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Biomedical subjects

M Costa

Publications and source records attributed to M Costa.

At least 91 records · Page 5Linked to original sources
PubMed2000

Overexpression of a dominant negative CREB protein in HT-1080 cells selectively disrupts plasminogen activator inhibitor type 2 but not tissue-type plasminogen activator gene expression.

The tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 2 (PAI-2) genes are differentially regulated by 12-phorbol 13-myristate acetate (PMA) in HT-1080 fibrosarcoma cells. PMA transcriptionally down-regulates the t-PA gene in HT-1080 cells, while the PAI-2 gene is simultaneously induced by this agonist. The t-PA and PAI-2 gene promoters harbour a cAMP-response element (CRE) which influences the expression of both genes. We have compared the binding activity of nuclear factors that recognise these CRE sites. We show that CREB (CRE binding protein) recognises each CRE and that the degree of constitutive Ser119-phosphorylated t-PA CRE-bound CREB was greater than for PAI-2 CRE bound CREB. Stable transfection of HT-1080 cells with a plasmid containing a CREB that could not be phosphorylated on Ser119 (pCI-CREB(ala119)) did not influence PMA-mediated suppression of t-PA mRNA, but markedly impaired PMA-mediated induction of PAI-2 mRNA. Our results demonstrate that the Ser119 residue of CREB plays a crucial role in PMA-mediated induction of PAI-2 gene expression, whereas PMA-mediated suppression of t-PA in HT-1080 cells requires a different process.

Cyclic AMP Response Element Modulator↗
PubMed2000

A three-dimensional perspective on exon binding by a group II self-splicing intron.

We have used chemical footprinting, kinetic dissection of reactions and comparative sequence analysis to show that in self-splicing introns belonging to subgroup IIB, the sites that bind the 5' and 3' exons are connected to one another by tertiary interactions. This unanticipated arrangement, which contrasts with the direct covalent linkage that prevails in the other major subdivision of group II (subgroup IIA), results in a unique three-dimensional architecture for the complex between the exons, their binding sites and intron domain V. A key feature of the modeled complex is the presence of several close contacts between domain V and one of the intron-exon pairings. These contacts, whose existence is supported by hydroxyl radical footprinting, provide a structural framework for the known role of domain V in catalysis and its recently demonstrated involvement in binding of the 5' exon.

Base Sequence↗
PubMed2000

Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization.

Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.

Amino Acid Sequence↗
PubMed2000

Interaction of Ni(II) and Cu(II) with a metal binding sequence of histone H4: AKRHRK, a model of the H4 tail.

Chromatin proteins are believed to represent reactive sites for nickel binding. The unique structure of the N-terminal tail of histone H4 contains sites for post-translational modification close to a histidine residue capable of anchoring binding sites for metal ions. We have analyzed as a minimal model for the H4 tail, the blocked peptide CH(3)CO-AKRHRK-CONH(2) for nickel and copper binding. Ultraviolet-visible, circular dichroism, electron paramagnetic resonance and nuclear magnetic resonance spectroscopic analysis showed that histidine acts as an anchoring metal binding site. A 1N complex is formed between pH=5-7 and 4-6 for Ni(II) and Cu(II), respectively, while at a higher pH a series of 4N complexes are formed. Above pH 8, the 2N high-spin octahedral resulted in a 4N low-spin planar Ni(II) complex. The stability constants of the Cu(II) (3N, 4N) and Ni(II) (4N) complexes with the peptide model of the H4 were distinctly higher than those for a similar blocked peptide with a histidine in the fourth position. Significant shifts in the alphaproton region in the 1H NMR spectrum of the 4N Ni-complex showed that the conformation of the peptide had been dramatically affected following Ni(II) complexation.

Amino Acid Sequence↗
PubMed2000

Carcinogenic metals induce hypoxia-inducible factor-stimulated transcription by reactive oxygen species-independent mechanism.

Nickel (Ni2+) and cobalt (Co2+) mimic hypoxia and were used as a tool to study the role of oxygen sensing and signaling cascades in the regulation of hypoxia-inducible gene expression. These metals can produce oxidative stress; therefore, it was conceivable that reactive oxygen species (ROS) may trigger signaling pathways resulting in the activation of the hypoxia-inducible factor (HIF)-1 transcription factor and up-regulation of hypoxia-related genes. We found that the exposure of A549 cells to Co2+ or Ni2+ produced oxidative stress, and although Co2+ was a more potent producer of ROS than Ni2+, both metals equally increased the expression of Cap43, a hypoxia-regulated gene. The coadministration of hydrogen peroxide with metals induced more ROS; however, this did not further increase the expression of Cap43 mRNA. The free radical scavenger 2-mercaptoethanol completely suppressed ROS generation by CoCl2 and NiCl2 but did not diminish the induced Cap43 gene expression. The activity of the HIF-1 transcription factor as assessed in transient transfection assays was stimulated by Ni2+, hypoxia, and desferrioxamine, but this activation was not diminished when oxidative stress was attenuated nor was HIF-dependent transcription enhanced by hydrogen peroxide. We conclude that ROS are produced during the exposure of cells to metals that mimic hypoxia, but the formation of ROS was not involved in the activation of HIF-1-dependent genes.

Carcinogens↗
PubMed2000

Methylene blue photosensitized oxidation of cysteine sulfinic acid and other sulfinates: the involvement of singlet oxygen and the azide paradox.

The methylene blue photosensitized oxidation of cysteine sulfinic acid is investigated. Enhancement of the oxygen consumption rate in deuterium oxide suggests the involvement of singlet oxygen ((1)O(2)) in oxidation. Addition of the (1)O(2) quencher azide produced an unusual enhancement of the oxidation rate of all the sulfinates assayed. It is assumed that azide works as a one-electron carrier between (1)O(2) and the sulfur compounds. Analyses of the products indicate that the photochemical oxidation of cysteine sulfinic acid proceeds through two simultaneous mechanisms. The Type II (singlet oxygen) mechanism is responsible for oxidation of the sulfinic group to the sulfonic group with production of cysteic acid, stable to the photooxidation system, whereas the Type I (electron transfer) mechanism is involved in the degradation of cysteine sulfinic acid to acetaldehyde. Other products detected were ammonia, sulfate, and hydrogen peroxide which account for the degradation of cysteine sulfinic acid and for the excess of oxygen consumption detected during the oxidative reaction.

Aerobiosis↗
PubMed2000

Synthesis of 2-oxazolidinones by direct palladium-catalyzed oxidative carbonylation of 2-amino-1-alkanols

[reaction: see text] 2-Oxazolidinones 2 are obtained in excellent yields (up to 100%) and with unprecedented catalytic efficiencies (up to 2000 mol of product/mol of catalyst used) by direct PdI2/KI-catalyzed oxidative carbonylation of the readily available 2-amino-1-alkanols 1. Reactions are carried out in MeOH as the solvent at 100 degrees C using a 1/6/5 CO/O2/air mixture (60 atm total pressure at 25 degrees C).

Journal Article↗
PubMed2000

Intraarterial beta irradiation induces smooth muscle cell apoptosis and reduces medial cellularity in a hypercholesterolemic rabbit restenosis model.

PURPOSE: Ionizing radiation has been shown to be a powerful inhibitor of neointimal hyperplasia following arterial injury in several animal models of post-percutaneous transluminal coronary angioplasty (post-PTCA) restenosis. This was previously shown to be associated with a reduction in smooth muscle cell (SMC) mitotic activity. This study evaluated the effect of intraarterial beta irradiation on the arterial wall SMC density and apoptosis. METHODS AND MATERIALS: Twenty-five carotid and 7 iliac arteries of hypercholesterolemic New Zealand white rabbits were injured using the Baumgartner technique. The impact of an 18 Gy beta radiation dose administered after balloon injury was studied and compared to a nonirradiated injured control group. The medial SMC density as well as the percentage of apoptotic cells were determined at 8 days, 21 days, and 6 weeks after injury using an automated computer-based software. Apoptotic cells were identified using in situ end-labeling of fragmented DNA. RESULTS: The values for medial apoptosis in control vs. irradiated arteries were: 0.014 +/- 0.023 vs. 0.23 +/- 0.28%, p = NS, at 8 days; 0.012 +/- 0.018 vs. 0.07 +/- 0.07%, p = 0.05, at 21 days; and 0 +/- 0 vs. 0.16 +/- 0.11%, p = 0.03, at 6 weeks. The overall incidence of medial apoptotic cells at all time points was 0.01 +/- 0.017 vs. 0.13 +/- 0.14% in controls and irradiated arteries respectively, p = 0.004. Medial SMC density was significantly decreased in irradiated arteries in comparison with controls (p < 0.01 at all time-points). CONCLUSIONS: Intraarterial beta irradiation stimulates medial SMC apoptosis in balloon-injured arteries. This, together with a decrease in SMC mitotic activity, contributes to a decrease in the arterial wall cellularity.

Animals↗
PubMed2000

Nickel compounds are novel inhibitors of histone H4 acetylation.

Environmental factors influence carcinogenesis by interfering with a variety of cellular targets. Carcinogenic nickel compounds, although generally inactive in most gene mutation assays, induce chromosomal damage in heterochromatic regions and cause silencing of reporter genes when they are located near telomere or heterochromatin in either yeast or mammalian cells. We studied the effects of nickel on the lysine acetylation status of the NH2-terminal region of histone H4. At nontoxic levels, nickel decreased the levels of histone H4 acetylation in vivo in both yeast and mammalian cells, affecting only lysine 12 in mammalian cells and all of the four lysine residues in yeast. In yeast, lysine 12 and 16 were more greatly affected than lysine 5 and 8. Interestingly, a histidine Ni2+ anchoring site is found at position 18 from the NH2-terminal tail of H4. Nickel was also found to inhibit the acetylation of H4 in vitro using purified recombinant histone acetyltransferase. To our knowledge, this is the first agent shown to decrease histone H4 acetylation at nontoxic levels.

Acetylation↗
PubMed2000

Carcinogenic nickel induces genes involved with hypoxic stress.

Carcinogenic nickel compounds alter the program of gene expression in normal cells and induce a pattern of gene expression similar to that found in nickel-induced cancers. Here we have demonstrated that nickel exposure induced hypoxic signaling pathways by inducing hypoxia-inducible transcription factor-1 (HIF-1), which mediated the induction of genes required by cells to survive hypoxia. We also show that a new gene, Cap43, is dependent upon HIF-1 because only HIF-1-proficient cells induced Cap43 when exposed to either hypoxia or nickel. We also show that glyceraldehyde-3-phosphate dehydrogenase, a gene induced by hypoxia through HIF-1, was similar to Cap43 in that it required HIF-1-proficient cells to be induced by either nickel or hypoxia. These data demonstrate that nickel exposure turns on signaling for hypoxic stress, which may be important in its carcinogenesis.

Animals↗
PubMed2000

Determination of Ibuprofen in human plasma by high-performance liquid chromatography: validation and application in pharmacokinetic study.

A specific method for the simultaneous determination of S-(+)Ibuprofen and R-(-)Ibuprofen enantiomers in human plasma is described. Adopting a high-performance liquid chromatographic (HPLC) system with spectrofluorometer detector, the compounds were extracted from plasma in alcohol medium and were separated on C18 column, using a solution of acetonitrile-water-acetic acid-triethylamine as mobile phase. The limit of quantitation was 0.1 microg/mL for both compounds. The method was validated by intra-day assays at three concentration levels and was used in a kinetic study in healthy volunteers. During the study we carried out inter-day assays to confirm the feasibility of the method.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Hypotaurine and superoxide dismutase: protection of the enzyme against inactivation by hydrogen peroxide and peroxidation to taurine.

Hypotaurine is able to prevent the inactivation of SOD by H2O2. The protection is concentration-dependent: at 20 mM hypotaurine the inactivation of SOD is completely prevented. It is likely that hypotaurine exerts this effect by reacting with hydroxyl radicals, generated during the inactivation process, in competition with the sensitive group on the active site of the enzyme. According to this, spectral studies indicate that in presence of hypotaurine the integrity of the active site of SOD is preserved by the disruptive action of H2O2. An interesting outcome of the SOD/H2O2/hypotaurine interaction is that SOD catalyzes the peroxidation of hypotaurine to taurine. Indeed, the formation of taurine increases with the reaction time and with the enzyme concentration. Although the peroxidase activity of SOD is not specific and relatively slow compared to the dismutation of superoxide, it might represent another valuable mechanism of production of taurine.

Animals↗
PubMed2000

Incidence of RET mutations in patients with Hirschsprung's disease.

BACKGROUND: RET mutations have been reported variously to affect 7% to 41% of Hirschsprung's disease (HSCR) patients depending on familial or sporadic occurrence of the disease, length of aganglionosis and possible association with other disease phenotypes. The authors report a study of the incidence of RET mutations in unselected HSCR patients from two regional centers and correlate their genotypes and phenotypes. METHODS: The records of HSCR patients treated in 2 regional centers with a combined population of 5 million were reviewed, and blood samples were obtained from 57 patients. During the same period, 39 patients with similar demographic data refused or provided insufficient blood for study. DNA was extracted, and the 21 exons of the RET protooncogene were screened for mutations using denaturing gradient gel electrophoresis (DGGE). RESULTS: Of 57 patients, 48 were sporadic, and 9 were familial. Lengths of aganglionosis were total colonic, 4; long, 11; short, 39; ultrashort, 1; unclassified, 2. Associated anomalies were present in 20. Causative mutations were identified in 4 (7%): missense or "silent" in 3 (exons 5, 11, 13) and deletion in 1. The silent mutation of exon 11 recently has been shown to have effects on correct RET mRNA splicing. One mutation occurred in total colonic aganglionosis (25%), 1 in long segment dysganglionosis (9%), and 2 in short segment aganglionosis (5%). Surprisingly, all these mutations occurred in sporadic cases (10%). Five patients (9%) had rare polymorphic alleles at exon 14 (n = 1) and exon 18 (n = 4). Fifty patients (88%) showed common polymorphic alleles (sequence variants) in 1 or more exons (> 4, n = 5). CONCLUSIONS: RET mutation as a primary cause for Hirschsprung's disease in the general surgical population is less frequent than previously thought. This observation is compatible with the hypothesis that HSCR could be a polygenic disease caused by additive subclinical effects of more than one gene, including RET.

Drosophila Proteins↗
PubMed2000

Synthesis of methyl dihydrohardwickiate and its C-4 epimer. Structural amendment of natural crolechinic acid.

Reduction of the alpha, beta-unsaturated ester moiety of (+)-methyl hardwickiate with magnesium in methanol afforded methyl (4aS,6S,8aS,1R,5R)-5, 6,8a-trimethyl-5-[2'-(3"-oxoyl)-ethyl-perhydro-1-naphthalenyl]- carboxaylate, while reduction with sodium in n-propanol, followed by esterification with diazomethane, furnished its C-4 epimer. After comparison of the 1H- and 13C-NMR data of these compounds with those reported for crolechinic acid isolated from Croton lechleri, a stereochemical revision for the natural product is suggested.

Diterpenes↗
PubMed2000

Synthesis of 16,18-dihydroxycleroda-3,13Z-dien-16,15-olide, (+)-16-hydroxycleroda-3,13Z-dien-16,15-olide, and (-)-hydroxyhalima-5(10),13-dien-16,15-olide from (+)-hardwickiic acid.

Syntheses of three enantiomers of natural hydroxybutenolide diterpenes, 16,18-dihydroxycleroda-3,13Z-dien-16,15-olide (4), (+)-16-hydroxycleroda-3,13Z-dien-16,15-olide (5), and (-)-16-hydroxyhalima-5(10),13Z-dien-16,15-olide (6), via a furan photosensitized oxygenation reaction of (+)-hardwickiic acid (2), are described.

Diterpenes↗
PubMed2000

Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients withtype-1 autoimmune hepatitis.

We previously described autoantibodies against a UGA serine tRNA-protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The presence of anti-tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47.5% of patients were positive compared with none of the control subjects. To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones (19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48.8 kD. Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35.9-kD protein. Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex. Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec.

Adolescent↗
PubMed2000

Metabolic effects of L-carnitine on prepubertal rat Sertoli cells.

The role of carnitine on Sertoli cell metabolism was investigated. Carnitine effects on Sertoli cell lipid metabolism were evaluated by measuring the intracellular levels of non-esterified fatty acids (NEFA) and ketone bodies. The concentration of NEFA in Sertoli cell cultured in the presence of carnitine is significantly reduced as compared to control, while, no significant changes were observed in the concentration of ketone bodies. The functional parameters evaluated to assess the influence of carnitine on Sertoli cell carbohydrate metabolism, i.e., lactate and pyruvate production, lactate dehydrogenase activity and hexose transport, were all significantly increased following carnitine in vitro supplementation. Thus, carnitine appears to drive Sertoli cell intermediary metabolism in an intimately interrelated way, stimulating both fatty acid breakdown and glycolysis. Our results indicate that Sertoli cells are a possible target for a widespread metabolic action of carnitine and strongly support the involvement of carnitine in the regulation of Sertoli cell functions which are related with germ cell "nutrition", convincingly suggesting a direct influence of the compound at testis level.

Animals↗