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BACKGROUND: In the past 10-20 years, knowledge of both thiopurine pharmacology and -pharmacogenetics has been extended dramatically and used to develop new strategies to improve efficacy and reduce toxicity. AIM: To review thiopurine efficacy, toxicity, pharmacology, pharmacogenetics, interactions in patients with inflammatory bowel disease. Special attention was paid to new strategies for optimization of pharmacotherapy. METHODS: To collect relevant scientific articles, a Pubmed search was performed from 1966 through January 2006 with the following key words (MeSH terms preferentially) in multiple combinations: 'azathioprine', '6-mercaptopurine', '6-MP', '6-thioguanine', '6-TG', 'thiopurine(s)', 'metabolites', 'level(s)', 'TDM', 'TMPT', 'ITPA', 'genotype(s)', 'phenotype(s)', 'inflammatory bowel disease', 'Crohn('s) disease', 'ulcerative colitis'. RESULTS: Strategies for optimization of pharmacotherapy include therapeutic drug monitoring of thiopurine metabolites, geno- or phenotyping crucial enzymes in thiopurine metabolism like thiopurine S-methyltransferase and inosine triphosphate pyrophosphatase, and the use of thioguanine as such. CONCLUSIONS: Thiopurine S-methyltransferase genotyping and therapeutic drug monitoring are useful instruments for individualizing thiopurine pharmacotherapy of inflammatory bowel disease. Inosine triphosphate pyrophosphatase genotyping may be helpful in case of unexplainable myelotoxicity. In case of azathioprine- or mercaptopurine-intolerance, thioguanine seems a promising alternative. However, more knowledge needs to be gathered about its potential hepatotoxicity.
Inosine triphosphate (ITP) and xanthosine triphosphate (XTP) are formed upon deamination of ATP and GTP as a result of exposure to chemical mutagens and oxidative damage. Nucleic acid synthesis requires safeguard mechanisms to minimize undesired lethal incorporation of ITP and XTP. Here, we present the crystal structure of YjjX, a protein of hitherto unknown function. The three-dimensional fold of YjjX is similar to those of Mj0226 from Methanococcus janschii, which possesses nucleotidase activity, and of Maf from Bacillus subtilis, which can bind nucleotides. Biochemical analyses of YjjX revealed it to exhibit specific phosphatase activity for inosine and xanthosine triphosphates and have a possible interaction with elongation factor Tu. The enzymatic activity of YjjX as an inosine/xanthosine triphosphatase provides evidence for a plausible protection mechanism by clearing the noncanonical nucleotides from the cell during oxidative stress in E. coli.
During transcription of DNA templates in vitro, Escherichia coli RNA polymerase pauses at certain sequences before resuming elongation. Previous studies have established that some pausing events are brought about by the formation of RNA hairpin structures in the nascent transcript; however, it is not known whether this is an invariant and causal relationship. We have mapped and characterized almost 200 distinct pause sites located within the early region of bacteriophage T7 DNA using a collection of T7 deletion mutant DNAs and taking advantage of a procedure that permits synchronous transcription from the T7 A1 promoter. The pausing pattern is sensitive both to the overall concentration of nucleotide substrates and to the relative concentrations of the four nucleotides. The apparent Ks value for a particular nucleoside triphosphate can vary over a 500-fold range depending on the nucleotide sequence, and pausing at some sites can be induced by modest reductions in substrate concentrations. However, pausing is not solely a consequence of substrate limitation. Pausing at certain sites is caused by some feature of the template or of the transcript itself. Substitution of inosine triphosphate (ITP) for GTP during transcription strongly affects the pattern and strength of pausing events, suggesting that base-pairing interactions involving the RNA strand are important for some pausing events. Other pauses are determined by sequences downstream from the elongation site that have not yet been transcribed, and pausing at these sites is generally insensitive to substitution of IMP for GMP in the nascent transcript. Pausing at one particular site on T7 DNA is strongly enhanced by the presence of E. coli gene nusA protein. These results confirm that there are multiple classes of sites that lead to transcriptional pausing, and provide a collection of sites for further study. Using selected pause sites in the early region of T7 DNA, we have tried to evaluate the possible roles of primary sequence, base composition and secondary structure in pausing. Computer analysis was used to compare primary sequences and potential RNA hairpin structures in transcripts for pauses known to share similar biochemical properties. We see no correlation of pause sites with regions of particular base composition or with specific primary sequences. While some pauses are correlated with the potential to form stable RNA hairpins just upstream from the growing point of the RNA chain, there is not a strict one-to-one relationship between predicted RNA hairpins and the location of pause sites.(ABSTRACT TRUNCATED AT 400 WORDS)
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Guanyl nucleotides are known to play a dual role in the activation of the adenylate cyclase system of the rat corpus luteum, being required for human choriogonadotropin (hCG) stimulation of the enzyme and modulating hCG binding to some hormone receptors. Current models of adenylate cyclase activation require that guanyl nucleotide binding be enhanced by hormones, and we have examined this binding in rat luteal membrane preparations known to contain guanyl nucleotide-modulated hCG receptors. [3H] Guanylyl-imidodiphosphate (GMPPnP), a nonhydrolyzable analog of guanosine triphosphate (GTP), was used to investigate binding to urea-washed, heavy rat luteal membranes. Binding was found to be linear, with respect to the amount of membranes added, in the range of 2-10 mg wet wt. tissue equivalents, and equilibrium was reached after a 30-min incubation at 30 degrees C. Analysis of equilibrium binding experiments gave a Ka of 1.2.10(7) +/- 0.9.10(7) M-1, with 460 +/- 430 fmol binding sites per mg tissue in the absence of hormone, Kinetic experiments showed an association rate constant of 2.6.10(5) +/- 0.5.10(5) M-1 min-1 and a dissociation rate constant of 1.8.10(-2) +/- 0.9.10(-2) min-1. In the presence of hCG, the Ka was unchanged; however, the number of binding sites increased by 50-120%. Competitive binding assays utilizing other nucleotides revealed that a hierarchy of GMPPnP = GTP greater than guanosine diphosphate (GDP) greater than inosine triphosphate (ITP) in displacing labeled GMPPnP. A similar hierarchy was also found for hCG-stimulated adenylate cyclase activity (GMPPnP = GTP greater than ITP) and for modulation of hCG binding (GMPPnP greater than GTP greater than ITP).(ABSTRACT TRUNCATED AT 250 WORDS)
1. Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2. The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3. In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4. ATP and UTP (ED50 both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5. The order of potency for the purine nucleotides in stimulating InsP3 formation was ATP = ATP-gamma-S greater than ADP greater than 2-methylthio-ATP greater than AMP = a,beta methylene-ATP = adenosine. 6. The increase of InsP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (greater than 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) M was observed. 7. Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) M) on InsP3 formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP.8. The results indicate that an increase of [Ca2+]i and inositol phosphate breakdown can be mediated via activation of a P2 receptor in human GEC.
Rat peritoneal mast cells have been permeabilised by treatment with streptolysin O which generates membrane lesions of macromolecular dimensions. In the presence of Ca2+ buffered at concentrations in the micromolar range, the permeabilised mast cells release histamine, beta-N-acetylglucosaminidase and lactate dehydrogenase. Release of the two secretory components (but not lactate dehydrogenase) has an obligatory requirement for a nucleoside triphosphate and micromolar concentrations of Ca2+. Inosine triphosphate (ITP) supports the release reaction better than ATP does. It is concluded that the secretory materials are released from the cells by an exocytotic mechanism, while lactate dehydrogenase leaks from the cells through the toxin-generated lesions. By initially withholding and then supplying Ca2+ to the permeabilised cells, it is shown that the exocytotic secretory reaction can persist even when the cytosol is depleted of the bulk of soluble proteins. The streptolysin O treated mast cell preparation represents a simplified system with which to study the mechanism of exocytosis.
Human erythrocytes incubated in inosine-pyruvate-phosphate (IPP) medium are able to accumulate great amounts of inosine monophosphate (IMP) and inosine triphosphate (ITP). Accumulation of ITP is low in freshly drawn cells. It increases during blood storage in acid-citrate-dextrose solution. Addition of labelled inosine or hypoxanthine to the IPP medium allowed to show the synthesis of small amounts of inosine diphosphate and the appearance of radioactivity in the ATP fraction. Conditions of incubation and the pathways of hypoxanthine nucleotides synthesis are discussed.
Drapeau, Gabriel R., (Macdonald College of McGill University, Quebec, Canada) and Robert A. MacLeod. Nutrition and metabolism of marine bacteria. XII. Ion activation of adenosine triphosphatase in membranes of marine bacterial cells. J. Bacteriol. 85:1413-1419. 1963.-Isolated membranes of two species of marine bacteria, a Pseudomonas and a Cytophaga, have been shown to possess adenosine triphosphatase activity. The optimal pH for enzyme action of both organisms was 8.8. The enzyme system was found to be capable of splitting inorganic o-phosphate from adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate, and inosine triphosphate but not from inorganic pyrophosphate. Mg(++) was required for enzyme activity; with the Pseudomonas species, the optimal Mg(++) to ATP ratio was 1:1. Ca(++) could not replace Mg(++). In the presence of the optimal concentration of Mg(++), the enzyme system was further stimulated, nonspecifically, by a number of different salts. Maximal activation was achieved at an ionic strength of 0.3 to 0.4. No evidence of an adenosine triphosphatase specifically activated by a combination of Na(+) and K(+) was obtained with either organism. No effect of ouabain on either the membrane adenosine triphosphatase activity or Na(+) transport by whole cells could be detected. The results suggest that the mechanism of ion regulation in marine bacterial cells is different from that in animal cells.
Using a reliable 2-step colorimetric assay, some catalytic properties of human erythrocyte ITPase (inosine triphosphate pyrophosphohydrolase) (EC 3.6.1.19) were determined. The enzyme hydrolyses ITP, dITP (deoxyinosine triphosphate) and XTP (xanthine triphosphate) but not other nucleotides. IDP is a competitive inhibitor. Subcellular fractionation of cultured lymphoid-line cells showed ITPase activity to be in the cytosol. ATP and GTP hydrolysing activities were not enriched in the cytosol fraction. The range of activity was investigated in erythrocytes, peripheral leukocytes, amniotic fluid cells, skin fibroblasts, long term lymphoid lines and bone marrow fibroblasts. The mean ITPase activity was found to vary in different cell types from 4.9 to 294 units per mg protein. Fractionation of red blood cells by age revealed that the enzyme decays exponentially with erythrocyte age with a half-life of 35 days. The enzyme had a wide range of specific activity in erythrocytes from 150 normal randomly selected individuals (15--722 pmol/h/g Hb). Within the low range of erythrocyte specific activities (from 15 to 200) a correlation was found between an individual's erythrocyte activity and the activity in peripheral leukocytes. This correlation was not found at higher erythrocyte ITPase activities. Two individuals were detected who had erythrocyte ITPase activities which were close to the lowest ITPase activities of all samples tested. Thus, low or absent ITPase activity is an individual characteristic which may be expressed both in erythrocytes and in other cell types.
We have demonstrated that reduced glomerular adenosine diphosphatase (ADPase) activity within the rat kidney is associated with an increased thrombotic tendency. To establish a possible causal relationship between these intraglomerular events, experiments were conducted to inhibit adenosine diphosphate (ADP) degradation without influencing other glomerular prothrombotic or antithrombotic mechanisms. Concurrently, we studied intraglomerular platelet aggregation. Two ways of selective inhibition of glomerular ADPase activity were applied: (1) by competitive substrates (ie, uridine diphosphate [UDP]), and (2) by the nondegradable ADP analogue ADP-beta-S. Both strategies were used during ex vivo alternate perfusion of kidneys with platelets and ADP (to test intraglomerular thrombotic tendency). Each group (n = 6) received different substrates or a combination of substrates. A significant increase in platelet aggregation was observed in kidneys after perfusion with platelets and ADP together with the competitive substrate UDP as compared to perfusions with platelets and ADP alone (78.5% +/- 9.8% v 27.9% +/- 11.4% glomeruli staining positive for platelets, P less than .005). In contrast, UDP alone had no effect on platelet aggregation. Other nucleoside polyphosphates (guanosine diphosphate and inosine triphosphate) were also effective as competitive substrates in the ex vivo perfusion model (n = 4). None of these substrates was capable of increasing ADP-induced aggregation when studied in vitro. In addition, ADP- beta-S also increased platelet aggregation in the perfusion model as compared with native ADP (P less than .005). These results show that selective reduction of ADP degradation in intact kidneys strongly promotes the intraglomerular proaggregatory condition. It can be concluded that glomerular ADPase exerts potent antithrombotic activity within the normal rat kidney.
BACKGROUND: Scleroderma patients exhibit increased chromosomal instability due to circulating clastogenic plasma factors (CF). Formation and action mechanisms of CF are mediated by superoxide. In addition, previous work detected inosine triphosphate (ITP) in the plasma of 2 patients, and the enzyme adenosine deaminase (ADA) was found to be increased. OBJECTIVE: To study correlations between CF, ITP and ADA levels, CF and disease activity, as well as other biomarkers of oxidative stress. METHODS: Clastogenic activity was evaluated by means of cytogenetic methods in 48 patients and 55 healthy subjects. ITP was detected by mass spectrometry and electrospray ionisation. ADA was measured with a colorimetric assay and malondialdehyde using the Yagi method. RESULTS: Clastogenic activity was significantly increased in patients' plasma compared to controls. In 10 patients CF, ITP and ADA were studied simultaneously. All three parameters were increased in the 7 patients of subgroups 2 (skin and esophagus involvement) and 3 (skin plus multiple organ involvement). ITP was not detected in 2 patients of subgroup 1 (skin involvement only) with low ADA and CF values. CONCLUSION: ITP, the deamination product of ATP, is one of the clastogenic and superoxide generating components of CF. The formation of this deamination product of ATP is probably related to the increase in ADA. CF are biomarkers of oxidative stress and can be used for evaluation of antioxidant treatments in scleroderma.
The glutamine synthetase of Bacillus licheniformis has been obtained at about 15% purity. Sucrose gradient centrifugation gave a molecular weight value of approximately 612,000. Both l- and d-glutamate can be utilized as substrates in the biosynthetic reaction, although the l isomer was five times more active. The requirement for adenosine triphosphate (ATP) can be partially replaced by guanosine or inosine triphosphates, but not by cytidine or uridine triphosphates. The Mn(++) was required for activity, and the requirement cannot be satisfied with Mg(++). Maximal activity of the biosynthetic reaction was observed when ATP and Mn(++) were present in equimolar amounts. An excess of either reactant gave less activity. However, other purine and pyrimidine nucleotides, when added in combination with ATP, can partially substitute for ATP in attaining the equimolar ratio of nucleotide to Mn(++). A complex of ATP and Mn(++) is the preferred form of substrate. The B. licheniformis enzyme catalyzes the glutamyl transfer reaction but at a much slower rate than the Escherichia coli glutamine synthetase. Either adenosine diphosphate (ADP) or ATP can activate the glutamotransferase, although ADP is more active.
The kinetics of elongation of the mRNA that initiates from the lambda PR promoter has been examined using specific restriction fragments as template, and the locations at which significant pausing of the RNA polymerase occurs during in vitro transcription have been established. Major pausing of polymerase, in the absence of rho, occurs at the five rho-dependent termination sites (located between 290 and 450 base pairs downstream from PR) that are described in the accompanying article (Morgan, W. D., Bear, D. G., and von Hippel, P. H. (1983) J. Biol. Chem. 258, 9553-9564). The replacement of guanosine by inosine triphosphate in the transcription mix results in the appearance of new pausing sites; these pausing sites correspond, in part, to the new rho-dependent termini between 100 and 260 base pairs from PR identified in the preceding article (cited above) when inosine replaces guanosine in the transcript. The effects of variations in nucleoside triphosphate and salt concentrations on pausing have also been determined. Analysis of the base pair sequences of pausing sites shows that pausing may result from the presence of dyad symmetry, GC-rich sequences, or (for inosine-substituted transcripts) C-rich sequences in the RNA-DNA hybrid region. Quantitation of RNA polymerase pausing at termination loci indicates that pausing sites with relaxation times of 10 to 25 s (at 37 degrees C and 100 to 200 mM KCl) can lead to significant rho-dependent termination. In addition, increasing the length of "natural" pauses by lowering the concentrations of specific nucleoside triphosphate substrates can lead to increased termination efficiency, but only at sites that correspond to rho-dependent termini in elongation experiments conducted at standard concentrations of nucleoside triphosphates. These results, and the findings of the article cited above, are interpreted in terms of a two-component model for rho-dependent termination. Required are: (i) a significant pause in transcript elongation due to sequence and/or structural features at the termination site(s); and (ii) a rho-binding site(s) on the nascent mRNA that is long (70-90 nucleotide residues) and relatively free of secondary structure, and that contains appropriate sequences of cytidine residues.
Inosine triphosphate pyrophosphatase (ITPase) deficiency occurs with polymorphic frequencies in Caucasians and results in the benign accumulation of the inosine nucleotide ITP. In 62 patients treated with azathioprine for inflammatory bowel disease, the ITPA 94C>A deficiency-associated allele was significantly associated with adverse drug reactions (OR 4.2, 95% CI 1.6-11.5, p = 0.0034). Significant associations were found for flu-like symptoms (OR 4.7, 95% CI 1.2-18.1, p = 0.0308), rash (OR 10.3, 95% CI 4.7-62.9, p = 0.0213) and pancreatitis (OR 6.2, CI 1.1-32.6, p = 0.0485). Polymorphism in the ITPA gene thus predicts AZA intolerance. Alternative immunosuppressive drugs, particularly 6-thioguanine, should be considered for AZA-intolerant patients with ITPase deficiency.
We have been using lysed cell models of teleost retinal cones to examine the mechanism of contraction in nonmuscle cells. We have previously reported that dark-adapted retinas can be lysed with the detergent Brij-58 to obtain cone motile models that undergo Ca++- and adenosine triphosphate (ATP)-dependent reactivated contraction. In this report we further dissect the roles of ATP and Ca++ in activation of contraction and force production by (a) characterizing the Ca++ and nucleotide requirements in more detail, (b) by analyzing the effects of inosine triphosphate (ITP) and the ATP analog ATP gamma S and (c) by testing effects of cyclic adenosine monophosphate (cAMP) on reactivated cone contraction. Exposing lysed cone models to differing free Ca++ concentrations produced reactivated contraction at rates proportional to the free Ca++ concentration between 3.16 X 10(-8) and 10(-6) M. A role for calmodulin (CaM) in this Ca++ regulation was suggested by the inhibition of reactivated contraction by the calmodulin inhibitors trifluoperazine and calmidazolium ( R24571 ). The results of analysis of nucleotide requirements in lysed cone models were consistent with those of smooth muscle studies suggesting a role for myosin phosphorylation in Ca++ regulation of contraction. ATP gamma S and ITP are particularly interesting in that ATP gamma S, on the one hand, can be used by kinases to phosphorylate proteins (e.g., myosin light chains) but resists cleavage by phosphatases or adenosine triphosphatases (ATPases), e.g., myosin ATPase. ITP, on the other hand, can be used by myosin ATPase but does not support Ca++/calmodulin mediated phosphorylation of myosin light chains by myosin light chain kinase. Thus, these nucleotides provide an opportunity to distinguish between the kinase and myosin ATPase requirements for ATP. When individual nucleotides were tested with cone motile models, the nucleotide requirement was highly specific for ATP; not only ITP and ATP gamma S, but also guanosine triphosphate, cytosine triphosphate, adenylyl-imidodiphosphate (AMPPNP) failed to support reactivated contraction when substituted for ATP throughout the incubation. However, if lysed cones were initially incubated with ATP gamma S and then subsequently incubated with ITP, the cones contracted to an extent that was comparable to that observed with ATP. As observed in skinned smooth muscle, adding cAMP to contraction medium strongly inhibited contraction in lysed cone models.