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S Ribeiro

Publications and source records attributed to S Ribeiro.

At least 37 records · Page 2Linked to original sources
PubMed1997

Brain gene regulation by territorial singing behavior in freely ranging songbirds.

To investigate the ecological relevance of brain gene regulation associated with singing behavior in songbirds, we challenged freely ranging song sparrows with conspecific song playbacks within their breeding territories. Males responded by approaching the speaker, searching for an intruder and actively singing. In situ hybridization of brain sections revealed significantly higher expression of the transcriptional regulator ZENK in challenged birds than in unstimulated controls in several auditory structures and song control nuclei. We conclude that singing behavior in the context of territorial defense is associated with gene regulation in brain centers that control song perception and production, and that behaviorally regulated gene expression can be used to investigate brain areas involved in the natural behaviors of freely ranging animals.

Animals↗
PubMed1997

Cell-surface carbohydrates of Entamoeba invadens.

Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc.

Animals↗
PubMed1997

Cardiac cephalgia: a treatable form of exertional headache.

We report two patients with exertional headaches beginning with vigorous exercise and relieved by rest. Neurologic evaluation and neuroimaging were normal in both. During exercise stress testing, the onset of the patients' typical headaches correlated with ECG changes indicative of myocardial ischemia. In both patients coronary angiography revealed three-vessel disease, and myocardial revascularization procedures were followed by complete resolution of headaches. Based on these patients, and a review of prior similar reports, we conclude that myocardial ischemia is a rare and treatable cause of exertional headache. Accurate diagnosis is critical to controlling headaches and preventing myocardial infarction.

Aged↗
PubMed1996

Structure of the Val122Ile variant transthyretin - a cardiomyopathic mutant.

The Val122Ile mutant transthyretin (TTR Ile122) is an amyloidogenic protein which has been described as the major protein component of amyloid fibrils isolated from patients with familial amyloidotic cardiomyopathy (FAC), a disease characterized by cardiac failure and amyloid deposits in the heart. The reasons for the deposition of TTR are still unknown and it is conceivable that a conformational alteration, resulting from the mutation, is fundamental for amyloid formation. The three-dimensional structure of TTR Ile122 was determined and refined to a crystallographic R factor of 15.8% at 1.9 A resolution. The r.m.s. deviation from ideality in bond distances is 0.019 A and in angle-bonded distances is 0.027 A. The presence of two crystallographically independent monomers in the asymmetric unit allowed additional means of estimation of atomic coordinate error. The structure of the mutant is essentially identical to that of the wild-type transthyretin (TTR). The largest deviations occur in surface loops and in the region of the substitution. The protein is a tetramer composed of identical subunits; each monomer has two four-stranded beta-sheets which are extended to eight-stranded beta-sheets when two monomers associate through hydrogen bonds forming a dimer, which is the crystallographic asymmetric unit. The replacement of valine for isoleucine introduces very small alterations in relation to the wild-type protein; nevertheless they seem to confirm a tendency for a less stable tetrameric structure. This would support the idea that the tetrameric structure might be disrupted in amyloid fibrils.

Journal Article↗
PubMed1996

Limited proteolysis and amino acid replacements in the effector region of Thermus thermophilus elongation factor Tu.

The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis. The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu. EF-Tu that was cleaved at the Arg59-Gly60 peptide bond [EF-Tu-(1-59)/EF-Tu-(60-405)] bound GDP, EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis. However, the GTPase activity of EF-Tu-(1-59)/EF-Tu-(60-405) was not stimulated by T. thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu. EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1-59)/EF-Tu-(60-405). By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T. thermophilus EF-Tu. These amino acid substitutions did not substantially affect either the affinity of EF-Tu. GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction in the ribosome-induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity. The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.

Amino Acid Sequence↗
PubMed1996

Inappropriate use of naloxone in cancer patients with pain.

Opioid overdose is rarely the primary cause of altered mental status in cancer patients receiving opioid therapy. The inappropriate administration of naloxone to reverse an abnormal mental status can cause severe withdrawal symptoms and pain. To illustrate this problem, we report the case of a patient inappropriately treated with naloxone and the results of a retrospective review of the medical records of 15 consecutive patients with cancer treated with naloxone in the emergency department over a 5-month period. We offer guidelines for a more thoughtful approach to the management of patients with cancer who present with encephalopathy.

Aged↗
PubMed1995

Properties of isolated domains of the elongation factor Tu from Thermus thermophilus HB8.

The relative contributions of the three domains of elongation factor Tu (EF-Tu) to the factor's function and thermal stability were established by dissecting the domains apart with recombination techniques. Domain I (EF-TuI), domains I/II (EF-TuI/II) and domain III (EF-TuIII) of the EF-Tu from Thermus thermophilus HB8 comprising the amino acids 1-211, 1-312 and 317-405, respectively, were overproduced in Escherichia coli and purified. A polypeptide consisting of domain II and III (EF-TuII/III) was prepared by limited proteolysis of native EF-Tu with V8 protease from Staphylococcus aureus [Peter, M. E., Reiser, C. O. A., Schirmer, N. K., Kiefhaber, T., Ott, G., Grillenbeck, N. W. & Sprinzl, M. (1990) Nucleic Acids Res. 18, 6889-6893]. As determined by circular dichroism spectrometry, the isolated domains have the secondary structure elements found in the native EF-Tu. GTP and GDP binding as well as GTPase activity are maintained by the EF-TuI and EF-TuI/II; however, the rate of GDP dissociation from EF-TuI . GDP and EF-TuI/II . GDP complex is increased as compared to native EF-Tu . GDP, reflecting a constraint imposed by domain III on the ability to release the nucleotide from its binding pocket located in domain I. A weak interaction of Tyr-tRNATyr with the EF-TuI . GTP suggests that domain I provides a part of the structure interacting with aminoacyl-tRNA. The domain III is capable of regulating the rate of GTPase in EF-Tu, since the polypeptide consisting only of domains I/II has a 39-fold higher intrinsic GTPase compared to the native EF-Tu. No in vitro poly(U)-dependent poly(Phe) synthesis was detectable with a mixture of equimolar amounts of domains I/II and domain III, demonstrating the necessity of covalent linkage between the domains of EF-Tu for polypeptide synthesis. In contrast to native EF-Tu and EF-TuII/III, EF-TuI and, to a lesser extent the polypeptide consisting of domains I/II, are unstable at elevated temperatures. This indicates that domains II/III strongly contribute to the thermal stability of this T. thermophilus EF-Tu. Deletion of amino acid residues 181-190 from domain I of T. thermophilus EF-Tu decreases the thermostability to that of EF-Tu from E. coli, which does not have these residues. Interdomain interactions must be important for the stabilisation of the structure of domain I, since isolated T. thermophilus EF-TuI is thermolabile despite the presence of the 181-190 loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Purification of aminoacyl-tRNA by affinity chromatography on immobilized Thermus thermophilus EF-Tu.GTP.

Elongation factor Tu from Thermus thermophilus containing six histidine residues on its C-terminus, EF-Tu(CHis6), was used for purification of aminoacyl-tRNA isoacceptors, from aminoacylated bulk tRNA, by affinity chromatography. Preformed aminoacyl-tRNA.EF-Tu(CHis6).GTP ternary complexes were immobilized on Ni(2+)-nitriloacetic acid agarose and the aminoacyl-tRNA was eluted at high ionic strength or with buffers containing GDP. Compared to alternative methods, the reported immobilization by C-terminal histidine residues does not lead to loss of EF-Tu's affinity for aminoacyl-tRNA. The method is well suited for preparative isolation of aminoacylated tRNA isoacceptors and as an analytical tool to test tRNA composition and aminoacylation of tRNA in crude cellular extracts.

Base Sequence↗
PubMed1995

Site-directed mutagenesis of Thermus thermophilus elongation factor Tu. Replacement of His85, Asp81 and Arg300.

His85 in Thermus thermophilus elongation factor Tu (EF-Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF-Tu, [H85L] EF-Tu and [H85G]EF-Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF-Tu, and replacement of Arg300 provided [R300I]EF-Tu. Glycine in position 85 of domain I induces a protease-sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF-Tu.GTP, inducing a protease-sensitive cleavage site in domain II. [H85Q]EF-Tu, which can be effectively overproduced in Escherichia coli, is slower in poly(U)-dependent poly(Phe) synthesis, has lower affinity to aminoacyl-tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl-tRNA and ribosomes. The slow GTPase of [H85Q]EF-Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)-dependent ribosomal translation. Replacement of His85 in T. thermophilus EF-Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl-tRNA binding. [H85L]EF-Tu is inactive in poly(U)-dependent poly(Phe)-synthesis. The rate of nucleotide dissociation is highest for [H85L]EF-Tu, followed by [H85Q]EF-Tu and native T. thermophilus EF-Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF-Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.

Amino Acid Sequence↗
PubMed1994

Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system.

Transforming growth factor-beta (TGF-beta) is a potent growth regulatory protein normally secreted by cells in a latent form. Primary regulation of TGF-beta activity occurs through factors which control the processing of the latent to the biologically active molecule. Thrombospondin (TSP), a platelet alpha-granule and extracellular matrix protein, forms specific complexes with active TGF-beta in platelet releasate and activates endogenous latent TGF-beta secreted by endothelial cells via a cell- and protease-independent mechanism. In order to better understand TSP-mediated activation of cell-secreted latent TGF-beta, we examined the consequences of interactions of the large (platelet-derived) and small (recombinant) forms of latent TGF-beta with TSP in a chemically defined system. Data from these studies show that interactions between TSP and both forms of latent TGF-beta result in the generation of biologically active TGF-beta as assayed by the ability of NRK-49F cells to form colonies in soft agar, by the ability to compete for binding to TGF-beta receptors on endothelial cells, and by an enzyme-linked immunosorbent assay selective for the active form of TGF-beta. Activation of latent TGF-beta by TSP stripped of associated TGF-beta activity (sTSP) is time- and concentration-dependent, but temperature-independent. The mechanism whereby sTSP activates latent TGF-beta appears to involve the direct binding of sTSP to the latent molecule as shown by gel permeation chromatography. In addition, a polyclonal antibody specific for the amino-terminal region of the latency-associated peptide (amino acids 81-94) inhibits sTSP-mediated activation of latent TGF-beta in both the chemically defined system and in endothelial cell conditioned medium. These data and the observation that similar concentrations of sTSP activate latent TGF-beta in both the chemically defined system and in the endothelial cell system indicate that there is a common mechanism by which TSP activates the small, large, and endothelial cell-derived latent TGF-beta complexes. The ability of TSP to convert latent TGF-beta to biologically active TGF-beta suggests that TSP is a major regulatory factor in the control of TGF-beta activity.

Animals↗
PubMed1994

Minimalist aminoacylated RNAs as efficient substrates for elongation factor Tu.

We demonstrate here, using RNA variants derived from tRNAAsp, that the minimalist aminoacylated structure able to interact efficiently with elongation factor Tu comprises a 10 base-pair helix linked to the 3'-terminal NCCA sequence. Shorter structures can interact with the elongation factor, but with significantly decreased affinity. Conserved features in the aminoacyl acceptor branch of tRNAs, such as base pair G53-C61 and the T-loop architecture, could be replaced respectively by the inverted base pair C53-G61 and by unusual anticodon loop or tetraloop sequences. Variants of whole tRNAAsp or of the 12 base-pair aspartate minihelix, with enlarged 13 base-pair long aminoacyl acceptor branches, as in selenocysteine-inserting tRNAs that are not recognized by elongation factor Tu, keep their binding ability to this factor. These functional results are well accounted for by the crystallographic structure of the Thermus thermophilus binary EF-Tu.GTP complex, which possesses a binding cleft accommodating the minimalist 10 base-pair domain of the tRNA aminoacyl acceptor branch.

Acylation↗
PubMed1993

Ternary complex between elongation factor Tu.GTP and Phe-tRNA(Phe).

The effect of aminoacylation and ternary complex formation with elongation factor Tu.GTP on the tertiary structure of yeast tRNA(Phe) was examined by 1H-NMR spectroscopy. Esterification of phenylalanine to tRNA(Phe) does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA(Phe) with elongation factor Tu.GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA(Phe) upon binding to elongation factor Tu.GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA(Phe)-elongation factor Tu-GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA(Phe) preserves its typical L-shaped tertiary structure in the complex. At high (> 10(-4) M) ligand concentrations a complex between tRNA(Phe) and elongation factor Tu-GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA(Phe) and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.

Acylation↗
PubMed1991

Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.

Base Sequence↗
PubMed1989

Sialic acid is a cell surface component of Entamoeba invadens trophozoites.

The surface anionic groups of Entamoeba invadens were analysed by cell electrophoresis, by ultrastructural cytochemistry, and by identification of sialic acids using paper and gas-liquid chromatography. Binding of colloidal iron hydroxide (CIH) and of cationized ferritin (CF) particles at pH 1.8 and 7.2, respectively, was observed on the cell surface. E. invadens has a highly negative surface charge (-0.96 microns s-1 V-1 cm). Treatment of the cells with trypsin and neuraminidase significantly reduced the electrophoretic mobility by 24% and 40%, respectively. Treatment of the amoebae with neuraminidase also markedly decreased the binding of CIH to the cell surface. This finding suggests that sialic acid residues are the major anionogenic groups exposed on the surface of E. invadens. Paper and gas-liquid chromatography showed that N-acetylneuraminic acid was the only derivative characterized in E. invadens.

Animals↗