Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?
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The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis. (+info)
Effects of acute anoxia on heart function in crucian carp: importance of cholinergic and purinergic control.
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The objective of this study was to characterize the effects of acute anoxia on contractile and electrical activity in the heart of an anoxia-tolerant fish species, the crucian carp (Carassius carassius L.). Responses of atrial and ventricular tissue or isolated cells to NaCN, adenosine, and carbachol were determined to examine the effects of anoxia on cardiac performance and to clarify the possible role of local purinergic modulation and parasympathetic nervous control in the function of the anoxic fish heart. The contractility of the crucian carp heart is strongly decreased by acute anoxia. A rapid reduction in cardiac contractility is attained by reflex bradycardia and suppression of atrial contractility. These responses are mediated by muscarinic cholinergic receptors through the opening of inwardly rectifying potassium channels and are likely to protect the cardiac muscle from hypoxic/anoxic damage. The depletion of tissue oxygen content also directly depresses heart rate and cardiac force. Ultimately, an increase in cytosolic Ca(2+) concentration occurs that activates sarcolemmal Ca(2+) extrusion through the Na(+)-Ca(2+)-exchange and generates an inward exchange current with consequent depolarization of the resting membrane potential and possible cell death. At physiological concentration, the effects of adenosine on contractile and electrical activity were relatively weak, suggesting that the purinergic system is not involved in the acute anoxia response of the crucian carp heart. (+info)
Inhibition of HIV-1 replication by a two-strand system (FTFOs) targeted to the polypurine tract.
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Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3'-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication. (+info)
Glutathione-dependent metabolism of cis-3-(9H-purin-6-ylthio)acrylic acid to yield the chemotherapeutic drug 6-mercaptopurine: evidence for two distinct mechanisms in rats.
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cis-3-(9H-Purin-6-ylthio)acrylic acid (PTA) is a structural analog of azathioprine, a prodrug of the antitumor and immunosuppressive drug 6-mercaptopurine (6-MP). In this study, we examined the in vitro and in vivo metabolism of PTA in rats. Two metabolites of PTA, 6-MP and the major metabolite, S-(9H-purin-6-yl)glutathione (PG), were formed in a time- and GSH-dependent manner in vitro. Formation of 6-MP and PG occurred nonenzymatically, but 6-MP formation was enhanced 2- and 7-fold by the addition of liver and kidney homogenates, respectively. Purified rat liver glutathione S-transferases enhanced 6-MP formation from PTA by 1.8-fold, whereas human recombinant alpha, mu, and pi isozymes enhanced 6-MP formation by 1.7-, 1.3-, and 1.3-fold, respectively. In kidney homogenate incubations, PG accumulation was only observed during the first 15 min because of further metabolism by gamma-glutamyltranspeptidase, dipeptidase, and beta-lyase to yield 6-MP, as indicated by the use of the inhibitors acivicin and aminooxyacetic acid. Based on these results and other lines of evidence, two different GSH-dependent pathways are proposed for 6-MP formation: an indirect pathway involving PG formation and further metabolism to 6-MP, and a direct pathway in which PTA acts as a Michael acceptor. HPLC analyses of urine of rats treated i.p. with PTA (100 mg/kg) showed that 6-MP was formed in vivo and excreted in urine without apparent liver or kidney toxicity. Collectively, these studies show that PTA is metabolized to 6-MP both in vitro and in vivo and may therefore be a useful prodrug of 6-MP. (+info)
Prevention of depurination during elution facilitates the reamplification of DNA from differential display gels.
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DNA fragments that show a pattern of differential expression on differential display gels must be eluted from the gel matrix and reamplified to enable further analysis. Elution is usually achieved by heating excised gel slices in a small volume of either water or TE. Here we show that this elution step can adversely affect the ability of the eluted DNA to act as a template for PCR reamplification, probably via the process of depurination. Simply switching to an elution solvent designed to minimise depurination (PCR buffer) facilitates the elution of intact DNA fragments. This improvement is likely to be most beneficial when eluting higher molecular weight fragments (e.g. those >500 bp), in situations where the amount of DNA in an excised gel slice is limited or when contaminating differential display products co-migrate with the differentially expressed product. (+info)
The identification of nuclear proteins that bind the homopyrimidine strand of d(GA.TC)n DNA sequences, but not the homopurine strand.
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Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences. (+info)
Site-selective electron transfer from purines to electrocatalysts: voltammetric detection of a biologically relevant deletion in hybridized DNA duplexes.
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BACKGROUND: The one-electron oxidation of guanine nucleobases is of interest for understanding the mechanisms of mutagenesis, probing electron-transfer reactions in DNA, and developing sensing schemes for nucleic acids. The electron-transfer rates for oxidation of guanine by exogenous redox catalysts depend on the base paired to the guanine. An important goal in developing the mismatch sensitivity is to identify a means for monitoring the current resulting from electron transfer at a single base in the presence of native oligonucleotides that contain all four bases. RESULTS: The nucleobase 8-oxo-guanine (8G) is selectively oxidized by the redox catalyst Os(bpy)(3)(3+/2+) (bpy = 2,2'-bipyridine) in the presence of native guanine. Cyclic voltammograms of Os(bpy)(3)(2+) show current enhancements indicative of nucleobase oxidation upon addition of oligonucleotides that contain 8G, but not in the presence of native guanine. As expected, similar experiments with Ru(bpy)(3)(2+) show enhancement with both guanine and 8G. The current enhancements for the 8G/Os(III) reaction increase in the order 8G-C approximately 8G.T < 8G.G < 8G.A < 8G, the same order as that observed for guanine/Ru(III). This site-selective mismatch sensitivity can be applied to detection of a TTT deletion, which is important in cystic fibrosis. CONCLUSIONS: The base 8G can be effectively used in conjunction with a low-potential redox catalyst as a probe for selective electron transfer at a single site. Because of the high selectivity for 8G, rate constants can be obtained that reflect the oxidation of only one base. The mismatch sensitivity can be used to detect biologically relevant abnormalities in DNA. (+info)
Expression of the soxR gene of Pseudomonas aeruginosa is inducible during infection of burn wounds in mice and is required to cause efficient bacteremia.
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Burn wounds are prone to infection by Pseudomonas aeruginosa, which is an opportunistic pathogen causing various human diseases. During infection, the bacterium senses environmental changes and regulates the expression of genes appropriate for survival. A purine-auxotrophic mutant of P. aeruginosa was unable to replicate efficiently on burn wounds, suggesting that burn wounds are purine-deficient environments. An in vivo expression technology based on purEK gene expression was applied to the burned mouse infection model to isolate P. aeruginosa genes that are specifically induced during infection. Four such in vivo-inducible (ivi) genetic loci were identified, including the gene for a superoxide response regulator (soxR), the gene for a malate synthase G homologue (glcG), an antisense transcript of a putative regulator responding to copper (copR), and an uncharacterized genetic locus. SoxR of Escherichia coli is known to regulate genes involved in protecting the bacterium against oxidative stress. The expression of soxR was proven to be highly inducible during the infection of burned mice and also inducible by treatment with paraquat, which is a redox-cycling reagent generating intracellular superoxide. The SoxR protein functions as an autorepressor in the absence of paraquat, whereas in the presence of paraquat, this autorepression is diminished. Furthermore, a soxR null mutant was shown to be much more sensitive than wild-type P. aeruginosa to macrophage-mediated killing. In support of this observation, a soxR null mutant exhibited a significant delay in causing systemic infections in the burned mice. Since most mortality in burn patients is caused by systemic infection, the defect in the ability to cause efficient bacteremia in burned mice suggests an important role of the soxR gene in the infection of burn wounds. (+info)