Deoxynucleotide-polymerizing enzyme activities in T- and B-cells of acute lymphoblastic leukemia origin.
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All 5 thymus-dependent cell (T-cell) lines (Molt-3; Molt-4; RPMI-8402; CCRF-CEM; CCRF-HSB-2) and 7 thymus-independent cell (B-cell) lines (RPMI-8382, RPMI-8392, RPMI-8412, RPMI-8422, RPMI-8432, RPMI-8442, CCRF-SB) established so far from acute lymphoblastic leukemia patients were examined for deoxynucleotide polymerizing enzymes. All T- and B-cells had DNA polymerase gamma, DNA polymerase beta, and terminal deoxynucleotidyl transferase both in the soluble (the latter 2 enzymes only in small amounts) and chromatin fraction, whereas DNA polymerase alpha was found only in the soluble fraction. With respect to their sedimentation and chromatographic behavior, template-primer requirements, Km for deoxythymidine triphosphate or deoxyguanosine triphosphate divalent cation preference, effect of NaCI and inhibitors, the enzymes from T- and B-cells resembled each other and those from other mammalian cells. DNA polymerase alpha, beta, and gamma from T-cells like those from "fresh" acute lymphoblastic leukemia cells, were more thermolabile than those from B-cells or phytohemagglutinin-stimulated normal lymphocytes. In addition, the terminal deoxynucleotidyl transferase from the above cells was completely inactivated in 5 to 6 min at 50 degrees, whereas the DNA polymerase alpha, beta, and gamma retained considerable activity even after heating for 25 min at 50 degrees. DNA polymerase activity of the soluble fraction from T-cells was of the same magnitude as in B-cells when expressed on a DNA basis but twice that of B-cells when expressed on a protein basis. High terminal deoxynucleotidyl transferase activity, equivalent to that observed in acute lymphoblastic leukemia cells, was found in all T-cell lines that, when expressed on a DNA basis, was 30 to 100 times higher than the B-cell lines tested. These results support the suggestion of earlier investigators that T-cell lines examined here may have originated from leukemic cells. (+info)
P2 purinoceptor-mediated control of rat cerebral (pial) microvasculature; contribution of P2X and P2Y receptors.
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Purine and pyrimidine nucleotides evoke changes in the vascular tone of medium to large cerebral vessels through the activation of P2 purinoceptors. We have applied P2 receptor drugs to rat pial arterioles and measured changes in arteriole diameter (o.d. 40-84 micrometer at rest), and recorded currents from arteriolar smooth muscle cells using patch-clamp techniques. Transient vasoconstrictions and rapidly inactivating currents were evoked by alpha,beta-methylene ATP (0.1-30 micrometer) and were sensitive to the P2 receptor antagonists suramin and iso-PPADS. UTP and UDP (0.1-1000 micrometer) evoked sustained suramin-sensitive vasoconstrictions. ATP (0.1-1000 micrometer) and 2-methylthioATP (2MeSATP, 300 micrometer) evoked transient vasoconstrictions followed by sustained vasodilatations. ADP application resulted in only vasodilatation (EC50 approximately 4 micrometer). Vasodilator responses to ATP, 2MeSATP or ADP were unaffected by suramin (100 micrometer). RT-PCR analysis indicated that P2X1-7 and P2Y1,2,6 RNA can be amplified from the pial sheet. Our results provide direct evidence for the presence of functional P2X receptors with a phenotype resembling the P2X1 receptor subtype on cerebral resistance arterioles. The pharmacological properties of the pyrimidine-evoked responses suggest that a combination of P2Y2- and P2Y6-like receptors are responsible for the sustained vasoconstrictions. It is therefore likely that the nucleotides and their associated receptors are involved in a complicated regulatory system to control cerebral blood pressure. (+info)
Selective regulatory effects of purine and pyrimidine nucleotides on vacuolar transport of amino acids.
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The release of amino acids from their vacuolar store was studied in situ, i.e. in cells with selectively permeabilized plasma membrane and functionally intact vacuoles. As we previously described [Roos et al., J. Biol. Chem. 272 (1997) 15849-15855], this transport process is regulated by extravacuolar adenylates at their physiological concentrations. We now show, using our test object Penicillium cyclopium, that not only purine but also pyrimidine nucleotides are involved in the control of efflux of vacuolar phenylalanine. At 0.1 mM adenosine or guanosine phosphates inhibit, whereas cytidine or uridine phosphates stimulate the rate of efflux. At 1 mM the same nucleotides have no measurable impact on efflux but abolish the effects of other nucleotides present at 0.1 mM. This argues for at least two interacting binding sites with different nucleotide affinities. The minimum structural requirement for any of the observed effects is a non-cyclic ribonucleoside monophosphate. In intact cells, cytosolic concentrations of ATP (representing purine nucleotides) and CTP (representing pyrimidine nucleotides) are 1-2 mM and 0.05-0.2 mM, respectively. ATP is therefore assumed to dominate transport control and allow optimum efflux (and uptake) rates. Short-time starvation of carbon and nitrogen adjusts CTP and ATP at levels that cause declining efflux rates. During prolonged starvation both nucleotides fall below their transport-controlling concentrations and thus allow increasing rates of efflux from the still maintained vacuolar pool. Hence, efflux control under nutrient limitation includes an interplay of purine and pyrimidine nucleotides which precisely regulates the release of vacuolar amino acids and enables flexible adjustment to either amino acid saving or cell survival. (+info)
Acid-soluble degradation products of ribonucleic acid in Escherichia coli and the role of nucleotidases in their catabolism.
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The fate of the internally formed nucleotides resulting from the degradation of ribonucleic acid was studied. Prelabeled Escherichia coli cells were submitted to carbon starvation, and the acid-soluble products were separated by thin-layer chromatography. It was determined that free bases constitute some 75% of the end product, the balance consisting of nucleoside diphosphates, 5'-nucleoside monophosphates, 3'-nucleoside monophosphates, and nucleosides. The majority of degradation products, including phosphorylated derivatives, were excreted into the medium. The amount of products in the pool remained constant. The soluble products formed by E. coli mutants lacking either 5'-nucleotidase (Ush-) or 3'-nucleotidase (Cpd-) were compared with those produced by the parental strain with both enzymes. The results obtained indicated that 5'-nucleotidase is involved in the degradation of internally foromed nucleotides but that 3'-nucleotidase takes no part in the process. (+info)
Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction.
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GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction. (+info)
CART classification of human 5' UTR sequences.
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A nonredundant database of 2312 full-length human 5'-untranslated regions (UTRs) was carefully prepared using state-of-the-art experimental and computational technologies. A comprehensive computational analysis of this data was conducted for characterizing the 5' UTR features. Classification and regression tree (CART) analysis was used to classify the data into three distinct classes. Class I consists of mRNAs that are believed to be poorly translated with long 5' UTRs filled with potential inhibitory features. Class II consists of terminal oligopyrimidine tract (TOP) mRNAs that are regulated in a growth-dependent manner, and class III consists of mRNAs with favorable 5' UTR features that may help efficient translation. The most accurate tree we found has 92.5% classification accuracy as estimated by cross validation. The classification model included the presence of TOP, a secondary structure, 5' UTR length, and the presence of upstream AUGs (uAUGs) as the most relevant variables. The present classification and characterization of the 5' UTRs provide precious information for better understanding the translational regulation of human mRNAs. Furthermore, this database and classification can help people build better computational models for predicting the 5'-terminal exon and separating the 5' UTR from the coding region. (+info)
Kinetics of formation of hypoxanthine containing base pairs by HIV-RT: RNA template effects on the base substitution frequencies.
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Hypoxanthine (H), the deamination product of adenine, has been implicated in the high frequency of A to G transitions observed in retroviral and other RNA genomes. Although H.C base pairs are thermodynamically more stable than other H.N pairs, polymerase selection may be determined in part by kinetic factors. Therefore, the hypoxanthine induced substitution pattern resulting from replication by viral polymerases may be more complex than that predicted from thermodynamics. We have examined the steady-state kinetics of formation of base pairs opposite template H in RNA by HIV-RT, and for the incorporation of dITP during first- and second-strand synthesis. Hypoxanthine in an RNA template enhances the k(2app) for pairing with standard dNTPs by factors of 10-1000 relative to adenine at the same sequence position. The order of base pairing preferences for H in RNA was observed to be H.C >> H.T > H.A > H.G. Steady-state kinetics of insertion for all possible mispairs formed with dITP were examined on RNA and DNA templates of identical sequence. Insertion of dITP opposite all bases occurs 2-20 times more frequently on RNA templates. This bias for higher insertion frequencies on RNA relative to DNA templates is also observed for formation of mispairs at template A. This kinetic advantage afforded by RNA templates for mismatches and pairing involving H suggests a higher induction of mutations at adenines during first-strand synthesis by HIV-RT. (+info)
Local changes in GC/AT substitution biases and in crossover frequencies on Drosophila chromosomes.
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I present here evidence of remarkable local changes in GC/AT substitution biases and in crossover frequencies on Drosophila chromosomes. The substitution pattern at 10 loci in the telomeric region of the X chromosome was studied for four species of the Drosophila melanogaster species subgroup. Drosophila orena and Drosophila erecta are clearly the most closely related species pair (the erecta complex) among the four species studied; however, the overall data at the 10 loci revealed a clear dichotomy in the silent substitution patterns between the AT-biased- substitution melanogaster and erecta lineages and the GC-biased-substitution yakuba and orena lineages, suggesting two or more independent changes in GC/AT substitution biases. More importantly, the results indicated a between- loci heterogeneity in GC/AT substitution bias in this small region independently in the yakuba and orena lineages. Indeed, silent substitutions in the orena lineage were significantly biased toward G and C at the consecutive yellow, lethal of scute, and asense loci, but they were significantly biased toward A and T at sta. The substitution bias toward G and C was centered in different areas in yakuba (significantly biased at EG:165H7.3, EG:171D11.2, and suppressor of sable). The similar silent substitution patterns in coding and noncoding regions, furthermore, suggested mutational biases as a cause of the substitution biases. On the other hand, previous study reveals that Drosophila yakuba has about 20-fold higher crossover frequencies in the telomeric region of the X chromosome than does D. melanogaster; this study revealed that the total genetic map length of the yakuba X chromosome was only about 1.5 times as large as that of melanogaster and that the map length of the X-telomeric y-sta region did not differ between Drosophila yakuba and D. erecta. Taken together, the data strongly suggested that an approximately 20- fold reduction in the X-telomeric crossover frequencies occurred in the ancestral population of D. melanogaster after the melanogaster-yakuba divergence but before the melanogaster-simulans divergence. (+info)