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PubMed2004

Structures of human purine nucleoside phosphorylase complexed with inosine and ddI.

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2('),3(')-dideoxyinosine, refined to 2.8A resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design.

Binding Sites↗
PubMed2005

Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency in the Japanese population.

Inosine triphosphate pyrophosphohydrolase (ITPase) is an enzyme that catalyzes the conversion of inosine triphosphate (ITP) to inosine monophosphate and pyrophosphate. In Caucasian populations it is reported that the frequency of cases showing decreased ITPase activity is 5%. The structure of ITPA gene along with five single nucleotide polymorphisms has been reported in Caucasians. We examined ITPase activity and frequency of two polymorphisms (94C>A and IVS2+21A>C) in 100 Japanese individuals. Among these individuals, we observed that three cases with zero activity were homozygote for 94C>A, and were accompanied by abnormal accumulation of ITP in erythrocytes. The cases included in the low ITPase activity group were heterozygote for 94C>A polymorphism. The activity of the heterozygote cases was approximately 27% of the mean value of the wild type. The allele frequency of the 94C>A polymorphism was 0.155, which was 2.6 times higher than that of the Caucasians (0.06). The IVS2+21A>C was not detected in Japanese cases, although it occurred with a frequency of 0.130 in Caucasians. Furthermore, we identified a novel mutation IVS2+68T>G in intron 2 in the case with the lowest enzyme activity in the 94C>A wild type. Since the frequency of ITPA 94C>A polymorphism is higher in the Japanese population than that in Caucasians, it is more important to examine ITPA 94C>A polymorphism in the Japanese population to prevent thiopurine drug toxicity. Pretherapeutic screening of individuals for ITPA polymorphisms should be considered for safer and more tolerable treatment with thiopurine drugs.

Adult↗
PubMed2004

Comparison of the effect of water release on the interaction of the Saccharomyces cerevisiae TATA binding protein (TBP) with "TATA Box" sequences composed of adenosine or inosine.

The formation of sequence-specific complexes of TATA binding protein (TBP) with the minor groove of DNA results in the burial of large nonpolar surfaces and the exclusion of water from these interfaces. The release of water is thus expected to provide a significant entropic driving force for formation of the transcription-preinitiated complexes mediated by the binding of TBP to specific sequences. In this article are described equilibrium-binding studies of Saccharomyces cerevisiae TBP to 14 bp oligonucleotides bearing either the tightly bound and efficiently transcribed adenovirus major late promoter (TATAAAAG) or its inosine-substituted derivative (TITIIIIG) as a function of neutral osmolyte concentration. These two DNA sequences present the same pattern of minor groove hydrogen-bond donors and acceptors to the protein. TBP-DNA complex formation was monitored by steady-state fluorescence resonance energy transfer measurements of the oligonucleotides end-labeled with fluorescein (donor) and TAMRA (acceptor). Correct interpretation of the results obtained with the inosine-substituted sequence required careful consideration of the optical properties of the dyes as a function of osmolyte concentration to demonstrate that the relative change in the end-to-end distances for TATAAAAG- and TITIIIIG-bearing oligonucleotides is the same upon TBP binding. Although the affinity of TBP is slightly greater for the adenosine compared with the inosine-substituted TATA sequence in the absence of osmolyte, the end-to-end distances of the bound DNA in complex with TBP, the enthalpic and electrostatic components of binding, are identical within experimental precision. However, approximately 18 additional molecules of water are released upon TBP binding the TATAAAAG as compared with the TITIIIIG sequence resulting in an entropic advantage to the binding of the natural promoter sequence. These results are considered with regard to differences in the flexibility and hydration of the two DNA sequences.

Adenosine↗
PubMed1991

Quantitation of adenosine, inosine and hypoxanthine in biological samples by microbore-column isocratic high-performance liquid chromatography.

This paper describes a simple and sensitive high-performance liquid chromatographic method for measuring adenosine, inosine and hypoxanthine in cell suspensions. The method involves direct injection of the filtered sample on a microbore C18 reversed-phase column with UV detection at 259 nm. The mobile phase consisted of 125 mM potassium dihydrogenphosphate, 1.0 mM tetrabutylammonium hydrogen-sulfate, 1.5% acetonitrile and 20 mM triethylamine, pH 6.5. The minimum detectable amounts (signal-to-noise ratio of 3:1) were 2.0 pmol of adenosine, 2.5 pmol inosine and 3.5 pmol of hypoxanthine. The limits of quantitation were 2.9 +/- 0.2 pmol for adenosine, 4.2 +/- 0.3 pmol for inosine and 4.9 +/- 0.4 pmol for hypoxanthine. This method was used to quantitate adenosine release by dispersed rat renal outer medullary cells (tubules) under conditions of normoxia and hypoxia.

Adenosine↗
PubMed1999

Ribose 1-phosphate and inosine activate uracil salvage in rat brain.

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.

Animals↗
PubMed1986

Measurement of adenosine, inosine and hypoxanthine in human term placenta by reversed-phase high-performance liquid chromatography.

An analytical method was developed for measuring adenosine, inosine and hypoxanthine in freshly delivered human term placentas. Representative freeze-clamped samples were taken from the sub-maternal surface of each placenta. Acid-soluble extracts of the samples were analyzed by reversed-phase high-performance liquid chromatography on columns packed with 10-micron porous octadecylsilica, using gradient elution with a linear increase in methanol concentration in ammonium phosphate buffer. Resolution of hypoxanthine from xanthine and adenosine from adenine, and quantitation of hypoxanthine and adenosine were achieved using 0.05 M ammonium dihydrogen phosphate, pH 6.5, as the low-strength eluent. Resolution of inosine from a prominent peak of beta-NAD was optimized using 0.02 M ammonium dihydrogen phosphate, pH 5.6, as low-strength eluent. Recovery of standards was greater than 90%. Mean contents (+/- S.D.) of the analytes in placentas from seven normal deliveries were, adenosine 30.6 +/- 11.5 nmol/g, inosine 68.0 +/- 25.8 nmol/g and hypoxanthine 217 +/- 127.5 nmol/g.

Adenosine↗
PubMed2000

Inosine and 2'-deoxyinosine and their synthetic analogues: lipophilicity in the relation to their retention in reversed-phase liquid chromatography and the stability characteristics.

The purines and among them inosine synthetic nucleoside derivatives and analogues belong to a group of compounds to which the attention is being paid because of their biological activities. Relationships of their various parameters are being investigated because of their effect on biological (antineoplastic, virostatic, immunosuppressive) properties. Hydrophobicity parameters expressed as the logarithm of the partition coefficient (log P) and the capacity factor k' for naturally occurring inosine, 2'-deoxyinosine, 2'-deoxyadenosine and 2'-deoxyguanosine and for inosine synthetic analogues 5'-deoxyinosine, 5'-chloro-5'-deoxyinosine and 2',3'-dideoxyinosine were measured. The effect of methanol percentage in the mobile phase and its pH on the retention of the studied compounds in a reversed-phase system was also examined. There was a good correlation between the lipophilicity expressed as log P and capacity factor k'. It was also determined that dissociation has a marginal effect on capacity factor k' in this group of nucleoside derivatives as the k' values were almost unchanged at various pH of the mobile phase used. The stability of the all investigated compounds was investigated in basic, neutral and acidic conditions. The values of the reaction constant k1 were calculated and effects of nucleoside structural characteristic on stability are discussed.

Chromatography, High Pressure Liquid↗
PubMed1997

The modified wobble base inosine in yeast tRNAIle is a positive determinant for aminoacylation by isoleucyl-tRNA synthetase.

Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity. Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively. We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase. We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost. As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity. The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript. The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E. coli isoleucyl-tRNA synthetase. In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine [Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S. (1988) Nature 336, 179-181], pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS. Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity.

Acylation↗
PubMed1998

Binding of daunomycin to diaminopurine- and/or inosine-substituted DNA.

The binding of the anticancer drug daunomycin to double-helical DNA has been investigated by DNase I footprinting and fluorescence titration, using a series of polymerase chain reaction (PCR) synthesized DNA fragments that contained systematic base substitutions to alter the disposition of functional groups within the minor groove. The 160 bp tyrT DNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substituted for adenine, and (iii) both inosine and diaminopurine were substituted for guanosine and adenine, respectively, were studied. These fragments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experiments confirmed that daunomycin binds preferentially to 5'(A/T)GC and 5'(A/T)CG triplets in the normal fragment. Substitution of inosine for guanosine, with the concomitant loss of the N-2 in the minor groove, weakened binding affinity but did not dramatically alter the sequence preference associated with daunomycin binding. Complete reversal of the location of the N-2 group by the double substitution, however, completely altered the sequence preference of daunomycin and shifted its binding from the canonical triplets to ones with a 5'IDD motif. These results have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating binding to preferred sites. In a parallel study, both macroscopic and microscopic binding to the normal tyrT fragment were investigated, experiments made possible by using PCR to prepare large quantities of the long, defined DNA sequence. The results of these experiments underscored the complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different binding sites. A class of high-affinity sites, most probably corresponding to the 5'(A/T)GC and 5'(A/T)CG triplets, was identified and characterized in macroscopic binding isotherms.

Adenosine↗
PubMed1981

Uridine and inosine: pressor nucleosides from man, rat and dog.

Short-acting pressor compounds isolated from rat kidney, brain, heart and spleen have been identified as inosine and uridine by gas chromatography, mass spectrometry, high pressure liquid chromatography and analysis of ultraviolet spectra. Inosine was further identified by nuclear magnetic resonance. These compounds have also been found in kidneys from hypertensive man, rejected renal transplants and dog and beef kidney. Tissues were extracted by acid ethanol extraction followed by gel filtration and high voltage paper electrophoresis. Compounds found to be pressor in the anaesthetised rat resisted proteolytic enzymes, boiling for 10 min, extremes of pH and incubation with plasma from the source species for up to 20 min. The pressor effects of bolus injections of active gel filtration fractions, uridine and inosine were short-lived with a maximum effect at 5-6 s. Intravenous (I.V.) infusions of extracts gave a sustained pressor response without a concurrent change in heart rate. The effect on blood pressure was not accompanied by increased heart rate and persisted when the pressor effects of angiotensin II, noradrenaline and 5-hydroxy-tryptamine were blocked pharmacologically.

Animals↗
PubMed2006

Cellular effects and metabolic stability of N1-cyclic inosine diphosphoribose and its derivatives.

BACKGROUND AND PURPOSE: Recently, a number of mimics of the second messenger cyclic ADP-ribose (cADPR) with replacement of adenosine by inosine were introduced. In addition, various alterations in the molecule ranging from substitutions at C8 of the base up to full replacement of the ribose moieties still retained biological activity. However, nothing is known about the metabolic stability and cellular effects of these novel analogues. EXPERIMENTAL APPROACH: cADPR and the inosine-based analogues were incubated with CD38, ADP-ribosyl cyclase and NAD-glycohydrolase and metabolism was analysed by RP-HPLC. Furthermore, the effect of the analogues on cytokine expression and proliferation was investigated in primary T-lymphocytes and T-lymphoma cells. KEY RESULTS: Incubation of cADPR with CD38 resulted in degradation to adenosine diphosphoribose. ADP-ribosyl cyclase weakly catabolised cADPR whereas NAD-glycohydrolase showed no such activity. In contrast, N1-cyclic inosine 5'-diphosphoribose (N1-cIDPR) was not hydrolyzed by CD38. Three additional N1-cIDPR analogues showed a similar stability. Proliferation of Jurkat T-lymphoma cells was inhibited by N1-cIDPR, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (N1-cIDP-DE) and N1-ethoxymethyl-cIDPR (N1-cIDPRE). In contrast, in primary T cells neither proliferation nor cytokine expression was affected by these compounds. CONCLUSIONS AND IMPLICATIONS: The metabolic stability of N1-cIDPR and its analogues provides an advantage for the development of novel pharmaceutical compounds interfering with cADPR mediated Ca2+ signalling pathways. The differential effects of N1-cIDPR and N1-cIDPRE on proliferation and cytokine expression in primary T cells versus T-lymphoma cells may constitute a starting point for novel anti-tumor drugs.

ADP-ribosyl Cyclase↗
PubMed2004

Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1.

We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 [e.g. NBMPR [S6-(4-nitrobenzyl)-mercaptopurine riboside], dilazep or dipyridamole] were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92-->Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260+/-16 mM) when compared with the wild-type transporter (65+/-7.8 mM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.

Adenosine↗
PubMed1987

Effect of adenosine and inosine on ureagenesis in hepatocytes.

Adenosine and inosine produced a dose-dependent stimulation of ureagenesis in isolated rat hepatocytes. Hypoxanthine, xanthine and uric acid were without effect. Half-maximally effective concentrations were 0.08 microM for adenosine and 5 microM for inosine. Activation of ureagenesis by both nucleosides had the following characteristics: (a) it was observed with either glutamine or (NH4)2CO3, provided that glucose was present; (b) it was not detected when glucose was replaced by lactate plus oleate; (c) it was mutually antagonized by glucagon, but not by adrenaline; and (d) it was dependent on Ca2+. We suggest that the action of adenosine and inosine on ureagenesis might be of physiological significance.

Adenosine↗
PubMed2002

Pericardial concentrations of adenosine, inosine and hypoxanthine in an experimental canine model of spastic ischaemia.

It has been shown that the adenosine concentration in the pericardial fluid of the normal heart is higher by one order of magnitude than that of the venous plasma. A further increase in the pericardial adenosine concentration was also demonstrated in myocardial ischaemia or hypoxia. It was proposed that pericardial nucleoside levels may represent the interstitial concentrations of the adenine nucleosides. An experimental model was designed to determine the intrapericardial concentrations of adenosine, inosine and hypoxanthine during coronary spasm provoked by intracoronary administration of endothelin-1 (ET-1; 0.08+/-0.02 nmol/g of myocardial tissue). In the in situ dog heart (n=10), adenosine, inosine and hypoxanthine concentrations were determined by HPLC in fluid samples collected from the closed pericardial sac before and after ET-1 administration, and from the systemic arterial blood. Systemic blood pressure, heart rate and standard ECG were registered continuously. We found that the nucleoside concentrations in the infusate samples increased significantly during coronary spasm [adenosine, 1.49+/-0.44 compared with 0.37+/-0.07 microM (P<0.05); inosine, 27.43+/-11.51 compared with 0.47+/-0.11 microM (P<0.05); hypoxanthine, 21.17+/-6.49 compared with 4.91+/-1.24 microM (P<0.05)], while a significant decrease in blood pressure and an elevation in ECG ST segments were observed. The levels of the purine metabolites did not change in the systemic blood. The data indicate that changes in adenine nucleoside levels measured in pericardial infusate samples reflect activation of coronary metabolic adaptation in this model of spastic ischaemia, and that pericardial nucleoside levels may characterize alterations in interstitial adenine nucleoside concentrations.

Adenosine↗
PubMed2003

Widespread inosine-containing mRNA in lymphocytes regulated by ADAR1 in response to inflammation.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional modification of pre-mRNA catalysed by an RNA-specific adenosine deaminase (ADAR). A-to-I RNA editing has been previously reported in the pre-mRNAs of brain glutamate and serotonin receptors and in lung tissue during inflammation. Here we report that systemic inflammation markedly induces inosine-containing mRNA to approximately 5% of adenosine in total mRNA. Induction was the result of up-regulation of A-to-I RNA editing as both dsRNA editing activity and ADAR1 expression were increased in the spleen, thymus and peripheral lymphocytes from endotoxin-treated mice. Up-regulation of ADAR1 was confirmed in vitro in T lymphocytes and macrophages stimulated with a variety of inflammatory mediators including tumour necrosis factor-alpha and interferon-gamma. A late induction of RNA editing was detected in concanavalin A-activated splenocytes stimulated with interleukin-2 in vitro. Taken together, these data suggest that a large number of inosine-containing mRNAs are produced during acute inflammation via up-regulation of ADAR1-mediated RNA editing. These events may affect the inflammatory and immune response through modulation of protein production.

Acute Disease↗
PubMedDate not supplied

Blood storage XXII. Improvement in red blood cell 2,3-DPG levels at six weeks by 20 mM PO4 in CPD-adenine-inosine.

Inorganic phosphate has been known to assist red blood cell maintenance of ATP and in the presence of inosine to assist in the maintenance of 2,3-DPG. High concentrations of phosphate, while helping ATP maintenance, were found to be deleterious to 2,3-DPG maintenance in CPD-adenine preservatives. However, in the presence of inosine, concentrations of phosphate as high as 10 mM were advantageous to 2,3-DPG maintenance. The present study extends the observations on ATP and 2,3-DPG maintenance in CPD-adenine-inosine preservatives from the previous 10 mM to 20 mM phosphate. A high phosphate (20 mM) effect has been seen as improved maintenance of 2,3-DPG levels during the fifth and sixth weeks of storage of whole blood at 4C. This supports the previously reported observation of improved maintenance of 2,3-DPG in a 10 mM phosphate preservative. This is ten times the 2 mM phosphate concentration in CPD-adenine. In the low phosphate preservative (2 mM), 2,3-DPG maintenance is less than that in all of the higher phosphate preservatives after the second week of storage. ATP concentrations in this experiment show good maintenance throughout six weeks of storage.

Adenine↗
PubMedDate not supplied

Blood preservation 42: improvement of ascorbate's ability to maintain 2,3-DPG with inosine.

Inosine and ascorbate have been shown to maintain normal 2,3-DPG levels during three to four weeks of blood storage. With the introduction of CPD-adenine, which allows five weeks of storage, the desire for 2,3-DPG maintenance may receive new emphasis. Red blood cell 2,3-DPG remained at normal or higher levels for six weeks whenever 10 or 15 mM inosine and 10 mM vitamin C (L-ascorbate) or D-ascorbate were present in the CPD-adenine preservative. Provision by inosine of a five-carbon sugar for 2,3-DPG synthesis, bypassing the rate-limiting phosphofructokinase reaction, may allow NADH oxidation by ascorbate to provide an increased supply of substrate for the Rappoport-Luebering shunt, thus affecting the net increase and maintenance of 2,3-DPG.

Ascorbic Acid↗
PubMed1990

Intravenous adenosine but not its first metabolite inosine provokes chest pain in healthy volunteers.

Intravenous (i.v.) bolus administration of adenosine causes increased ventilation and an angina pectoris-like chest pain. Whether adenosine per se or one of its metabolites such as inosine mediates these effects is not clear. Bolus doses of adenosine, inosine, or saline were administered i.v. blindly to six volunteers. Spirometry, ECG recordings, and pain ratings were taken. Adenosine induced both an increase in tidal volume and respiration rate, a dose-dependent chest pain and, at higher doses, various degrees of atrioventricular (AV) block. None of these effects were noted after equimolar injections of inosine or saline. The findings indicate that the angina pectoris-like pain and increased ventilation is induced by adenosine per se and is not produced by adenosine metabolites.

Adenosine↗