0.6 nmol/mg/30 min (mean +/- SD). IP3 was able to induce release of calcium only in the absence of oxalate. At 6 microM ambient free calcium, half-maximal effect of IP3 was attained at 2 microM and maximal calcium release was produced by IP3 concentrations over 5 microM. Barium and strontium did not modify calcium uptake by microsomes but markedly inhibited the action of IP3.

Adenosine Triphosphate↗

PubMed1988

Regulation of fructose 2,6-bisphosphate levels in cold-acclimated brown adipose tissue of rat.

The effects of cold exposure and T4 administration on fructose 2,6-bisphosphate levels, phosphofructokinase-2 and pyruvate kinase activities were examined in rat brown adipose tissue. Cold adaptation (14 days) gave rise to a 2-fold increase in the amount of fructose 2,6-bisphosphate and phosphofructokinase-2 activity, and increased the pyruvate kinase activity 4-fold. If, in addition, the cold-acclimated rats were treated with T4, these parameters were again significantly enhanced. The effect on phosphofructokinase-2 was on the Vmax, without modification of the Km (for both fructose 6-phosphate and ATP) of the enzyme. In the hypothyroid state, however, the activity of pyruvate kinase remains unchanged. These data support previous observations on stimulation of glycolytic flux during cold adaptation in brown adipose tissue, and a permissive role of thyroid hormones in the process.

Adaptation, Physiological↗

PubMed1987

Ca2+- and voltage-dependent K+ conductance in dispersed parathyroid cells.

The membrane ionic conductances of dispersed parathyroid cells kept in primary culture were studied using the "whole-cell" and "inside-out excised patch" variants of the patch-clamp technique. The major component of the total current was a voltage-dependent outward K+ current without an appreciable inward current. The amplitude of the K+ current was markedly reduced when free internal Ca2+ was buffered by addition of 10 mM EGTA. Recordings of single-channel current in excised membrane patches revealed the presence of K+ channels with large unitary conductance (200 pS in symmetrical 130 mM K+ solutions) which were also activated by depolarization when internal Ca2+ concentration was about 10(-5)-10(-6) M. At any membrane voltage these channels were closed most of the time at internal Ca2+ concentrations lower than 10(-10) M. These results demonstrate the existence of a Ca2+- and voltage-dependent K+ permeability in parathyroid cells which may participate in the unusual membrane potential changes induced by alterations of external Ca2+ and, possibly, in the regulation of parathormone secretion.

Animals↗

PubMed1987

Effect of protein kinase C activation on mast cell histamine release.

The role of protein Kinase C activators in the process of histamine secretion has been studied in rat peritoneal mast cells purified by a density gradient. TPA (12-O-tetradecanoyl-phorbol-13-acetate), a tumor promoter which activates protein kinase C, induced histamine release in the presence and in the absence of external free Ca2+. TPA and the calcium ionophore A23187 have an additive effect on secretion. Histamine release induced by TPA is energy-dependent. In the presence of 100 microM KCN secretion was moderately inhibited, however when glucose was removed from the incubation medium TPA-induced histamine release in the presence of KCN was strongly depressed.

Animals↗