A phylogenetic analysis reveals an unusual sequence conservation within introns involved in RNA editing.
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Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines within cellular and viral RNAs. Certain glutamate receptor (gluR) pre-mRNAs are substrates for the enzymes in vivo. For example, at the R/G editing site of gluR-B, -C, and -D RNAs, ADARs change an arginine codon (AGA) to a glycine codon (IGA) so that two protein isoforms can be synthesized from a single encoded mRNA; the highly related gluR-A sequence is not edited at this site. To gain insight into what features of an RNA substrate are important for accurate and efficient editing by an ADAR, we performed a phylogenetic analysis of sequences required for editing at the R/G site. We observed highly conserved sequences that were shared by gluR-B, -C, and -D, but absent from gluR-A. Surprisingly, in contrast to results obtained in phylogenetic analyses of tRNA and rRNA, it was the bases in paired, helical regions whose identity was conserved, whereas bases in nonhelical regions varied, but maintained their nonhelical state. We speculate this pattern in part reflects constraints imposed by ADAR's unique specificity and gained support for our hypotheses with mutagenesis studies. Unexpectedly, we observed that some of the gluR introns were conserved beyond the sequences required for editing. The approximately 600-nt intron 13 of gluR-C was particularly remarkable, showing >94% nucleotide identity between human and chicken, organisms estimated to have diverged 310 million years ago. (+info)
Tonic activation of A(2A) adenosine receptors unmasks, and of A(1) receptors prevents, a facilitatory action of calcitonin gene-related peptide in the rat hippocampus.
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1. We investigated how manipulations of the degree of activation of adenosine A(1) and A(2A) receptors influences the action of the neuropeptide, calcitonin gene-related peptide (CGRP) on synaptic transmission in hippocampal slices. Field excitatory post-synaptic potentials (EPSPs) from the CA1 area were recorded. 2. When applied alone, CGRP (1 - 30 nM) was without effect on field EPSPs. However, CGRP (10 - 30 nM) significantly increased the field EPSP slope when applied to hippocampal slices in the presence of the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopenthyl xanthine (DPCPX, 10 nM), or in the presence of the A(2A) adenosine receptor agonist CGS 21680 (10 nM). 3. The A(2A) receptor antagonist, ZM 241385 (10 nM) as well as adenosine deaminase (ADA, 2 U ml(-1)), prevented the enhancement of field EPSP slope caused by CGRP (30 nM) in the presence of DPCPX (10 nM), suggesting that this effect of CGRP requires the concomitant activation of A(2A) adenosine receptors by endogenous adenosine. 4. The protein kinase-A inhibitors, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 microM) and adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS, 50 microM), as well as the inhibitor of ATP-sensitive potassium (K(ATP)) channels, glibenclamide (30 microM), prevented the facilitation of synaptic transmission caused by CGRP (30 nM) in the presence of DPCPX (10 nM), suggesting that this effect of CGRP involves both K(ATP) channels and protein kinase-A. 5. It is concluded that the ability of CGRP to facilitate synaptic transmission in the CA1 area of the hippocampus is under tight control by adenosine, with tonic A(1) receptor activation by endogenous adenosine 'braking' the action of CGRP, and the A(2A) receptors triggering this action. (+info)
Evidence for control of adenosine metabolism in rat oxidative skeletal muscle by changes in pH.
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1. We investigated the effects of pH elevation or depression on adenosine output from buffer-perfused rat gracilis muscle, and kinetic properties of adenosine-forming enzymes, 5'-nucleotidase (5'N) and non-specific phosphatase (PT), and adenosine-removing enzymes, adenosine kinase (AK) and adenosine deaminase (AD), in homogenates of muscle. 2. Depression of the perfusion buffer pH from 7.4 to 6.8, by addition of sodium acetate, reduced arterial perfusion pressure from 8.44 +/- 1.44 to 7.33 +/- 0.58 kPa, and increased adenosine output from 35 +/- 5 to 56 +/- 6 pmol min-1 (g wet wt muscle)-1 and AMP output from 1.8 +/- 0.3 to 9.1 +/- 3.9 pmol min-1 (g wet wt muscle)-1. 3. Elevation of the buffer pH to 7.8, by addition of ammonium chloride, reduced arterial perfusion pressure from 8.74 +/- 0.57 to 6.96 +/- 1.37 kPa, and increased adenosine output from 25 +/- 5 to 47 +/- 8 pmol min-1 (g wet wt muscle)-1 and AMP output from 3.7 +/- 1.1 to 24.6 +/- 6.8 pmol min-1 (g wet wt muscle)-1. 4. Activity of membrane-bound 5'N was an order of magnitude higher than that of either cytosolic 5'N or PT: pH depression reduced the K(m) of 5'N, which increased its capacity to form adenosine by 10-20% for every 0.5 unit decrease inpH within the physiological range. PT was only found in the membrane fraction: its contribution to extracellular adenosine formation increased from about 5% at pH 7.0 to about 15% at pH 8.0. 5. Cytosolic 5'N had a low activity, which was unaffected by pH; the rate of intracellular adenosine formation was an order of magnitude lower than the rate of adenosine removal by adenosine kinase or adenosine deaminase, which were both exclusively intracellular enzymes. 6. We conclude that (i) adenosine is formed in the extracellular compartment of rat skeletal muscle, principally by membrane-bound 5'N, where it is protected from enzymatic breakdown; (ii) adenosine is formed intracellularly at a very low rate, and is unlikely to leave the cell; (iii) enhanced adenosine formation at low pH is driven by an increased extracellular AMP concentration and an increased affinity of membrane-bound 5'N for AMP; (iv) enhanced adenosine formation at high pH is driven solely by the elevated extracellular AMP concentration, since the catalytic capacity of membrane 5'N is reduced at high pH. (+info)
The role of adenosine receptors in the action of theophylline on human peripheral blood mononuclear cells from healthy and asthmatic subjects.
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1. The aim of the present study was to investigate the role of adenosine A2b receptors in the anti-proliferative action of theophylline in human peripheral blood mononuclear cells (HPBMC) from healthy and asthmatic subjects. 2. Theophylline significantly inhibited PHA-induced proliferation of HPBMC from both healthy and asthmatic donors but only at relatively high concentrations at 1 mM (P<0.05). Enprophylline, a drug which also acts as a non-selective phosphodiesterase (PDE) inhibitor and is a selective A2b receptor antagonist, had no significant effect on proliferation of cells from either group at concentrations up to 10 microM (P>0.05; n=6). 3. Adenosine deaminase (2 u ml(-1)), which metabolizes adenosine, had no significant effect on PHA-induced HPBMC proliferation over a range of concentrations (0 - 8 microg ml(-1)) in cells from either healthy or asthmatic subjects. 4. The adenosine receptor agonists N(6)-cyclopentyladenosine (CPA, A1-selective) and 5'-N-ethylcarboxamidoadenosine (NECA, A1/A2) produced a small but significant inhibition of PHA-induced proliferation of HPBMC from healthy and asthmatic subjects (10 microM, P<0.05; n=6). In contrast, 5'-N-ethylcarboxamido-2-[4-(2-]carboxyethyl)phenethyl]adenosine (CGS21680, A2a-selective) was without significant effect (P>0.05; n=6). 5. The adenosine receptor antagonist alloxazine (A2b-selective) had no significant effect, while 8(3-chlorostyryl)caffeine,(CSC, A2a-selective) significantly inhibited PHA-induced proliferation of HPBMC from both groups (P<0.05; n=6). 6. Our results suggest that endogenous or exogenous adenosine has little effect on the proliferation of HPBMC obtained from healthy or asthmatic subjects. Thus it would appear that the effect of high concentrations of theophylline is not related to adenosine receptor antagonism. (+info)
Absence of oroticaciduria in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency.
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Orotic acid excretion was normal when tested by three methods in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. These results do not support the speculation, based on the oroticaciduria observed by others, that the immunodeficiency in these disorders results from the inhibition of pyrimidine biosynthesis. An alternative hypothesis is discussed. (+info)
Selective disruption of protein aggregation by cyclodextrin dimers.
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Beta-cyclodextrin (CD) dimers (n = 11) were synthesized and tested against eight enzymes, seven of which were dimeric or tetrameric, for inhibitor activity. Initial screening showed that only L-lactate dehydrogenase and citrate synthase were inhibited but only by two specific CD dimers in which two beta-CDs were linked on the secondary face by a pyridine-2,6-dicarboxylic group. Further investigation suggested that these CD dimers inhibit the activity of L-lactate dehydrogenase and citrate synthase at least in part by disruption of protein-protein aggregation. (+info)
First-pass disposition of (-)-6-aminocarbovir in rats: II. Inhibition of intestinal first-pass metabolism.
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A CBV [(-)-carbovir, (-)-carbocyclic 2',3'-didehydro-2', 3'-dideoxyguanosine] prodrug, 6AC [(-)-6-aminocarbovir, (-)-carbocyclic 2',3'-didehydro-2', 3'-dideoxy-6-deoxy-6-aminoguanosine], was previously evaluated in rats, and it exhibited superiority to the parent drug in increasing systemic and central nervous system exposure to CBV. The gut wall was determined to be the dominant site of the first-pass activation of 6AC after lumenal administration. If subsequent delivery to the brain is desired, then such a first-pass effect might not be viewed favorably. Because the first-pass conversion of 6AC primarily takes place in the intestine by adenosine deaminase (ADA), quenching of the intestinal activation of 6AC by oral administration of ADA inhibitors may result in an increased 6AC bioavailability, and thus an improved brain exposure to CBV. The objectives of the study were to determine whether the ADA inhibitors 2'-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)adenine were capable of achieving a substantial and selective inhibition of gut wall activation of 6AC, and to determine whether the systemic concentrations of 6AC would be thus increased. Thirty-nine male Sprague-Dawley rats were divided into two groups. One group received 6AC by either the portal vein or intralumenally with the coadministration of intralumenal 2'-deoxycoformycin. Similarly, the other group received 6AC with coadministration of erythro-9-(2-hydroxy-3-nonyl)adenine. Substantial suppression of the first-pass conversion of 6AC was achieved with both inhibitors. This inhibition appeared to be relatively selective, allowing the choice of dose of inhibitor that would sufficiently inhibit the first-pass metabolism while leaving the activation capacity in the systemic circulation unaltered. The systemic level of 6AC increased with the escalating dose of inhibitors, thus increasing the driving force for passive uptake into the brain. (+info)
Morphological and molecular evidence of differentiation during etoposide-induced apoptosis in human lymphoblastoid cells.
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The relationship between apoptosis and cell differentiation has been a subject for continuous debate, with evidence showing leukaemic cell differentiation and drug-induced apoptosis have reciprocal, interdependent and a highly schedule-dependent relationship. We have addressed this relationship in terms of a widely-used model for apoptosis induced by cytotoxic drugs: namely the effect of etoposide on CEM cells. In respect of commitment toward differentiation, we assessed changes in expression of marker genes and the level of CD3 antigenicity. Changes in these parameters following exposure of CEM cells to etoposide was similar to that observed following treatment of the same cells with phorbol 12-myristate 13-acetate (PMA), though this latter treatment did not cause cell death. Similarities in response to etoposide and PMA also included generation of 50 kilobase fragmentation of DNA and convolution of nuclei as assessed by transmission electron microscopy. However, condensation of chromatin and externalization of phosphatidylserine were only recorded in response to the cytotoxic drug and not in response to PMA. The data are consistent with apoptosis in these lymphoblastoid cells being accompanied by activation of specific markers of T-cell differentiation, but ultimately involving processes unequivocally associated with cell death. (+info)