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Sample records for pronounced purine sequence

  1. Complete genome sequence of Bacillus amyloliquefaciens XH7, which exhibits production of purine nucleosides.

    PubMed

    Yang, Huilin; Liao, Yuling; Wang, Bin; Lin, Ying; Pan, Li

    2011-10-01

    Here, we report the complete annotated genome sequence of Bacillus amyloliquefaciens XH7, which is used to produce purine nucleosides in industry. The genome sequence will allow for the characterization of the molecular mechanisms underlying its beneficial properties.

  2. Association of poly-purine/poly-pyrimidine sequences with meiotic recombination hot spots

    PubMed Central

    Bagshaw, Andrew TM; Pitt, Joel PW; Gemmell, Neil J

    2006-01-01

    Background Meiotic recombination events have been found to concentrate in 1–2.5 kilo base regions, but these recombination hot spots do not share a consensus sequence and why they occur at specific sites is not fully understood. Some previous evidence suggests that poly-purine/poly-pyrimidine (poly-pu/py) tracts (PPTs), a class of sequence with distinctive biochemical properties, could be involved in recombination, but no general association of PPTs with meiotic recombination hot spots has previously been reported. Results We used computational methods to investigate in detail the relationship between PPTs and hot spots. We show statistical associations of PPT frequency with hot spots of meiotic recombination initiating lesions, double-strand breaks, in the genome of the yeast S. cerevisiae and with experimentally well characterized human meiotic recombination hot spots. Supporting a possible role of poly-pu/py-rich sequences in hot spot recombination, we also found that all three single nucleotide polymorphisms previously shown to be associated with human hot spot activity changes occur within sequence contexts of 14 bp or longer that are 85% or more poly-pu/py and at least 70% G/C. These polymorphisms are all close to the hot spot mid points. Comparing the sequences of experimentally characterized human hot spots with the orthologous regions of the chimpanzee genome previously shown not to contain hot spots, we found that in all five cases in which comparisons for the hot spot central regions are possible with publicly available sequence data, there are differences near the human hot spot mid points within sequences 14 bp or longer consisting of more than 80% poly-pu/py and at least 50% G/C. Conclusion Our results, along with previous evidence for the unique biochemical properties and recombination-stimulating potential of poly-pu/py-rich sequences, suggest that the possible functional involvement of this type of sequence in meiotic recombination hot spots

  3. Factors associated with a purine-rich exonic splicing enhancer sequence in Xenopus oocyte nucleus

    SciTech Connect

    Masuyama, Kaoru; Taniguchi, Ichiro; Okawa, Katsuya; Ohno, Mutsuhito . E-mail: hitoohno@virus.kyoto-u.ac.jp

    2007-08-03

    Purine-rich exonic splicing enhancers (ESEs) stimulate splicing of the adjacent introns with suboptimal splice sites. To elucidate the mechanism regarding ESEs, factors specifically associated with ESEs in HeLa cell nuclear extracts were previously investigated, and shown to include SR (serine/arginine-rich) proteins. However, factors associated with ESEs in vivo have not yet been explored. Here we show that a GAA repeat RNA sequence, a typical ESE, is associated in Xenopus oocyte nuclei with at least one SR protein, SF2/ASF, as was expected. Moreover, components of SF3a/b complexes, U2 snRNA, and U2AF{sup 65} were also found to be associated with the ESE in the nucleus. Since SF3a/b complexes are the constituents of the 17S U2 snRNP, these results suggest that the 17S U2 snRNP is associated with the ESE in the nucleus, probably through bridging interactions of U2AF and SR proteins. The identified factors may represent a functional splicing enhancer complex in vivo.

  4. Structure/Function Analysis of DNA-glycosylases That Repair Oxidized Purines and Pyrimidines and the Influence of Surrounding DNA Sequence on Their Interactions

    SciTech Connect

    Wallace, Susan S.

    2005-08-22

    The overall goal of this project was to elucidate the structure/function relationships between oxidized DNA bases and the DNA repair enzymes that recognize and remove them. The NMR solution structure of formamidopyrimidine DNA glycosylase (Fpg) that recognizes oxidized DNA purines was to be determined. Furthermore, the solution structures of DNA molecules containing specific lesions recognized by Fpg was to be determined in sequence contexts that either facilitate or hinder this recognition. These objectives were in keeping with the long-term goals of the Principal Investigator's laboratory, that is, to understand the basic mechanisms that underpin base excision repair processing of oxidative DNA lesions and to elucidate the interactions of unrepaired lesions with DNA polymerases. The results of these two DNA transactions can ultimately determine the fate of the cell. These objectives were also in keeping with the goals of our collaborator, Dr. Michael Kennedy, who is studying the repair and recognition of damaged DNA. Overall the goals of this project were congruent with those of the Department of Energy's Health Effects and Life Sciences Research Program, especially to the Structural Biology, the Human Genome and the Health Effects Programs. The mission of the latter Program includes understanding the biological effects and consequences of DNA damages produced by toxic agents in the many DOE waste sites so that cleanup can be accomplished in a safe, effective and timely manner.

  5. Triple, MPEG-conjugated, helix-forming oligonucleotides (TRIPEGXs): liquid-phase synthesis of natural and chimeric "all-purine" sequences linked to high molecular weight poly(ethylene glycols).

    PubMed

    Ballico, M; Drioli, S; Morvan, F; Xodo, L; Bonora, G M

    2001-01-01

    Long "all-purine" oligonucleotides, up to the 20mer, known to be active as antigene effectors, conjugated to high molecular weight monomethoxy poly(ethylene glycol)s (MPEG)s, were successfully synthesized. Through a liquid-phase, MPEG-supported process, both natural and chimeric sequences containing selected phosphorothioate backbone modifications were obtained, purified, and characterized. To follow their cellular trafficking, a fluorescent probe was linked by soluble supported organic reactions to the 5'-terminus, and the efficiency of the different synthetic procedures for the introduction of a fluorescein moiety was compared. The usefulness of the fluorescent marker was estimated by laser confocal microscopy that ascertains that the MPEG-conjugation enhances the oligonucleotide capacity to cross the cellular membranes and to be accumulated inside the nuclei.

  6. Purine alkaloids in Paullinia.

    PubMed

    Weckerle, Caroline S; Stutz, Michael A; Baumann, Thomas W

    2003-10-01

    Among the few purine alkaloid-containing genera consumed as stimulants, Paullinia is the least investigated with respect to both chemotaxonomy and within-the-plant allocation of caffeine and its allies. Since purine alkaloids (PuA) have been proved to be valuable marker compounds in chemotaxonomy, 34 species of Paullinia and related genera were screened for them, but only one, P. pachycarpa, was positive in addition to the already known P. cupana and P. yoco. The PuA allocation in P. pachycarpa was examined and found to be restricted to theobromine in the stem, leaves and flowers. Moreover, the theobromine concentration in the stem cortex increased significantly towards the base of the plant. Since the stem cortex of P. yoco is traditionally used by the natives of Colombia and Ecuador to prepare a caffeine-rich beverage, we suspected that within the genus Paullinia the PuA are preferentially allocated to the older parts of the stem and not to young shoots like e.g., in the coffee plant (Coffea spp.). Indeed, the axis (greenhouse) of P. cupana (guaraná), known for its caffeine-rich seeds, exhibited a basipetal PuA gradient (0.005-0.145%). Moreover, the analysis of young cortex samples (herbarium) and of one piece of old stem (museum collection) revealed the same for P. yoco, even though we found much less (0.5 vs 2.5%) caffeine in the old cortex as compared to the only two analyses in 1926 of similar material. However, this discrepancy may be explained by the high variability of the PuA pattern we detected among yoco, the diversity of which the Indians take advantage.

  7. Importance of purine and pyrimidine content of local nucleotide sequences (six bases long) for evolution of the human immunodeficiency virus type 1.

    PubMed Central

    Doi, H

    1991-01-01

    Human immunodeficiency virus type 1 evolves rapidly, and random base change is thought to act as a major factor in this evolution. However, segments of the viral genome differ in their variability: there is the highly variable env gene, particularly hypervariable regions located within env, and, in contrast, the conservative gag and pol genes. Computer analysis of the nucleotide sequences of human immunodeficiency virus type 1 isolates reveals that base substitution in this virus is nonrandom and affected by local nucleotide sequences. Certain local sequences 6 base pairs long are excessively frequent in the hypervariable regions. These sequences exhibit base-substitution hotspots at specific positions in their 6 bases. The hotspots tend to be nonsilent letters of codons in the hypervariable regions--thus leading to marked amino acid substitutions there. Conversely, in the conservative gag and pol genes the hotspots tend to be silent letters because of a difference in codon frame from the hypervariable regions. Furthermore, base substitutions in the local sequences that frequently appear in the conservative genes occurred at a low level, even within the variable env. Thus, despite the high variability of this virus, the conservative genes and their products could be conserved. These may be some of the strategies evolved in human immunodeficiency virus type 1 to allow for positive-selection pressures, such as the host immune system, and negative-selection pressures on the conservative gene products. Images PMID:1924392

  8. Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.

    PubMed

    Eide, L; Bjørås, M; Pirovano, M; Alseth, I; Berdal, K G; Seeberg, E

    1996-10-01

    One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.

  9. Dephosphorylation of purine mononucleotides by alkaline phosphatases. Substrate specificity and inhibition patterns.

    PubMed

    Jensen, M H

    1979-11-09

    Three purine mononucleotides, adenosine-, inosine- and guanosine monophosphate, were used as substrates at pH 7.4 and at 10.4 for three alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.1) containing similar phosphate-binding serine groups at their esteratic sites. Substrate specificity was found for the enzymes from calf intestine and bovine liver. Alkaline phosphatase from Escherichia coli was nonspecific. A substrate-dependent and pronounced inhibition with the purine analogue 1,3-dimethyl xanthine was found for the enzymes from intestine and liver, but not for alkaline phosphatase from E. coli. A substrate-independent and pronounced inhibition was found for all three enzymes with the phosphomonoester p-nitrophenol phosphate as the inhibitor. Alkaline phosphatases may play an important role in the regulation of the intracellular content of purine mononucleotides.

  10. Was adenine the first purine?

    NASA Technical Reports Server (NTRS)

    Schwartz, Alan W.; Bakker, C. G.

    1989-01-01

    Oligomerization of HCN (1 molar) in the presence of added formaldehyde (0.5 molar) produced an order of magnitude more 8-hydroxymethyladenine than adenine or any other biologically significant purine. This result suggests that on the prebiotic earth, nucleoside analogs may have been synthesized directly in more complex mixtures of HCN with other aldehydes.

  11. Purine metabolism in Toxoplasma gondii

    SciTech Connect

    Krug, E.C.; Marr, J.J.; Berens, R.L.

    1989-06-25

    We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.

  12. Structure and mechanism of purine binding riboswitches

    PubMed Central

    Batey, Robert T.

    2013-01-01

    A riboswitch is a non-protein coding sequence capable of directly binding a small molecule effector without the assistance of accessory proteins to regulate expression of the mRNA in which it is embedded. Currently, over 20 different classes of riboswitches have been validated in bacteria with the promise of many more to come, making them an important means of regulation of the genome in the bacterial kingdom. Strikingly, half of the known riboswitches recognize effector compounds that contain a purine or related moiety. In the last decade significant progress has been made to determine how riboswitches specifically recognize these compounds against the background of many other similar cellular metabolites and transduce this signal into a regulatory response. Of the known riboswitches, the purine family containing guanine, adenine, and 2’-deoxyguanosine binding classes are the most extensively studied, serving as a simple and useful paradigm for understanding how these regulatory RNAs function. This review provides a comprehensive summary of the current state of knowledge regarding the structure and mechanism of these riboswitches, as well as insights into how they might be exploited as therapeutic targets and novel biosensors. PMID:22850604

  13. Comparison of the effects of UV irradiation on 5-methyl-substituted and unsubstituted pyrimidines in alternating pyrimidine-purine sequences in DNA.

    PubMed

    Zuo, S; Boorstein, R J; Cunningham, R P; Teebor, G W

    1995-09-12

    We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5-methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions.

  14. Microwave-assisted synthesis of C-8 aryl and heteroaryl inosines and determination of their inhibitory activities against Plasmodium falciparum purine nucleoside phosphorylase.

    PubMed

    Gigante, Alba; Priego, Eva-María; Sánchez-Carrasco, Paula; Ruiz-Pérez, Luis Miguel; Vande Voorde, Johan; Camarasa, María-José; Balzarini, Jan; González-Pacanowska, Dolores; Pérez-Pérez, María-Jesús

    2014-07-23

    8-Arylinosines have been scarcely studied for therapeutic purposes, probably due to difficulties in their synthesis. The recently described direct arylation reaction at position 8 of purine nucleosides has been employed to synthesize a series of 8-aryl and 8-pyridylinosines. These compounds have been studied for hydrolytic stability and subjected to biological evaluation. Three compounds have shown a pronounced specific inhibition of Plasmodium falciparum-encoded purine nucleoside phosphorylase, an important target for antimalarial chemotherapy.

  15. Kinetics of luminol sonochemiluminescence quenched by purines.

    PubMed

    Wang, Jian; Lai, Yongquan; Chen, Meili; Jiang, Zhou; Chen, Guonan

    2013-01-01

    A homogeneous chemiluminescence (CL) reaction was initiated by ultrasound irradiation. Luminol sonochemiluminescence (SCL) reaction kinetics were determined under pseudo-first-order conditions, and the reaction followed the model for simple rise-fall kinetics. In addition, SCL quenching reactions induced by purines were also investigated in which the interactions between luminol and purines were analysed using the Stern-Volmer (S-V) mechanism. The results implied that the high rate constant of luminol CL quenched by purines may be attributed to ground state interactions originating from hydrogen bonding.

  16. Genetics Home Reference: purine nucleoside phosphorylase deficiency

    MedlinePlus

    ... patients with purine nucleoside phosphorylase deficiency. Nucleosides Nucleotides Nucleic Acids. 2004 Oct;23(8-9):1411-5. Erratum in: Nucleosides Nucleotides Nucleic Acids. 2005;24(4):303. Citation on PubMed Nyhan ...

  17. Riboswitch Structure: an Internal Residue Mimicking the Purine Ligand

    SciTech Connect

    Delfosse, V.; Bouchard, P; Bonneau, E; Dagenais, P; Lemay, J; Lafontaine, D; Legault, P

    2009-01-01

    The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson-Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39-C65 and A39-U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.

  18. Prebiotic syntheses of purines and pyrimidines.

    PubMed

    Basile, B; Lazcano, A; Oró, J

    1984-01-01

    The work done in many laboratories during the last two decades has confirmed that hydrogen cyanide and cyanoacetylene are the two major precursors for the prebiotic synthesis of purines and pyrimidines, respectively. Although several different pathways for the synthesis of purines have been described, they are all variations of the initial mechanism proposed by Oró and Kimball, where hydrogen cyanide leads first to the formation of a 4,5-di-substituted imidazole derivative, and then to the closing of the purine ring with a C1 compound. A number of experiments have shown that purines and pyrimidines can also be obtained from methane, ammonia (nitrogen), and water mixtures, provided an activating source of energy (radiation, electric discharges, etc.) is available. However, in this case the yields are lower by about two orders of magnitude because of the intermediate formation of hydrogen cyanide and cyanoacetylene. The latter two compounds have been found in interstellar space, Titan and other bodies of the solar system. They were probably present in the primordial parent bodies from the solar nebula in concentrations of 10(-2) to 10(-3) M as inferred from recent calculations by Miller and coworkers obtained for the Murchison meteorite. These concentrations should have been sufficient to generate relatively large amounts of purine and pyrimidine bases on the primitive Earth.

  19. Prebiotic syntheses of purines and pyrimidines

    NASA Technical Reports Server (NTRS)

    Basile, B.; Oro, J.; Lazcano, A.

    1984-01-01

    The results of experimental and theoretical investigations of the prebiotic synthesis of purines and pyramidines are surveyed. Topics examined include the synthesis of purines from HCN via 4,5-disubstituted imidazole derivatives in aqueous solutions or liquid NH3, simultaneous formation of amino acids and purines by electron irradiation of CH4-NH3-H2O mixtures, synthesis of pyrimadines from cynoacetylene, energetics, formation of bases under anhydrous or concentrated conditions, formation of bases under dilute conditions, Fischer-Tropsch-type reactions, and the role of activated intermediates. It is pointed out that the precursor compounds have been detected in the interstellar medium, on Titan, and in other solar-system bodies, and that solar-nebula HCN concentrations of the order of 1-10 mM have been estimated on the basis of meteorite measurements.

  20. Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

    PubMed Central

    Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

    2016-01-01

    Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a

  1. Radical-induced purine lesion formation is dependent on DNA helical topology.

    PubMed

    Terzidis, Michael A; Prisecaru, Andreea; Molphy, Zara; Barron, Niall; Randazzo, Antonio; Dumont, Elise; Krokidis, Marios G; Kellett, Andrew; Chatgilialoglu, Chryssostomos

    2016-11-01

    Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO(•)) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5',8-cyclo-2'-deoxyguanosine (5',8-cdG) and 5',8-cyclo-2'-deoxyadenosine (5',8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]4 (TG4T), d[AGGG(TTAGGG)3] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)3A] (mutTel24) were exposed to HO(•) radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L > OC ≫ SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (∼50% solution folded) and mutTel24 (∼90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33 Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.

  2. A New HPLC Purine Assay for Quantifying Microbial Flow

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A HPLC method was developed to quantify the purines adenine and guanine and their metabolites xanthine and hypoxanthine in hydrolysates of isolated bacteria and omasal digesta and to assess the effect of using either purines only, or purines plus metabolites, as microbial markers for estimating micr...

  3. Total purine and purine base content of common foodstuffs for facilitating nutritional therapy for gout and hyperuricemia.

    PubMed

    Kaneko, Kiyoko; Aoyagi, Yasuo; Fukuuchi, Tomoko; Inazawa, Katsunori; Yamaoka, Noriko

    2014-01-01

    Purines are natural substances found in all of the body's cells and in virtually all foods. In humans, purines are metabolized to uric acid, which serves as an antioxidant and helps to prevent damage caused by active oxygen species. A continuous supply of uric acid is important for protecting human blood vessels. However, frequent and high intake of purine-rich foods reportedly enhances serum uric acid levels, which results in gout and could be a risk factor for cardiovascular disease, kidney disease, and metabolic syndrome. In Japan, the daily intake of dietary purines is recommended to be less than 400 mg to prevent gout and hyperuricemia. We have established an HPLC method for purine analysis and determined purines in a total of 270 foodstuffs. A relatively small number of foods contained concentrated amounts of purines. For the most part, purine-rich foods are also energy-rich foods, and include animal meats, fish meats, organs such as the liver and fish milt, and yeast. When the ratio of the four purine bases (adenine, guanine, hypoxanthine, and xanthine) was compared, two groups of foods were identified: one that contained mainly adenine and guanine and one that contained mainly hypoxanthine. For patients with gout and hyperuricemia, the amount of total purines and the types of purines consumed, particularly hypoxanthine, are important considerations. In this context, the data from our analysis provide a purine content reference, and thereby clinicians and patients could utilize that reference in nutritional therapy for gout and hyperuricemia.

  4. Characterization of purine catabolic pathway genes in coelacanths.

    PubMed

    Forconi, Mariko; Biscotti, Maria Assunta; Barucca, Marco; Buonocore, Francesco; De Moro, Gianluca; Fausto, Anna Maria; Gerdol, Marco; Pallavicini, Alberto; Scapigliati, Giuseppe; Schartl, Manfred; Olmo, Ettore; Canapa, Adriana

    2014-09-01

    Coelacanths are a critically valuable species to explore the gene changes that took place in the transition from aquatic to terrestrial life. One interesting and biologically relevant feature of the genus Latimeria is ureotelism. However not all urea is excreted from the body; in fact high concentrations are retained in plasma and seem to be involved in osmoregulation. The purine catabolic pathway, which leads to urea production in Latimeria, has progressively lost some steps, reflecting an enzyme loss during diversification of terrestrial species. We report the results of analyses of the liver and testis transcriptomes of the Indonesian coelacanth Latimeria menadoensis and of the genome of Latimeria chalumnae, which has recently been fully sequenced in the framework of the coelacanth genome project. We describe five genes, uricase, 5-hydroxyisourate hydrolase, parahox neighbor B, allantoinase, and allantoicase, each coding for one of the five enzymes involved in urate degradation to urea, and report the identification of a putative second form of 5-hydroxyisourate hydrolase that is characteristic of the genus Latimeria. The present data also highlight the activity of the complete purine pathway in the coelacanth liver and suggest its involvement in the maintenance of high plasma urea concentrations.

  5. Structural Biology of the Purine Biosynthetic Pathway

    PubMed Central

    Zhang, Yang; Morar, Mariya; Ealick, Steven E.

    2008-01-01

    Purine biosynthesis requires ten enzymatic transformations to generate inosine monophosphate. PurF, PurD, PurL, PurM, PurC, and PurB are common to all pathways, while PurN or PurT, PurK/PurE-I or PurE-II, PurH or PurP, and PurJ or PurO catalyze the same steps in different organisms. X-ray crystal structures are available for all 15 purine biosynthetic enzymes, including seven ATP-dependent enzymes, two amidotransferases and two tetrahydrofolate-dependent enzymes. Here we summarize the structures of the purine biosynthetic enzymes, discuss similarities and differences, and present arguments for pathway evolution. Four of the ATP-dependent enzymes belong to the ATP-grasp superfamily and two to the PurM superfamily. The amidotransferases are unrelated with one utilizing an NTN-glutaminase and the other utilizing a triad glutaminase. Likewise the tetrahydrofolate-dependent enzymes are unrelated. Ancestral proteins may have included a broad specificity enzyme instead of PurD, PurT, PurK, PurC, and PurP, and a separate enzyme instead of PurM and PurL. PMID:18712276

  6. Purines: forgotten mediators in traumatic brain injury.

    PubMed

    Jackson, Edwin K; Boison, Detlev; Schwarzschild, Michael A; Kochanek, Patrick M

    2016-04-01

    Recently, the topic of traumatic brain injury has gained attention in both the scientific community and lay press. Similarly, there have been exciting developments on multiple fronts in the area of neurochemistry specifically related to purine biology that are relevant to both neuroprotection and neurodegeneration. At the 2105 meeting of the National Neurotrauma Society, a session sponsored by the International Society for Neurochemistry featured three experts in the field of purine biology who discussed new developments that are germane to both the pathomechanisms of secondary injury and development of therapies for traumatic brain injury. This included presentations by Drs. Edwin Jackson on the novel 2',3'-cAMP pathway in neuroprotection, Detlev Boison on adenosine in post-traumatic seizures and epilepsy, and Michael Schwarzschild on the potential of urate to treat central nervous system injury. This mini review summarizes the important findings in these three areas and outlines future directions for the development of new purine-related therapies for traumatic brain injury and other forms of central nervous system injury. In this review, novel therapies based on three emerging areas of adenosine-related pathobiology in traumatic brain injury (TBI) were proposed, namely, therapies targeting 1) the 2',3'-cyclic adenosine monophosphate (cAMP) pathway, 2) adenosine deficiency after TBI, and 3) augmentation of urate after TBI.

  7. The Effects of Azathioprine (Imuran) on Purine Synthesis in Clinical Disorders of Purine Metabolism*

    PubMed Central

    Kelley, William N.; Rosenbloom, Frederick M.; Seegmiller, J. Edwin

    1967-01-01

    Azathioprine, a purine analogue, significantly suppressed the purine synthesis de novo of two gouty patients manifesting overproduction of uric acid, as well as three of four gouty patients who showed normal uric acid production. This suppression is taken as evidence that phosphoribosyl-pyrophosphate amidotransferase, the rate-controlling step in purine synthesis de novo, has a normal sensitivity to feedback inhibitors in the patients who responded to the drug. Two children afflicted with the familial disorder of hyperuricemia, choreo-athetosis, and self-mutilation described by Lesch and Nyhan showed no reduction in the activity of the biosynthetic pathway in response to azathioprine. This inability to respond to azathioprine can be directly related to the absence in these patients of the enzyme hypoxanthine-guanine phosphoribosyltransferase which is required for conversion of the drug or its metabolites to the biochemically active ribonucleotide form. PMID:16695929

  8. Purines 2010: Adenine Nucleosides and Nucleotides in Biomedicine.

    PubMed

    Sereda, Michal J

    2010-08-01

    The Purines 2010: Adenine Nucleosides and Nucleotides in Biomedicine meeting, held in Tarragona, Spain, included topics covering new findings in the field of purinergic signaling and the development of purine-based drugs. This conference report highlights selected presentations on developments in purinerigic signaling, medicinal chemistry, the therapeutic potential of purine-based drugs, and the role of purines and adenosine receptors in neurodegenerative disorders, sickle cell disease, bone homeostasis, pulmonary fibrosis and pain. Investigational drugs discussed include CF-101 (Can-Fite BioPharma Ltd/NIH/Kwang Dong Pharmaceutical Co Ltd/Seikagaku Corp) and denufosol tetrasodium (Cystic Fibrosis Foundation Therapeutics Inc/Inspire Pharmaceuticals Inc).

  9. Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

    SciTech Connect

    Taylor,E.; Rinaldo-Matthis, A.; Li, L.; Ghanem, M.; Hazleton, K.; Cassera, M.; Almo, S.; Schramm, V.

    2007-01-01

    The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

  10. Distinct Purine Distribution in Carbonaceous Chondrites

    NASA Technical Reports Server (NTRS)

    Callahan, Michael P.; Smith, Karen E.; Cleaves, Henderson J.; Ruzicka, Josef; Stern, Jennifer C.; Glavin, Daniel P.; House, Christopher H.; Dworkin, Jason P.

    2011-01-01

    Carbonaceous chondrite meteorites are known to contain a diverse suite of organic compounds, many of which are essential components of biochemistry. Amino acids, which are the monomers of proteins, have been extensively studied in such meteorites (e.g. Botta and Bada 2002; Pizzarello et aI., 2006). The origin of amino acids in meteorites has been firmly established as extraterrestrial based on their detection typically as racemic mixtures of amino acids, the presence of many non-protein amino acids, and non-terrestrial values for compound-specific deuterium, carbon, and nitrogen isotopic measurements. In contrast to amino acids, nucleobases in meteorites have been far less studied. Nucleobases are substituted one-ring (pyrimidine) or two-ring (purine) nitrogen heterocyclic compounds and serve as the information carriers of nucleic acids and in numerous coenzymes. All of the purines (adenine, guanine, hypoxanthine, and xanthine) and pyrimidines (uracil) previously reported in meteorites are biologically common and could be interpreted as the result of terrestrial contamination (e.g. van del' Velden and Schwartz, 1974.) Unlike other meteoritic organics, there have been no observations of stochastic molecular diversity of purines and pyrimidines in meteorites, which has been a criterion for establishing extraterrestrial origin. Maltins et al. (2008) performed compound-specific stable carbon isotope measurements for uracil and xanthine in the Murchison meteorite. They assigned a non-terrestrial origin for these nucleobases; however, the possibility that interfering indigenous molecules (e.g. carboxylic acids) contributed to the 13C-enriched isotope values for these nucleobases cannot be completely ruled out. Thus, the origin of these meteoritic nucleobases has never been established unequivocally. Here we report on our investigation of extracts of II different carbonaceous chondrites covering various petrographic types (Cl, CM, and CR) and degrees of aqueous alteration

  11. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  12. Allosteric Modulation of Purine and Pyrimidine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Göblyös, Anikó; IJzerman, Adriaan P.

    2011-01-01

    Among the purine and pyrimidine receptors, the discovery of small molecular allosteric modulators has been most highly advanced for the A1 and A3 ARs. These AR modulators have allosteric effects that are structurally separated from the orthosteric effects in SAR studies. The benzoylthiophene derivatives tend to act as allosteric agonists, as well as selective positive allosteric modulators (PAMs) of the A1 AR. A 2-amino-3-aroylthiophene derivative T-62 has been under development as a PAM of the A1 AR for the treatment of chronic pain. Several structurally distinct classes of allosteric modulators of the human A3 AR have been reported: 3-(2-pyridinyl)isoquinolines, 2,4-disubstituted quinolines, 1H-imidazo-[4,5-c]quinolin-4-amines, endocannabinoid 2-arachidonylglycerol and the food dye Brilliant Black BN. Site-directed mutagenesis of A1 and A3 ARs has identified residues associated with the allosteric effect, distinct from those that affect orthosteric binding. A few small molecular allosteric modulators have been reported for several of the P2X ligand-gated ion channels and the G protein-coupled P2Y receptor nucleotides. Metal ion modulation of the P2X receptors has been extensively explored. The allosteric approach to modulation of purine and pyrimidine receptors looks promising for development of drugs that are event-specific and site-specific in action. PMID:21586360

  13. Extracellular purines, purinergic receptors and tumor growth

    PubMed Central

    Di Virgilio, F; Adinolfi, E

    2017-01-01

    Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host–tumor interaction and highlights novel therapeutic options stemming from recent advances in this field. PMID:27321181

  14. Prolonged fasting increases purine recycling in post-weaned northern elephant seals.

    PubMed

    Soñanez-Organis, José Guadalupe; Vázquez-Medina, José Pablo; Zenteno-Savín, Tania; Aguilar, Andres; Crocker, Daniel E; Ortiz, Rudy M

    2012-05-01

    Northern elephant seals are naturally adapted to prolonged periods (1-2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species.

  15. Acceleration of purine degradation by periodontal diseases.

    PubMed

    Barnes, V M; Teles, R; Trivedi, H M; Devizio, W; Xu, T; Mitchell, M W; Milburn, M V; Guo, L

    2009-09-01

    Periodontal diseases, such as gingivitis and periodontitis, are characterized by bacterial plaque accumulation around the gingival crevice and the subsequent inflammation and destruction of host tissues. To test the hypothesis that cellular metabolism is altered as a result of host-bacteria interaction, we performed an unbiased metabolomic profiling of gingival crevicular fluid (GCF) collected from healthy, gingivitis, and periodontitis sites in humans, by liquid and gas chromatography mass spectrometry. The purine degradation pathway, a major biochemical source for reactive oxygen species (ROS) production, was significantly accelerated at the disease sites. This suggests that periodontal-disease-induced oxidative stress and inflammation are mediated through this pathway. The complex host-bacterial interaction was further highlighted by depletion of anti-oxidants, degradation of host cellular components, and accumulation of bacterial products in GCF. These findings provide new mechanistic insights and a panel of comprehensive biomarkers for periodontal disease progression.

  16. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

    Mills, G C; Schmalstieg, F C; Trimmer, K B; Goldman, A S; Goldblum, R M

    1976-01-01

    Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides. PMID:1066699

  17. Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

    PubMed

    Dangoudoubiyam, Sriveny; Zhang, Zijing; Howe, Daniel K

    2014-09-01

    Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite.

  18. Determination and profiling of purines in foods by using HPLC and LC-MS.

    PubMed

    Inazawa, K; Sato, A; Kato, Y; Yamaoka, N; Fukuuchi, T; Yasuda, M; Mawatari, K; Nakagomi, K; Kaneko, K

    2014-01-01

    Purines in food are known to raise serum uric acid levels. We determined the purine content of sweet potato and beef by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The purine content of the samples was 118-1,034 μmol/100 g. The total purine content was also divided into purine bases, nucleosides, nucleotides, and nucleic acids. Our results suggest that differences in total purine content and in the ratio of purine types between vegetables and beef cause a difference in elevation of serum uric acid levels.

  19. Enzymic cleavage of purine ultraviolet photoproducts formed at biologically significant wavelengths

    SciTech Connect

    Gallagher, P.E.; Duker, N.J.

    1986-05-01

    A paradox of ultraviolet carcinogenesis research has been that maximal absorption and mutagenesis occurs at 254 nm irradiation, while the greatest tumor yield in irradiated animals has been at wavelengths between 275 and 300 nm. Ambient actinic radiation contains mostly wavelengths above 280 nm with no substantial 254 nm component. Therefore, the authors investigated formation of DNA damage by 250-400 nm irradiation. Irradiated, 3'-end-labeled, 92 base pair sequence of the human alphoid segment was incubated with endonuclease v, purified from T4-infected E. coli, or with a crude extract of M. luteus. Analysis by gel electrophoresis showed that besides pyrimidine photodimers, previously unreported photoproducts were incised. These are not 6-4'(pyrimidin-2'-one)-pyrimidines, apurinic or apyrimidinic sites, or ring-opened purines. The new products are at specific purine loci and are formed in quantities similar to pyridimine dimers. The optimal wavelengths for their formation are 275-295 nm, similar to the maximum peak of actinic carcinogenesis. The enzyme incising these products is inactivated by different heating conditions than the pyrimidine dimer-DNA glycosylase, and they appear to be separable by column chromatography. The authors propose that a novel family of photoproducts, possibly purine-containing dimers, are incised by previously uncharacterized DNA repair enzymes.

  20. Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs.

    PubMed

    Strom, Shane; Shiskova, Evgenia; Hahm, Yaeeun; Grover, Neena

    2015-07-01

    Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1- to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl(2) or 1 M KCl. The [Formula: see text] loop parameters for 1- to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ΔΔG° values relative to 1 M KCl were 0.47-2.06 kcal/mol more favorable for the RNA bulge loops. The [Formula: see text] loop parameters for 1- to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4- and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1- to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1- to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride.

  1. Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

    PubMed Central

    Strom, Shane; Shiskova, Evgenia; Hahm, Yaeeun

    2015-01-01

    Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1- to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl2 or 1 M KCl. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ΔΔG° values relative to 1 M KCl were 0.47–2.06 kcal/mol more favorable for the RNA bulge loops. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4- and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1- to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1- to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride. PMID:26022248

  2. Biosensor measurement of purine release from cerebellar cultures and slices.

    PubMed

    Wall, Mark; Eason, Robert; Dale, Nicholas

    2010-09-01

    We have previously described an action-potential and Ca(2+)-dependent form of adenosine release in the molecular layer of cerebellar slices. The most likely source of the adenosine is the parallel fibres, the axons of granule cells. Using microelectrode biosensors, we have therefore investigated whether cultured granule cells (from postnatal day 7-8 rats) can release adenosine. Although no purine release could be detected in response to focal electrical stimulation, purine (adenosine, inosine or hypoxanthine) release occurred in response to an increase in extracellular K(+) concentration from 3 to 25 mM coupled with addition of 1 mM glutamate. The mechanism of purine release was transport from the cytoplasm via an ENT transporter. This process did not require action-potential firing but was Ca(2+)dependent. The major purine released was not adenosine, but was either inosine or hypoxanthine. In order for inosine/hypoxanthine release to occur, cultures had to contain both granule cells and glial cells; neither cellular component was sufficient alone. Using the same stimulus in cerebellar slices (postnatal day 7-25), it was possible to release purines. The release however was not blocked by ENT blockers and there was a shift in the Ca(2+) dependence during development. This data from cultures and slices further illustrates the complexities of purine release, which is dependent on cellular composition and developmental stage.

  3. Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

    SciTech Connect

    Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika; Alfaro, Joshua F.; Yang, Feng; Burnum-Johnson, Kristin E.; Petyuk, Vladislav A.; Weitz, Karl K.; Camp, David G.; Smith, Richard D.; Wilmarth, Phillip A.; David, Larry L.; Ramasamy, Gowthaman; Myler, Peter J.; Carter, Nicola S.

    2014-02-27

    The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

  4. Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani

    PubMed Central

    Soysa, Radika; Alfaro, Joshua F.; Yang, Feng; Burnum-Johnson, Kristin E.; Petyuk, Vladislav A.; Weitz, Karl K.; Camp, David G.; Smith, Richard D.; Wilmarth, Phillip A.; David, Larry L.; Ramasamy, Gowthaman; Myler, Peter J.; Carter, Nicola S.

    2014-01-01

    The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6–48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms. PMID:24586154

  5. Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

    SciTech Connect

    Ting, L; Shi, W; Lewandowicz, A; Singh, V; Mwakingwe, A; Birck, M R; Taylor Ringia, E A; Bench, G; Madrid, D C; Tyler, P C; Evans, G B; Furneaux, R H; Schramm, V L; Kim, K

    2004-05-19

    Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of the polyamine pathway are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not previously been described. 5'-methylthio-Immucillin-H, a transition state analogue inhibitor that is selective for malarial over human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may have application as antimalarials.

  6. Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya

    PubMed Central

    2012-01-01

    Background The small RNAs (sRNA) are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant) from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. Results We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV). We also used the deep sequencing data to annotate the micro RNA (miRNA) in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff’s purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. Conclusions We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally functionally redundant. The

  7. Isolation of Purines and Pyrimidines from the Murchison Meteorite

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Bada, J. K.

    2003-01-01

    The origin of life on Earth, and possibly on other planets such as Mars, would have required the presence of liquid water and a continuous supply of prebiotic organic compounds. The delivery of organic matter by asteroids, comets, and carbonaceous meteorites could have contributed to the early Earth's prebiotic inventory by seeding the planet with biologically important organic compounds. A wide variety of prebiotic organic compounds have previously been detected in the Murchison CM type carbonaceous chondrite including amino acids, purines and pyrimidines'. These compounds play a major role in terrestrial biochemistry and are integral components of proteins, DNA and RNA. In this study we developed a new extraction technique using sublimation in order to isolate purines and pyrimidines from Murchison2, which is cleaner and more time efficient that traditional methods3. Several purines including adenine, guanine, hypoxanthine and xanthine were positively identified by high performance liquid chromatography and ultraviolet absorption detection in our Murchison extracts. The purines detected in Murchison do not correlate with the distribution of nucleobases found in geological environments on Earth4. Moreover, the abundance of extraterrestrial amino acids and the low level of terrestrial amino acid contaminants found in Murchison', support the idea that the purines in t h s meteorite are extraterrestrial in origin.

  8. Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts

    PubMed Central

    Goldman, Nanna; Chandler-Militello, Devin; Langevin, Helene; Nedergaard, Maiken; Takano, Takahiro

    2013-01-01

    Earlier studies have shown that activation of adenosine A1 receptors on peripheral pain fibers contributes to acupuncture-induced suppression of painful input. In addition to adenosine, acupuncture triggers the release of other purines, including ATP and ADP that may bind to purine receptors on nearby fibroblasts. We here show that purine agonists trigger increase in cytosolic Ca 2+ signaling in a cultured human fibroblasts cell line. The profile of agonist-induced Ca2+ increases indicates that the cells express functional P2yR2 and P2yR4 receptors, as well as P2yR1 and P2xR7 receptors. Unexpectedly, purine-induced Ca2+ signaling was associated with a remodeling of the actin cytoskeleton. ATP induced a transient loss in F-actin stress fiber. The changes of actin cytoskeleton occurred slowly and peaked at 10 min after agonist exposure. Inhibition of ATP-induced increases in Ca2+ by cyclopiazonic acid blocked receptor-mediated cytoskeleton remodeling. The Ca2+ ionophore failed to induce cytoskeletal remodeling despite triggering robust increases in cytosolic Ca2+. These observations indicate that purine signaling induces transient changes in fibroblast cytoarchitecture that could be related to the beneficial effects of acupuncture. PMID:23462235

  9. 8-Phosphorus substituted isosteres of purine and deazapurines.

    PubMed Central

    Khwaja, T A; Pande, H

    1979-01-01

    Synthesis of 8-phosphorus substituted isosteres of purine [pyrimidino (4,5-d)-1,3,2-diazaphosphole], 1-deazapurine [pyridino (2,3-d)-1,3,2-diazaphosphole] and 3-deazapurine [pyridino (4,5-d)-1,3,2-diazaphosphole] has been achieved by the reaction of equimolar amounts of triphenylphosphite and 4,5-diaminopyrimidine, 2,3-diaminopyridine and 3,4-diaminopyridine, respectively. These compounds hydrolyzed (cleavage of the phosphorus-nitrogen bounds) in aqueous solutions to provide the corresponding diaminopyrimidine or diaminopyridines. These three new basic ring systems constitute the first reported synthesis of purines in which ring carbon atom is substituted with a phosphorus atom. 8-Phosphorus substituted purine at a concentration of 4 X 10(-4)M caused a 50% inhibition in the growth of leukemia L1210 cells in culture. The biochemical rationale for the synthesis of these compounds is discussed. PMID:493140

  10. Arthrobacter oxydans as a biocatalyst for purine deamination.

    PubMed

    Médici, Rosario; Lewkowicz, Elizabeth S; Iribarren, Adolfo M

    2008-12-01

    Deaminases are enzymes that catalyze the hydrolysis of amino groups of nucleosides or their bases. Because these enzymes play important roles in nucleotide metabolism, they are relevant targets in anticancer and antibacterial therapies. Mammalian deaminases are commercially available but the use of bacterial whole cells, especially as biocatalysts, is continuously growing because of their economical benefits. Moreover, deaminases are useful for the preparative chemoenzymatic transformation of nucleoside and base analogues into a variety of derivatives. The purine deaminase activities of Arthrobacter oxydans, a gram-positive bacterium utilized widely in bioremediation, were studied. The presence of adenosine, adenine and guanine deaminases was demonstrated and some purine bases and nucleosides were analyzed as substrates. Using A. oxydans whole cells as the biocatalyst, different purine compounds such as the anti-HIV, 2',3'-dideoxyinosine (73%, 2 h) were obtained.

  11. Gene expression control by Bacillus anthracis purine riboswitches.

    PubMed

    Kirchner, Marion; Schneider, Sabine

    2017-02-16

    In all kingdoms of life, cellular replication relies on the presence of nucleosides and nucleotides, the building blocks of nucleic acids and the main source of energy. In bacteria, the availability of metabolites sometimes directly regulates the expression of enzymes and proteins involved in purine salvage, biosynthesis and uptake through riboswitches. Riboswitches are located in bacterial mRNAs and can control gene expression by conformational changes in response to ligand binding. We have established an inverse reporter gene system in Bacillus subtilis that allows us to monitor riboswitch-controlled gene expression. We used it to investigate the activity of five potential purine riboswitches from B. anthracis in response to different purines and pyrimidines. Furthermore, in vitro studies on the aptamer domains of the riboswitches reveal their variation in guanine binding affinity ranging from nM to µM. These data do not only provide insight into metabolite sensing but can also aid to engineer artificial cell regulatory systems.

  12. A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy.

    PubMed

    Bromley, Jennifer R; Warnes, Barbara J; Newell, Christine A; Thomson, Jamie C P; James, Celia M; Turnbull, Colin G N; Hanke, David E

    2014-03-01

    StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

  13. Molecular and Genetic Analyses of Drosophila Prat, Which Encodes the First Enzyme of De Novo Purine Biosynthesis

    PubMed Central

    Clark, D. V.

    1994-01-01

    The Drosophila Prat gene encodes phosphoribosylamidotransferase (PRAT), the enzyme that performs the first committed step of the de novo purine nucleotide biosynthesis pathway. Using information from amino acid sequence alignments of PRAT from other organisms, a polymerase chain reaction-based approach was employed to clone Prat. Amino acid sequence alignment of Drosophila PRAT with PRAT from bacteria, yeast, and vertebrates indicates that it is most identical (at least 60%) to the vertebrate PRATs. It shares putative amino-terminal propeptide and ironbinding domains seen only in Bacillus subtilis and vertebrate PRATs. Prat was localized to the right arm of chromosome 3 at polytene band 84E1-2. Owing to the fact that this region had been well characterized previously, Prat was localized to a 30-kilobase region between two deficiency break-points. By making the prediction that Prat would have a similar ``purine syndrome'' phenotype as mutations in the genes ade2 and ade3, which encode enzymes downstream in the pathway, five alleles of Prat were isolated. Three of the alleles were identified as missense mutations. A comparison of PRAT enzyme activity with phenotype in three of the mutants indicates that a reduction to 40% of the wild-type allele's activity is sufficient to cause the purine syndrome, suggesting that PRAT activity is limiting in Drosophila. PMID:8150282

  14. Purine utilization by Klebsiella oxytoca M5al: genes for ring-oxidizing and -opening enzymes.

    PubMed

    Pope, Scott D; Chen, Li-Ling; Stewart, Valley

    2009-02-01

    The enterobacterium Klebsiella oxytoca uses a variety of inorganic and organic nitrogen sources, including purines, nitrogen-rich compounds that are widespread in the biosphere. We have identified a 23-gene cluster that encodes the enzymes for utilizing purines as the sole nitrogen source. Growth and complementation tests with insertion mutants, combined with sequence comparisons, reveal functions for the products of these genes. Here, we report our characterization of 12 genes, one encoding guanine deaminase and the others encoding enzymes for converting (hypo)xanthine to allantoate. Conventionally, xanthine dehydrogenase, a broadly distributed molybdoflavoenzyme, catalyzes sequential hydroxylation reactions to convert hypoxanthine via xanthine to urate. Our results show that these reactions in K. oxytoca are catalyzed by a two-component oxygenase (HpxE-HpxD enzyme) homologous to Rieske nonheme iron aromatic-ring-hydroxylating systems, such as phthalate dioxygenase. Our results also reveal previously undescribed enzymes involved in urate oxidation to allantoin, catalyzed by a flavoprotein monooxygenase (HpxO enzyme), and in allantoin conversion to allantoate, which involves allantoin racemase (HpxA enzyme). The pathway also includes the recently described PuuE allantoinase (HpxB enzyme). The HpxE-HpxD and HpxO enzymes were discovered independently by de la Riva et al. (L. de la Riva, J. Badia, J. Aguilar, R. A. Bender, and L. Baldoma, J. Bacteriol. 190:7892-7903, 2008). Thus, several enzymes in this K. oxytoca purine utilization pathway differ from those in other microorganisms. Isofunctional homologs of these enzymes apparently are encoded by other species, including Acinetobacter, Burkholderia, Pseudomonas, Saccharomyces, and Xanthomonas.

  15. 40 CFR 721.4685 - Substituted purine metal salt (generic name).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Substituted purine metal salt (generic... Specific Chemical Substances § 721.4685 Substituted purine metal salt (generic name). (a) Chemical... as a substituted purine metal salt (PMN P-95-175) is subject to reporting under this section for...

  16. 40 CFR 721.4685 - Substituted purine metal salt (generic name).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Substituted purine metal salt (generic... Specific Chemical Substances § 721.4685 Substituted purine metal salt (generic name). (a) Chemical... as a substituted purine metal salt (PMN P-95-175) is subject to reporting under this section for...

  17. 40 CFR 721.4685 - Substituted purine metal salt (generic name).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Substituted purine metal salt (generic... Specific Chemical Substances § 721.4685 Substituted purine metal salt (generic name). (a) Chemical... as a substituted purine metal salt (PMN P-95-175) is subject to reporting under this section for...

  18. 40 CFR 721.4685 - Substituted purine metal salt (generic name).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Substituted purine metal salt (generic... Specific Chemical Substances § 721.4685 Substituted purine metal salt (generic name). (a) Chemical... as a substituted purine metal salt (PMN P-95-175) is subject to reporting under this section for...

  19. 40 CFR 721.4685 - Substituted purine metal salt (generic name).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted purine metal salt (generic... Specific Chemical Substances § 721.4685 Substituted purine metal salt (generic name). (a) Chemical... as a substituted purine metal salt (PMN P-95-175) is subject to reporting under this section for...

  20. Purines and neuronal excitability: links to the ketogenic diet.

    PubMed

    Masino, S A; Kawamura, M; Ruskin, D N; Geiger, J D; Boison, D

    2012-07-01

    ATP and adenosine are purines that play dual roles in cell metabolism and neuronal signaling. Acting at the A(1) receptor (A(1)R) subtype, adenosine acts directly on neurons to inhibit excitability and is a powerful endogenous neuroprotective and anticonvulsant molecule. Previous research showed an increase in ATP and other cell energy parameters when an animal is administered a ketogenic diet, an established metabolic therapy to reduce epileptic seizures, but the relationship among purines, neuronal excitability and the ketogenic diet was unclear. Recent work in vivo and in vitro tested the specific hypothesis that adenosine acting at A(1)Rs is a key mechanism underlying the success of ketogenic diet therapy and yielded direct evidence linking A(1)Rs to the antiepileptic effects of a ketogenic diet. Specifically, an in vitro mimic of a ketogenic diet revealed an A(1)R-dependent metabolic autocrine hyperpolarization of hippocampal neurons. In parallel, applying the ketogenic diet in vivo to transgenic mouse models with spontaneous electrographic seizures revealed that intact A(1)Rs are necessary for the seizure-suppressing effects of the diet. This is the first direct in vivo evidence linking A(1)Rs to the antiepileptic effects of a ketogenic diet. Other predictions of the relationship between purines and the ketogenic diet are discussed. Taken together, recent research on the role of purines may offer new opportunities for metabolic therapy and insight into its underlying mechanisms.

  1. Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase

    PubMed Central

    Donaldson, Teraya M.; Cassera, María B.; Ho, Meng-Chiao; Zhan, Chenyang; Merino, Emilio F.; Evans, Gary B.; Tyler, Peter C.; Almo, Steven C.; Schramm, Vern L.

    2014-01-01

    The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5′-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5′ groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5′-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2′-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5′-methylthioinosine and similarly 5′-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa. PMID:24585883

  2. Distinct Distribution of Purines in CM and CR Carbonaceous Chondrites

    NASA Technical Reports Server (NTRS)

    Callahan, Michael P.; Stern, Jennifer C.; Glavin, Daniel P.; Smith, Karen E.; Martin, Mildred G.; Dworkin, Jason P.

    2010-01-01

    Carbonaceous meteorites contain a diverse suite of organic molecules and delivered pre biotic organic compounds, including purines and pyrimidines, to the early Earth (and other planetary bodies), seeding it with the ingredients likely required for the first genetic material. We have investigated the distribution of nucleobases in six different CM and CR type carbonaceous chondrites, including fivc Antarctic meteorites never before analyzed for nucleobases. We employed a traditional formic acid extraction protocol and a recently developed solid phase extraction method to isolate nucleobases. We analyzed these extracts by high performance liquid chromatography with UV absorbance detection and tandem mass spectrometry (HPLC-UV -MS/MS) targeting the five canonical RNAIDNA bases and hypoxanthine and xanthine. We detected parts-per-billion levels of nucleobases in both CM and CR meteorites. The relative abundances of the purines found in Antarctic CM and CR meteorites were clearly distinct from each other suggesting that these compounds are not terrestrial contaminants. One likely source of these purines is formation by HCN oligomerization (with other small molecules) during aqueous alteration inside the meteorite parent body. The detection of the purines adenine (A), guanine (0), hypoxanthine (HX), and xanthine (X) in carbonaceous meteorites indicates that these compounds should have been available on the early Earth prior to the origin of the first genetic material.

  3. Purines and Neuronal Excitability: Links to the Ketogenic Diet

    PubMed Central

    Masino, SA; Kawamura, M; Ruskin, DN; Geiger, JD; Boison, D

    2011-01-01

    ATP and adenosine are purines that play dual roles in cell metabolism and neuronal signaling. Acting at the A1 receptor (A1R) subtype, adenosine acts directly on neurons to inhibit excitability and is a powerful endogenous neuroprotective and anticonvulsant molecule. Previous research showed an increase in ATP and other cell energy parameters when an animal is administered a ketogenic diet, an established metabolic therapy to reduce epileptic seizures, but the relationship among purines, neuronal excitability and the ketogenic diet was unclear. Recent work in vivo and in vitro tested the specific hypothesis that adenosine acting at A1Rs is a key mechanism underlying the success of ketogenic diet therapy and yielded direct evidence linking A1Rs to the antiepileptic effects of a ketogenic diet. Specifically, an in vitro mimic of a ketogenic diet revealed an A1R-dependent metabolic autocrine hyperpolarization of hippocampal neurons. In parallel, applying the ketogenic diet in vivo to transgenic mouse models with spontaneous electrographic seizures revealed that intact A1Rs are necessary for the seizure-suppressing effects of the diet. This is the first direct in vivo evidence linking A1Rs to the antiepileptic effects of a ketogenic diet. Other predictions of the relationship between purines and the ketogenic diet are discussed. Taken together, recent research on the role of purines may offer new opportunities for metabolic therapy and insight into its underlying mechanisms. PMID:21880467

  4. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

    PubMed

    Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

    2014-09-01

    Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

  5. Roles of the amino group of purine bases in the thermodynamic stability of DNA base pairing.

    PubMed

    Nakano, Shu-ichi; Sugimoto, Naoki

    2014-08-05

    The energetic aspects of hydrogen-bonded base-pair interactions are important for the design of functional nucleotide analogs and for practical applications of oligonucleotides. The present study investigated the contribution of the 2-amino group of DNA purine bases to the thermodynamic stability of oligonucleotide duplexes under different salt and solvent conditions, using 2'-deoxyriboinosine (I) and 2'-deoxyribo-2,6-diaminopurine (D) as non-canonical nucleotides. The stability of DNA duplexes was changed by substitution of a single base pair in the following order: G • C > D • T ≈ I • C > A • T > G • T > I • T. The apparent stabilization energy due to the presence of the 2-amino group of G and D varied depending on the salt concentration, and decreased in the water-ethanol mixed solvent. The effects of salt concentration on the thermodynamics of DNA duplexes were found to be partially sequence-dependent, and the 2-amino group of the purine bases might have an influence on the binding of ions to DNA through the formation of a stable base-paired structure. Our results also showed that physiological salt conditions were energetically favorable for complementary base recognition, and conversely, low salt concentration media and ethanol-containing solvents were effective for low stringency oligonucleotide hybridization, in the context of conditions employed in this study.

  6. Purine nucleoside phosphorylase from Pseudoalteromonas sp. Bsi590: molecular cloning, gene expression and characterization of the recombinant protein.

    PubMed

    Li, Xiaohui; Jiang, Xinyin; Li, Huirong; Ren, Daming

    2008-05-01

    The gene encoding purine nucleoside phosphorylase (PNP) from the cold-adapted marine bacterium Pseudoalteromonas sp. Bsi590 was identified, cloned and expressed in Escherichia coli. The gene encodes a polypeptide of 233 amino acids with a calculated molecular weight of 25,018 Da. Pseudoalteromonas sp. Bsi590 PNP (PiPNP) shares 60% amino sequence identity and conservation of amino acid residues involved in catalysis with mesophilic Escherichia coli deoD-encoded purine nucleoside phosphorylase (EcPNP). N-terminal his-tagged PiPNP and EcPNP were purified to apparent homogeneity using Ni2+-chelating column. Compared with EcPNP, PiPNP possessed a lower temperature optimum and thermal stability. As for PNP enzymes in general, PiPNP and EcPNP displayed complicated kinetic properties; PiPNP possessed higher Km and catalytic efficiency (kcat/Km) compared to EcPNP at 37 degrees C. Substrate specificity results showed PiPNP catalyzed the phosphorolytic cleavage of 6-oxopurine and 6-aminopurine nucleosides (or 2-deoxynucleosides), and to a lesser extent purine arabinosides. PiPNP showed a better activity with inosine while no activity toward pyrimidine nucleosides. The protein conformation was analyzed by temperature perturbation difference spectrum. Results showed that PiPNP had lower conformation transition point temperature than EcPNP; phosphate buffer and KCl had significant influence on PiPNP protein conformation stability and thermostability.

  7. Infrared Spectroscopy of Charge Transfer Complexes of Purines and Pyrimidines

    SciTech Connect

    Rathod, Pravinsinh I.; Oza, A. T.

    2011-10-20

    The FTIR spectra of charge transfer complexes of purines and pyrimidines with organic acceptors such as TCNQ, TCNE, DDQ, chloranil and iodine are obtained and studied in the present work. Adenine, guanine, thymine, cytosine and uracil are the purines and pyrimidines which are found as constituent of DNA and RNA. Charge transfer induced hydrogen bonding is concluded on the basis of indirect transitions observed in the infrared range in these CTCs. Some CTCs show gaussian bands revealing delocalization of charge carriers. The CTCs show interband transition in three-dimensions rather than two-dimensions unlike CTCs of amino acids. There is no extended hydrogen bonded network spanning the whole crystal. This leads to indirect transition due to locally deformed lattice furnishing a phonon-assisted transition.

  8. Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris.

    PubMed Central

    Surette, M; Gill, T; MacLean, S

    1990-01-01

    Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography. PMID:2111121

  9. Infrared Spectroscopy of Charge Transfer Complexes of Purines and Pyrimidines

    NASA Astrophysics Data System (ADS)

    Rathod, Pravinsinh I.; Oza, A. T.

    2011-10-01

    The FTIR spectra of charge transfer complexes of purines and pyrimidines with organic acceptors such as TCNQ, TCNE, DDQ, chloranil and iodine are obtained and studied in the present work. Adenine, guanine, thymine, cytosine and uracil are the purines and pyrimidines which are found as constituent of DNA and RNA. Charge transfer induced hydrogen bonding is concluded on the basis of indirect transitions observed in the infrared range in these CTCs. Some CTCs show gaussian bands revealing delocalization of charge carriers. The CTCs show interband transition in three-dimensions rather than two-dimensions unlike CTCs of amino acids. There is no extended hydrogen bonded network spanning the whole crystal. This leads to indirect transition due to locally deformed lattice furnishing a phonon-assisted transition.

  10. Enantioselective and Regiodivergent Addition of Purines to Terminal Allenes: Synthesis of Abacavir.

    PubMed

    Thieme, Niels; Breit, Bernhard

    2017-02-01

    The rhodium-catalyzed atom-economic asymmetric N-selective intermolecular addition of purine derivatives to terminal allenes is reported. Branched allylic purines were obtained in high yields, regioselectivity and outstanding enantioselectivity utilizing a Rh/Josiphos catalyst. Conversely, linear selective allylation of purines could be realized in good to excellent regio- and E/Z-selectivity with a Pd/dppf catalyst system. Furthermore, the new methodology was applied to a straightforward asymmetric synthesis of carbocyclic nucleoside abacavir.

  11. Evaluation of Purine Salvage as a Chemotherapeutic Target in the Plasmodium yoelii Rodent Model

    DTIC Science & Technology

    2008-03-01

    modification machinery to control gene expression. The sexual cycle of Plasmodium is a critical process required for transmission of malaria via the...for RNA and DNA and must salvage purines from their host. We are investigating whether the malaria purine salvage pathway can be exploited to develop...chemotherapy are needed to protect military personnel stationed in developing countries. Malaria parasites cannot make purines needed for RNA and DNA and

  12. Determination of purine contents of alcoholic beverages using high performance liquid chromatography.

    PubMed

    Kaneko, Kiyoko; Yamanobe, Tomoyo; Fujimori, Shin

    2009-08-01

    The purine contents of alcoholic beverages were determined in order to utilize them in the dietary care of gout and hyperuricemia. In the management of these diseases, restriction of both alcohol and purine intake are important. The method employed in this study is a quantitative determination of purine contents by HPLC. Alcoholic beverages were hydrolyzed to corresponding purine bases, which were then separated by HPLC, and base peaks were identified using an enzymatic peak-shift technique. This method is sufficiently accurate and reproducible to examine the purine contents of various alcoholic beverages that patients consume. Purine contents were as follows: spirits, 0.7-26.4 micromol/L; regular beer, 225.0-580.2 micromol/L; low-malt beer, 193.4-267.9 micromol/L; low-malt and low-purine beer, 13.3 micromol/L; other liquors, 13.1-818.3 micromol/L. Some local and low-alcohol beers were found to contain about 2.5 times more purines than regular beer. As some alcoholic beverages contain considerable amounts of purines, we recommend that excess consumption of these beverages be avoided. These data should be useful in the management of hyperuricemia and gout, not only for patients but also for physicians.

  13. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

    PubMed Central

    Yeakley, J M; Morfin, J P; Rosenfeld, M G; Fu, X D

    1996-01-01

    A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8755518

  14. Homochiral Selectivity in RNA Synthesis: Montmorillonite-catalyzed Quaternary Reactions of D, L-Purine with D, L- Pyrimidine Nucleotides

    NASA Astrophysics Data System (ADS)

    Joshi, Prakash C.; Aldersley, Michael F.; Ferris, James P.

    2011-06-01

    Selective adsorption of D, L-ImpA with D, L-ImpU on the platelets of montmorillonite demonstrates an important reaction pathway for the origin of homochirality in RNA synthesis. Our earlier studies have shown that the individual reactions of D, L-ImpA or D, L-ImpU on montmorillonite catalyst produced oligomers which were only partially inhibited by the incorporation of both D- and L-enantiomers. Homochirality in these reactions was largely due to the formation of cyclic dimers that cannot elongate. We investigated the quaternary reactions of D, L-ImpA with D, L-ImpU on montmorillonite. The chain length of these oligomers increased from 9-mer to 11-mer as observed by HPLC, with a concominant increase in the yield of linear dimers and higher oligomers in the reactions involving D, L-ImpA with D, L-ImpU as compared to the similar reactions carried out with D-enantiomers only. The formation of cyclic dimers of U was completely inhibited in the quaternary reactions. The yield of cyclic dimers of A was reduced from 60% to 10% within the dimer fraction. 12 linear dimers and 3 cyclic dimers were isolated and characterized from the quaternary reaction. The homochirality and regioselectivity of dimers were 64.1% and 71.7%, respectively. Their sequence selectivity was shown by the formation of purine-pyrimidine (54-59%) linkages, followed by purine-purine (29-32%) linkages and pyrimidine-pyrimidine (9-13%) linkages. Of the 16 trimers detected, 10 were homochiral with an overall homochirality of 73-76%. In view of the greater homochirality, sequence- and regio- selectivity, the quaternary reactions on montmorillonite demonstrate an unexpectedly favorable route for the prebiotic synthesis of homochiral RNA compared with the separate reactions of enantiomeric activated mononucleotides.

  15. Associations between purine metabolites and monoamine neurotransmitters in first-episode psychosis

    PubMed Central

    Yao, Jeffrey K.; Dougherty, George G.; Reddy, Ravinder D.; Matson, Wayne R.; Kaddurah-Daouk, Rima; Keshavan, Matcheri S.

    2013-01-01

    Schizophrenia (SZ) is a biochemically complex disorder characterized by widespread defects in multiple metabolic pathways whose dynamic interactions, until recently, have been difficult to examine. Rather, evidence for these alterations has been collected piecemeal, limiting the potential to inform our understanding of the interactions amongst relevant biochemical pathways. We herein review perturbations in purine and neurotransmitter metabolism observed in early SZ using a metabolomic approach. Purine catabolism is an underappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. We have observed a homeostatic imbalance of purine catabolism in first-episode neuroleptic-naïve patients with SZ (FENNS). Precursor and product relationships within purine pathways are tightly correlated. Although some of these correlations persist across disease or medication status, others appear to be lost among FENNS suggesting that steady formation of the antioxidant uric acid (UA) via purine catabolism is altered early in the course of illness. As is the case for within-pathway correlations, there are also significant cross-pathway correlations between respective purine and tryptophan (TRP) pathway metabolites. By contrast, purine metabolites show significant cross-pathway correlation only with tyrosine, and not with its metabolites. Furthermore, several purine metabolites (UA, guanosine, or xanthine) are each significantly correlated with 5-hydroxyindoleacetic acid (5-HIAA) in healthy controls, but not in FENNS at baseline or 4-week after antipsychotic treatment. Taken together, the above findings suggest that purine catabolism strongly associates with the TRP pathways leading to serotonin (5-hydroxytryptamine, 5-HT) and kynurenine metabolites. The lack of a significant correlation between purine metabolites and 5-HIAA, suggests alterations in key 5-HT pathways that may both be modified by and contribute to oxidative stress via purine

  16. Computer-generated Model of Purine Nucleoside Phosphorylase (PNP)

    NASA Technical Reports Server (NTRS)

    1987-01-01

    Purine Nucleoside Phosphorylase (PNP) is an important target enzyme for the design of anti-cancer and immunosuppressive drugs. Bacterial PNP, which is slightly different from the human enzyme, is used to synthesize chemotherapuautic agents. Knowledge of the three-dimensional structure of the bacterial PNP molecule is useful in efforts to engineer different types of PNP enzymes, that can be used to produce new chemotherapeutic agents. This picture shows a computer model of bacterial PNP, which looks a lot like a display of colorful ribbons. Principal Investigator was Charles Bugg.

  17. INTERMOLECULAR FORCES IN ASSOCIATION OF PURINES WITH POLYBENZENOID HYDROCARBONS.

    PubMed

    PULLMAN, B; CLAVERIE, P; CAILLET, J

    1965-03-12

    The interactions in solution between purine or pyrimidine bases and polybenzenoid aromatic hydrocarbons probably consist in a vertical, stacking-type physical association. By molecular orbital calculations the role of the Van der Waals-London intermolecular forces in these interactions is determined. The electrostatic dipole-dipole forces are negligible, the polarization (or induction) dipole-induced dipole forces are contributory, but most important are the dispersion (or fluctuation) forces. This loose, physical type of interaction should not show any specificity with respect to the carcinogenic activity of the hydrocarbons.

  18. Purine import into malaria parasites as a target for antimalarial drug development.

    PubMed

    Frame, I J; Deniskin, Roman; Arora, Avish; Akabas, Myles H

    2015-04-01

    Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage pathway enzymes to form the required purine nucleotides. The Plasmodium falciparum genome encodes four putative ENTs (PfENT1-4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (<10 μM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.

  19. The electrochemical properties of the purine bases : at the interface between biological conjugates to inorganic surfaces

    NASA Technical Reports Server (NTRS)

    Hays, Charles C.

    2003-01-01

    The study of the charge transfer and interfacial reactions of the purine bases in physiological solutions provides valuable knowledge, as these processes are relevant to the origins of life. It has been proposed that the adsorption of the adsorption of the purine bases on an inorganic surface could serve as a template for specifying the arrangement of amino acids in peptides.

  20. Morphine enhances the release of /sup 3/H-purines from rat brain cerebral cortical prisms

    SciTech Connect

    Wu, P.H.; Phillis, J.W.; Yuen, H.

    1982-10-01

    In vitro experiments have shown that /sup 3/H-purines can be released from /sup 3/H-adenosine preloaded rat brain cortical prisms by a KCl-evoked depolarization. The KCl-evoked release of /sup 3/H-purines is dependent on the concentration of KCl present in the superfusate. At concentrations of 10(-7) approximately 10(-5)M morphine did not influence the basal release of /sup 3/H-purines from the prisms, although it enhanced the KCl-evoked release of /sup 3/H-purines. The enhancement of KCl-evoked /sup 3/H-purine release by morphine was concentration-dependent and was antagonized by naloxone, suggesting the involvement of opiate receptors. Uptake studies with rat brain cerebral cortical synaptosomes show that morphine is a very weak inhibitor of adenosine uptake. Comparisons with dipyridamole, a potent inhibitor of adenosine uptake, suggest that this low level of inhibition of the uptake did not contribute significantly to the release of /sup 3/H-purine by morphine seen in our experiments. It is therefore suggested that morphine enhances KCl-evoked /sup 3/H-purine release by an interaction with opiate receptors and that the resultant increase in extracellular purine (adenosine) levels may account for some of the actions of morphine.

  1. Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

    PubMed Central

    Serafim, Tiago Donatelli; Figueiredo, Amanda Braga; Costa, Pedro Augusto Carvalho; Marques-da-Silva, Eduardo Almeida; Gonçalves, Ricardo; de Moura, Sandra Aparecida Lima; Gontijo, Nelder Figueiredo; da Silva, Sydnei Magno; Michalick, Marilene Suzan Marques; Meyer-Fernandes, José Roberto; de Carvalho, Roberto Paes; Uliana, Silvia Reni Bortolin; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos Crocco

    2012-01-01

    Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that “stressful conditions” will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines. PMID:23050028

  2. Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis

    PubMed Central

    2014-01-01

    Background Purine nucleotides are essential metabolites for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and biosynthesis of several amino acids and riboflavin. Owing to the pivotal roles of purines in cell physiology, the pool of intracellular purine nucleotides must be maintained under strict control, and hence the de novo purine biosynthetic pathway is tightly regulated by transcription repression and inhibition mechanism. Deregulation of purine pathway is essential for this pathway engineering in Bacillus subtilis. Results Deregulation of purine pathway was attempted to improve purine nucleotides supply, based on a riboflavin producer B. subtilis strain with modification of its rib operon. To eliminate transcription repression, the pur operon repressor PurR and the 5’-UTR of pur operon containing a guanine-sensing riboswitch were disrupted. Quantitative RT-PCR analysis revealed that the relative transcription levels of purine genes were up-regulated about 380 times. Furthermore, site-directed mutagenesis was successfully introduced into PRPP amidotransferase (encoded by purF) to remove feedback inhibition by homologous alignment and analysis. Overexpression of the novel mutant PurF (D293V, K316Q and S400W) significantly increased PRPP amidotransferase activity and triggered a strong refractory effect on purine nucleotides mediated inhibition. Intracellular metabolite target analysis indicated that the purine nucleotides supply in engineered strains was facilitated by a stepwise gene-targeted deregulation. With these genetic manipulations, we managed to enhance the metabolic flow through purine pathway and consequently increased riboflavin production 3-fold (826.52 mg/L) in the purF-VQW mutant strain. Conclusions A sequential optimization strategy was applied to deregulate the rib operon and purine pathway of B. subtilis to create genetic diversities and to improve riboflavin production

  3. Absorption and intermediary metabolism of purines and pyrimidines in lactating dairy cows.

    PubMed

    Stentoft, Charlotte; Røjen, Betina Amdisen; Jensen, Søren Krogh; Kristensen, Niels B; Vestergaard, Mogens; Larsen, Mogens

    2015-02-28

    About 20 % of ruminal microbial N in dairy cows derives from purines and pyrimidines; however, their intermediary metabolism and contribution to the overall N metabolism has sparsely been described. In the present study, the postprandial patterns of net portal-drained viscera (PDV) and hepatic metabolism were assessed to evaluate purine and pyrimidine N in dairy cows. Blood was sampled simultaneously from four veins with eight hourly samples from four multi-catheterised Holstein cows. Quantification of twenty purines and pyrimidines was performed with HPLC-MS/MS, and net fluxes were estimated across the PDV, hepatic tissue and total splanchnic tissue (TSP). Concentration differences between veins of fifteen purine and pyrimidine nucleosides (NS), bases (BS) and degradation products (DP) were different from zero (P≤ 0·05), resulting in the net PDV releases of purine NS (0·33-1·3 mmol/h), purine BS (0·0023-0·018 mmol/h), purine DP (7·0-7·8 mmol/h), pyrimidine NS (0·30-2·8 mmol/h) and pyrimidine DP (0·047-0·77 mmol/h). The hepatic removal of purine and pyrimidine was almost equivalent to the net PDV release, resulting in no net TSP release. One exception was uric acid (7·9 mmol/h) from which a large net TSP release originated from the degradation of purine NS and BS. A small net TSP release of the pyrimidine DP β-alanine and β-aminoisobutyric acid (-0·032 to 0·37 mmol/h) demonstrated an outlet of N into the circulating N pool. No effect of time relative to feeding was observed (P>0·05). These data indicate that considerable amounts of N are lost in the dairy cow due to prominent intermediary degradation of purines, but that pyrimidine N is reusable to a larger extent.

  4. A nuclear factor that binds purine-rich, single-stranded oligonucleotides derived from S1-sensitive elements upstream of the CFTR gene and the MUC1 gene.

    PubMed Central

    Hollingsworth, M A; Closken, C; Harris, A; McDonald, C D; Pahwa, G S; Maher, L J

    1994-01-01

    We have identified two regions of non-random purine/pyrimidine strand asymmetry that were nearly identical in sequence in the 5' flanking (promoter) regions of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene and the human MUC1 gene. These regions contain perfect mirror repeat elements, a sequence motif previously found to be associated with the formation of H-DNA conformations. In this report we demonstrate that a single-stranded non-B DNA conformation exists at low pH in supercoiled plasmids containing the similar mirror repeat elements, and that S1 nuclease digestion maps the single-stranded region to the position of the mirror repeats. In addition, we identify a nuclear protein of approximately 27 kD that binds to single-stranded oligonucleotides corresponding to the purine-rich strand of this region, but not to the pyrimidine-rich strands or to double-stranded oligonucleotides with corresponding purine/pyrimidine strand asymmetry. Images PMID:7513081

  5. Identification of genes containing expanded purine repeats in the human genome and their apparent protective role against cancer.

    PubMed

    Singh, Himanshu Narayan; Rajeswari, Moganty R

    2016-01-01

    Purine repeat sequences present in a gene are unique as they have high propensity to form unusual DNA-triple helix structures. Friedreich's ataxia is the only human disease that is well known to be associated with DNA-triplexes formed by purine repeats. The purpose of this study was to recognize the expanded purine repeats (EPRs) in human genome and find their correlation with cancer pathogenesis. We developed "PuRepeatFinder.pl" algorithm to identify non-overlapping EPRs without pyrimidine interruptions in the human genome and customized for searching repeat lengths, n ≥ 200. A total of 1158 EPRs were identified in the genome which followed Wakeby distribution. Two hundred and ninety-six EPRs were found in geneic regions of 282 genes (EPR-genes). Gene clustering of EPR-genes was done based on their cellular function and a large number of EPR-genes were found to be enzymes/enzyme modulators. Meta-analysis of 282 EPR-genes identified only 63 EPR-genes in association with cancer, mostly in breast, lung, and blood cancers. Protein-protein interaction network analysis of all 282 EPR-genes identified proteins including those in cadherins and VEGF. The two observations, that EPRs can induce mutations under malignant conditions and that identification of some EPR-gene products in vital cell signaling-mediated pathways, together suggest the crucial role of EPRs in carcinogenesis. The new link between EPR-genes and their functionally interacting proteins throws a new dimension in the present understanding of cancer pathogenesis and can help in planning therapeutic strategies. Validation of present results using techniques like NGS is required to establish the role of the EPR genes in cancer pathology.

  6. PRESENCE OF PURINE METABOLITES IN OMASAL DIGESTA AND BACTERIA: NEW ANALYTICAL METHOD AND EFFECTS ON MICROBIAL FLOWS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new HPLC method was developed to determine concentrations of purines [adenine (A) and guanine (G)], and their metabolites [xanthine (X) and hypoxanthine (HX)] in omasal digesta and bacterial samples and to assess the effect of using either purines (TP) or purines plus their metabolites (PM) as mic...

  7. Synthesis and cytostatic activity of purine nucleosides derivatives of allofuranose.

    PubMed

    Besada, Pedro; Costas, Tamara; Teijeira, Marta; Terán, Carmen

    2010-12-01

    Several new purine nucleosides derivatives of allofuranose were prepared according to Vorbrüggen method, starting from 1,2,5,6-di-O-isopropylidene-α-D-allofuranose and using 1,2,3,5,6-pentaacetoxy-β-D-allofuranose as key intermediate. The synthesized allofuranosyl nucleosides, as well as some acetyl derivatives, were evaluated for their cytotoxicity in vitro in three human cancer cell lines (MCF-7, Hela-229 and HL-60). Among the studied compounds the 9-(2,3,5,6-tetra-O-acetyl-β-D-allofuranosyl)-2,6-dichloropurine (9) was the most potent one on the three cell lines evaluated, being its activity against HL-60 cells similar to cisplatin.

  8. [Metformin impact on purine metabolism in breast cancer].

    PubMed

    Shatova, O P; Butenko, Eu V; Khomutov, Eu V; Kaplun, D S; Sedakov, I Eu; Zinkovych, I I

    2016-03-01

    Large-scale epidemiological and clinical studies have demonstrated the efficacy of metformin in oncology practice. However, the mechanisms of implementation of the anti-tumor effect of this drug there is still need understanding. In this study we have investigated the effect of metformin on the activity of adenosine deaminase and respectively adenosinergic immunosuppression in tumors and their microenvironment. The material of the study was taken during surgery of breast cacer patients receiveing metformin, and also patients which did not take this drug. The adenosine deaminase activity and substrate (adenosine) and products (inosine, hypoxanthine) concentrations were determined by HPLC. Results of this study suggest that metformin significantly alters catabolism of purine nucleotides in the node breast adenocarcinoma tisue. However, the metformin-induced increase in the adenosine deaminase activity is not sufficient to reduce the level of adenosine in cancer tissue. Thus, in metformin treated patients the adenosine concentration remained unchanged, and inosine and hypoxanthine concentration significantly increased.

  9. The Attachment of Amino Fragment to Purine: Inner-Shell Structures And Spectra

    SciTech Connect

    Saha, Saumitra; Wang, Feng; MacNaughton, Janay B.; Moewes, Alex; Chong, Denalo P.; /British Columbia U.

    2009-05-11

    The impact of the amino fragment (-NH{sub 2}) attachment on the inner-shell structures and spectra of unsubstituted purine and the purine ring of adenine are studied. Density functional theory calculations, using the LB94/TZ2P//B3LYP/TZVP model, reveal significant site-dependent electronic structural changes in the inner shell of the species. A condensed Fukui function indicates that all of the N and C sites, except for N{sub (1)} and C{sub (5)}, demonstrate significant electrophilic reactivity (f > 0.5 in |e|) in the unsubstituted purine. Once the amino fragment binds to the C{sub (6)} position of purine to form adenine, the electrophilic reactivity of these N and C sites is greatly reduced. As expected, the C{sub (6)} position experiences substantial changes in energy and charge transfer, owing to the formation of the C-NH{sub 2} bond in adenine. The present study reveals that the N1s spectra of adenine inherit the N1s spectra of the unsubstituted purine, whereas the C1s spectra experience significant changes although purine and adenine have geometrically similar carbon frames. The findings also indicate that the attachment of the NH{sub 2} fragment to purine exhibits deeply rooted influences to the inner-shell structures of DNA/RNA bases. The present study suggests that some fragment-based methods may not be applicable to spectral analyses in the inner shell.

  10. Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

    PubMed

    Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

    2014-09-01

    The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

  11. Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

    PubMed Central

    Schultz, Eric P.; Vasquez, Ernesto E.; Scott, William G.

    2014-01-01

    The hammerhead ribozyme catalyzes RNA cleavage via acid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK a of the substituted purine; in both cases inosine, which is similar to G in pK a and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the

  12. The role of purine degradation in methane biosynthesis and energy production in Methanococcus vannielii

    SciTech Connect

    DeMoll, E.

    1990-10-22

    Research continues on the role of purine degradation in methane biosynthesis and energy production in Methanococcus vannielii. This report summarizes current progress of the research. Topics include: A survey of other methanogens for the purine degradation pathway; isolate and characterize the enzyme and products of formiminoglycine cleavage; ascertain the fate of glycine from the formiminoglycine cleavage; elucidate the route of incorporation of the formyl moiety of formiminoglycine into methane biosynthesis; determine the percent methane and amino acid synthesis from purine degradation; and related studies on xanthine dehydrogenase and pyrimidine degradation of M. Vannielii. (SM)

  13. Novel N9-arenethenyl purines as potent dual Src/Abl tyrosine kinase inhibitors.

    PubMed

    Wang, Yihan; Shakespeare, William C; Huang, Wei-Sheng; Sundaramoorthi, Raji; Lentini, Scott; Das, Sasmita; Liu, Shuangying; Banda, Geeta; Wen, David; Zhu, Xiaotian; Xu, Qihong; Keats, Jeffrey; Wang, Frank; Wardwell, Scott; Ning, Yaoyu; Snodgrass, Joseph T; Broudy, Mark I; Russian, Karin; Dalgarno, David; Clackson, Tim; Sawyer, Tomi K

    2008-09-01

    Novel N(9)-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N(9) on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner-Wadsworth-Emmons reaction of N(9)-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.

  14. [Quantum-chemical investigation of the elementary molecular mechanisms of pyrimidine-purine transversions].

    PubMed

    Brovarets', O O; Govorun D M

    2010-01-01

    Purine-purine mispairs of DNA (thus involving template base in anti-conformation along the glycosidic bond and base of the incoming nucleotide - in syn-conformation) leading to pyrimidine-purine "transversions"-type point mutations were revealed and characterized at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory in vacuum approach adequately modeling hydrophobic environment of the active centre of high-fidelity replicative DNA-polymerases.

  15. Pronounceability: a measure of language samples based on children's mastery of the phonemes employed in them.

    PubMed

    Whissell, Cynthia

    2003-06-01

    56 samples (n > half a million phonemes) of names (e.g., men's, women's jets'), song lyrics (e.g., Paul Simon's, rap, Beatles'), poems (frequently anthologized English poems), and children's materials (books directed at children ages 3-10 years) were used to study a proposed new measure of English language samples--Pronounceability-based on children's mastery of some phonemes in advance of others. This measure was provisionally equated with greater "youthfulness" and "playfulness" in language samples and with less "maturity." Findings include the facts that women's names were less pronounceable than men's and that poetry was less pronounceable than song lyrics or children's materials. In a supplementary study, 13 university student volunteers' assessments of the youth of randomly constructed names was linearly related to how pronounceable each name was (eta = .8), providing construct validity for the interpretation of Pronounceability as a measure of Youthfulness.

  16. Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

    SciTech Connect

    Grenha, Rosa; Levdikov, Vladimir M.; Fogg, Mark J.; Blagova, Elena V.; Brannigan, James A. Wilkinson, Anthony J.; Wilson, Keith S.

    2005-05-01

    The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis was solved by X-ray crystallography using molecular replacement and refined at a resolution of 2.24 Å. Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium.

  17. Versatile synthesis and biological evaluation of novel 3’-fluorinated purine nucleosides

    PubMed Central

    Ren, Hang; Hatala, Paul J; Stevens, William C; He, Baicheng

    2015-01-01

    Summary A unified synthetic strategy accessing novel 3'-fluorinated purine nucleoside derivatives and their biological evaluation were achieved. Novel 3’-fluorinated analogues were constructed from a common 3’-deoxy-3’-fluororibofuranose intermediate. Employing Suzuki and Stille cross-coupling reactions, fifteen 3’-fluororibose purine nucleosides 1–15 and eight 3’-fluororibose 2-chloro/2-aminopurine nucleosides 16–23 with various substituents at position 6 of the purine ring were efficiently synthesized. Furthermore, 3’-fluorine analogs of natural products nebularine and 6-methylpurine riboside were constructed via our convergent synthetic strategy. Synthesized nucleosides were tested against HT116 (colon cancer) and 143B (osteosarcoma cancer) tumor cell lines. We have demonstrated 3’-fluorine purine nucleoside analogues display potent tumor cell growth inhibition activity at sub- or low micromolar concentration. PMID:26734098

  18. Solvent effects on the photochemistry of 4-aminoimidazole-5-carbonitrile, a prebiotically plausible precursor of purines.

    PubMed

    Szabla, Rafał; Sponer, Judit E; Sponer, Jiří; Sobolewski, Andrzej L; Góra, Robert W

    2014-09-07

    4-Aminoimidazole-5-carbonitrile (AICN) was suggested as a prebiotically plausible precursor of purine nucleobases and nucleotides. Although it can be formed in a sequence of photoreactions, AICN is immune to further irradiation with UV-light. We present state-of-the-art multi-reference quantum-chemical calculations of potential energy surface cuts and conical intersection optimizations to explain the molecular mechanisms underlying the photostability of this compound. We have identified the N-H bond stretching and ring-puckering mechanisms that should be responsible for the photochemistry of AICN in the gas phase. We have further considered the photochemistry of AICN-water clusters, while including up to six explicit water molecules. The calculations reveal charge transfer to solvent followed by formation of an H3O(+) cation, both of which occur on the (1)πσ* hypersurface. Interestingly, a second proton transfer to an adjacent water molecule leads to a (1)πσ*/S0 conical intersection. We suggest that this electron-driven proton relay might be characteristic of low-lying (1)πσ* states in chromophore-water clusters. Owing to its nature, this mechanism might also be responsible for the photostability of analogous organic molecules in bulk water.

  19. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

    PubMed Central

    Schild, D; Brake, A J; Kiefer, M C; Young, D; Barr, P J

    1990-01-01

    Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme. Images PMID:2183217

  20. De novo purine biosynthesis by two pathways in Burkitt lymphoma cells and in human spleen.

    PubMed

    Reem, G H

    1972-05-01

    This study was designed to answer the question whether human lymphocytes and spleen cells were capable of de novo purine biosynthesis. Experiments were carried out in cell-free extracts prepared from human spleen, and from a cell line established from Burkitt lymphoma. Burkitt lymphoma cells and human spleen cells could synthesize the first and second intermediates of the purine biosynthetic pathway. Cell-free extracts of all cell lines studied contained the enzyme systems which catalyze the synthesis of phosphoribosyl-1-amine, the first intermediate unique to the purine biosynthetic pathway and of phosphoribosyl glycinamide, the second intermediate of this pathway. Phosphoribosyl-1-amine could be synthesized in cell-free extracts from alpha-5-phosphoribosyl-1-pyrophosphate (PRPP) and glutamine, from PRPP and ammonia, and by an alternative pathway, directly from ribose-5-phosphate and ammonia. These findings suggest that extrahepatic tissues may be an important source for the de novo synthesis of purine ribonucleotide in man. They also indicate that ammonia may play an important role in purine biosynthesis. The alternative pathway for the synthesis of phosphoribosyl-1-amine from ribose-5-phosphate and ammonia was found to be subject to inhibition by the end products of the purine synthetic pathway, particularly by adenylic acid and to a lesser degree by guanylic acid. The alternative pathway for phosphoribosyl-1-amine synthesis from ribose-5-phosphate and ammonia may contribute significantly towards the regulation of the rate of de novo purine biosynthesis in the normal state, in metabolic disorders in which purines are excessively produced and in myeloproliferative diseases.

  1. A Targeted Metabolomics Assay to Measure Eight Purines in the Diet of Common Bottlenose Dolphins, Tursiops truncatus

    PubMed Central

    Ardente, AJ; Garrett, TJ; Wells, RS; Walsh, M; Smith, CR; Colee, J; Hill, RC

    2016-01-01

    Bottlenose dolphins managed under human care, human beings and Dalmatian dogs are prone to forming urate uroliths. Limiting dietary purine intake limits urate urolith formation in people and dogs because purines are metabolized to uric acid, which is excreted in urine. Managed dolphins develop ammonium urate nephroliths, whereas free-ranging dolphins do not. Free-ranging dolphins consume live fish, whereas managed dolphins consume different species that have been stored frozen and thawed. Differences in the purine content of fish consumed by dolphins under human care versus in the wild may be responsible for the difference in urolith prevalence. Commercially available purine assays measure only four purines, but reported changes in purines during frozen storage suggest that a wider range of metabolites should be measured when comparing fresh and stored fish. A method using high performance liquid chromatography with tandem mass spectrometry was developed to quantify eight purine metabolites in whole fish and squid commonly consumed by dolphins. The coefficient of variation within and among days was sometimes high for purines present in small amounts but was acceptable (≤ 25%) for guanine, hypoxanthine, and inosine, which were present in high concentrations. This expanded assay identified a total purine content up to 2.5 times greater than the total that would be quantified if only four purines were measured. Assuming additional purines are absorbed, these results suggest that additional purine metabolites should be measured to better understand the associated risk when fish or other purine-rich foods are consumed by people or animals prone to developing uroliths. PMID:27904786

  2. From formamide to purine: an energetically viable mechanistic reaction pathway.

    PubMed

    Wang, Jing; Gu, Jiande; Nguyen, Minh Tho; Springsteen, Greg; Leszczynski, Jerzy

    2013-02-28

    A step-by-step mechanistic pathway following the transformation of formamide to purine through a five-membered ring intermediate has been explored by density functional theory computations. The highlight of the mechanistic route detailed here is that the proposed pathway represents the simplest reaction pathway. All necessary reactants are generated from a single starting compound, formamide, through energetically viable reactions. Several important reaction steps are involved in this mechanistic route: formylation-dehydration, Leuckart reduction, five- and six-membered ring-closure, and deamination. On the basis of the study of noncatalytic pathways, catalytic water has been found to provide energetically viable step-by-step mechanistic pathways. Among these reaction steps, five-member ring-closure is the rate-determining step. The energy barrier (ca. 42 kcal/mol) of this rate-control step is somewhat lower than the rate-determining step (ca. 44 kcal/mol) for a pyrimidine-based pathway reported previously. The mechanistic pathway reported herein is less energetically demanding than for previously proposed routes to adenine.

  3. Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites.

    PubMed

    Gero, A M; Scott, H V; O'Sullivan, W J; Christopherson, R I

    1989-04-01

    The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.

  4. Universal full-length nucleosome mapping sequence probe.

    PubMed

    Tripathi, Vijay; Salih, Bilal; Trifonov, Edward N

    2015-01-01

    For the computational sequence-directed mapping of the nucleosomes, the knowledge of the nucleosome positioning motifs - 10-11 base long sequences - and respective matrices of bendability, is not sufficient, since there is no justified way to fuse these motifs in one continuous nucleosome DNA sequence. Discovery of the strong nucleosome (SN) DNA sequences, with visible sequence periodicity allows derivation of the full-length nucleosome DNA bendability pattern as matrix or consensus sequence. The SN sequences of three species (A. thaliana, C. elegans, and H. sapiens) are aligned (512 sequences for each species), and long (115 dinucleotides) matrices of bendability derived for the species. The matrices have strong common property - alternation of runs of purine-purine (RR) and pyrimidine-pyrimidine (YY) dinucleotides, with average period 10.4 bases. On this basis the universal [R,Y] consensus of the nucleosome DNA sequence is derived, with exactly defined positions of respective penta- and hexamers RRRRR, RRRRRR, YYYYY, and YYYYYY.

  5. [Hyperuricemia and disorders in content of amino acids-purine precursors in patients with autoimmune diseases and gout].

    PubMed

    Nikolenko, Iu I; Siniachenko, O V; Anan'eva, M N; Nikolenko, V Iu; Dubiaga, V V; Shchukin, I N

    2005-06-01

    Patients with system lupus erythematosus, rheumatic arthritis, chronic active hepatitis and gout were found to have considerable hyperuricemia and be decreased in aminoacides content, which are the predecessors of purine. Hyperactivity of xanthineoxidase and POL content were also revealed. The close correlation relation of purine indices of such patients has been observed. The obtained data allow applying methods of correction of immunity system and purine metabolism to these patients by introducing in a complex therapy inhibitors of xanthineoxidase.

  6. Helicobacter pylori Salvages Purines from Extracellular Host Cell DNA Utilizing the Outer Membrane-Associated Nuclease NucT

    PubMed Central

    Liechti, George W.

    2013-01-01

    Helicobacter pylori is a bacterial pathogen that establishes life-long infections in humans, and its presence in the gastric epithelium is strongly associated with gastritis, peptic ulcer disease, and gastric cancer. Having evolved in this specific gastric niche for hundreds of thousands of years, this microbe has become dependent on its human host. Bioinformatic analysis reveals that H. pylori has lost several genes involved in the de novo synthesis of purine nucleotides, and without this pathway present, H. pylori must salvage purines from its environment in order to grow. While the presence and abundance of free purines in various mammalian tissues has been loosely quantified, the concentration of purines present within the gastric mucosa remains unknown. There is evidence, however, that a significant amount of extracellular DNA is present in the human gastric mucosal layer as a result of epithelial cell turnover, and this DNA has the potential to serve as an adequate purine source for gastric purine auxotrophs. In this study, we characterize the ability of H. pylori to grow utilizing only DNA as a purine source. We show that this ability is independent of the ComB DNA uptake system, and that H. pylori utilization of DNA as a purine source is largely influenced by the presence of an outer membrane-associated nuclease (NucT). A ΔnucT mutant exhibits significantly reduced extracellular nuclease activity and is deficient in growth when DNA is provided as the sole purine source in laboratory growth media. These growth defects are also evident when this nuclease mutant is grown in the presence of AGS cells or in purine-free tissue culture medium that has been conditioned by AGS cells in the absence of fetal bovine serum. Taken together, these results indicate that the salvage of purines from exogenous host cell DNA plays an important role in allowing H. pylori to meet its purine requirements for growth. PMID:23893109

  7. A link between impaired purine nucleotide synthesis and apoptosis in Drosophila melanogaster.

    PubMed

    Holland, Catherine; Lipsett, David B; Clark, Denise V

    2011-06-01

    The biosynthetic pathways and multiple functions of purine nucleotides are well known. However, the pathways that respond to alterations in purine nucleotide synthesis in vivo in an animal model organism have not been identified. We examined the effects of inhibiting purine de novo synthesis in vivo and in cultured cells of Drosophila melanogaster. The purine de novo synthesis gene ade2 encodes phosphoribosylformylglycinamidine synthase (EC 6.3.5.3). An ade2 deletion, generated by P-element transposon excision, causes lethality in early pupal development, with darkening, or necrosis, of leg and wing imaginal disc tissue upon disc eversion. Together with analysis of a previously isolated weaker allele, ade2(4), and an allele of the Prat gene, which encodes an enzyme for the first step in the pathway, we determined that the lethal arrest and imaginal disc phenotypes involve apoptosis. A transgene expressing the baculovirus caspase inhibitor p35, which suppresses apoptosis caused by other stresses such as DNA damage, suppresses both the imaginal disc tissue darkening and the pupal lethality of all three purine de novo synthesis mutants. Furthermore, we showed the presence of apoptosis at the cellular level in both ade2 and Prat mutants by detecting TUNEL-positive nuclei in wing imaginal discs. Purine de novo synthesis inhibition was also examined in tissue culture by ade2 RNA interference followed by analysis of genome-wide changes in transcript levels. Among the upregulated genes was HtrA2, which encodes an apoptosis effector and is thus a candidate for initiating apoptosis in response to purine depletion.

  8. Lactobacillus gasseri PA-3 Uses the Purines IMP, Inosine and Hypoxanthine and Reduces Their Absorption in Rats

    PubMed Central

    Yamada, Naruomi; Saito-Iwamoto, Chizuru; Nakamura, Marie; Soeda, Misato; Chiba, Yoshika; Kano, Hiroshi; Asami, Yukio

    2017-01-01

    Excessive intake of purine-rich foods elevates serum levels of uric acid. Animal and fish meats contain high amounts of inosine and its related purines, and the reduction of taking those purines is crucial for the improvement of serum uric acid levels. We previously showed that Lactobacillus gasseri PA-3 (PA-3) incorporates adenosine and its related purines and that oral treatment with PA-3 reduced adenosine absorption in rats. This study investigated whether PA-3 also incorporates IMP (inosine 5′-monophosphate), inosine, and hypoxanthine, and whether it reduces their absorption in rats. PA-3 was incubated in vitro with radioisotope (RI)-labeled IMP, inosine, and hypoxanthine, and the incorporation of these compounds by PA-3 was evaluated. In addition, rats were orally administered PA-3 along with RI-labeled inosine 5′-monophosphate, inosine, or hypoxanthine, and the ability of PA-3 to attenuate the absorption of these purines was determined. PA-3 incorporated all three purines and displayed greater proliferation in the presence than in the absence of these purines. Oral administration of PA-3 to rats reduced the absorption of IMP, inosine, and hypoxanthine. These results indicate that PA-3 reduces the absorption of purines contained in foods and it is expected that PA-3 contributes attenuation of the excessive intake of dietary purines. PMID:28282902

  9. Autoimmune dysregulation and purine metabolism in adenosine deaminase deficiency.

    PubMed

    Sauer, Aisha Vanessa; Brigida, Immacolata; Carriglio, Nicola; Aiuti, Alessandro

    2012-01-01

    Genetic defects in the adenosine deaminase (ADA) gene are among the most common causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive, and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-ADA), or hematopoietic stem cell gene therapy (HSC-GT). Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment. A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T- and B-cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.

  10. Effects of NAD at purine receptors in isolated blood vessels.

    PubMed

    Alefishat, E; Alexander, S P H; Ralevic, V

    2015-03-01

    Nicotinamide adenine dinucleotide (NAD) belongs to the family of naturally occurring adenine dinucleotides, best known for their various intracellular roles. However, there is evidence that they can also be released from cells to act as novel extracellular signalling molecules. Relatively little is known about the extracellular actions of NAD, especially in the cardiovascular system. The present study investigated the actions of NAD in the rat thoracic aorta, porcine coronary artery and porcine mesenteric arteries, mounted in organ baths for isometric tension recording. In the rat thoracic aorta and porcine coronary artery, NAD caused endothelium-independent concentration-dependent vasorelaxations which were unaffected by palmitoylCoA, a P2Y1 receptor antagonist, but which were blocked by CGS15943, a non-selective adenosine receptor antagonist. In the porcine coronary artery, NAD-evoked relaxations were abolished by SCH58261, a selective A2A receptor antagonist. In the rat thoracic aorta, NAD-evoked relaxations were attenuated by A2A receptor antagonism with SCH58261 but were unaffected by an A2B receptor antagonist, MRS1754. In contrast, in the porcine mesenteric artery, NAD-evoked endothelium-independent contractions, which were unaffected by a P2 receptor antagonist, suramin, or by NF449, a P2X1 receptor antagonist, but were attenuated following P2X receptor desensitisation with αβ-meATP. In conclusion, the present results show that NAD can alter vascular tone through actions at purine receptors in three different arteries from two species; its molecular targets differ according to the type of blood vessel.

  11. Identification of a chemoreceptor that specifically mediates chemotaxis toward metabolizable purine derivatives.

    PubMed

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Krell, Tino

    2016-01-01

    Chemotaxis is an essential mechanism that enables bacteria to move toward favorable ecological niches. Escherichia coli, the historical model organism for studying chemotaxis, has five well-studied chemoreceptors. However, many bacteria with different lifestyle have more chemoreceptors, most of unknown function. Using a high throughput screening approach, we identified a chemoreceptor from Pseudomonas putida KT2440, named McpH, which specifically recognizes purine and its derivatives, adenine, guanine, xanthine, hypoxanthine and uric acid. The latter five compounds form part of the purine degradation pathway, permitting their use as sole nitrogen sources. Isothermal titration calorimetry studies show that these six compounds bind McpH-Ligand Binding Domain (LBD) with very similar affinity. In contrast, non-metabolizable purine derivatives (caffeine, theophylline, theobromine), nucleotides, nucleosides or pyrimidines are unable to bind McpH-LBD. Mutation of mcpH abolished chemotaxis toward the McpH ligands identified - a phenotype that is restored by complementation. This is the first report on bacterial chemotaxis to purine derivatives and McpH the first chemoreceptor described that responds exclusively to intermediates of a catabolic pathway, illustrating a clear link between metabolism and chemotaxis. The evolution of McpH may reflect a saprophytic lifestyle, which would have exposed the studied bacterium to high concentrations of purines produced by nucleic acid degradation.

  12. Which Electronic and Structural Factors Control the Photostability of DNA and RNA Purine Nucleobases?

    NASA Astrophysics Data System (ADS)

    Pollum, Marvin; Reichardt, Christian; Crespo-Hernández, Carlos E.; Martínez-Fernández, Lara; Corral, Inés; Rauer, Clemens; Mai, Sebastian; Marquetand, Philipp; González, Leticia

    2015-06-01

    Following ultraviolet excitation, the canonical purine nucleobases, guanine and adenine, are able to efficiently dissipate the absorbed energy within hundreds of femtoseconds. This property affords these nucleobases with great photostability. Conversely, non-canonical purine nucleobases exhibit high fluorescence quantum yields or efficiently populate long-lived triplet excited states from which chemistry can occur. Using femtosecond broadband transient absorption spectroscopy in combination with ab initio static and surface hopping dynamics simulations we have determined the electronic and structural factors that regulate the excited state dynamics of the purine nucleobase derivatives. Importantly, we have uncovered that the photostability of the guanine and adenine nucleobases is not due to the structure of the purine core itself and that the substituent at the C6 position of the purine nucleobase plays a more important role than that at the C2 position in the ultrafast relaxation of deleterious electronic energy. [The authors acknowledge the CAREER program of the National Science Foundation (Grant No. CHE-1255084) for financial support.

  13. Extracellular conversion of guanine-based purines to guanosine specifically enhances astrocyte glutamate uptake.

    PubMed

    Frizzo, Marcos Emílio dos Santos; Antunes Soares, Félix Alexandre; Dall'Onder, Leonara Patrícia; Lara, Diogo Rizzato; Swanson, Raymond A; Souza, Diogo Onofre

    2003-05-16

    Guanosine (GUO) has been shown to stimulate glutamate uptake in primary astrocyte cultures. The purpose of this study was to determine the effect and specificity of guanine- or adenine-based purines on glutamate and GABA uptake in cultured astrocytes. Stimulatory effect on glutamate uptake was observed with GUO, GMP or GTP. Simultaneous exposure with these guanine-based purines did not show an additive effect. We also investigated a possible interconversion of guanine-based purines during incubation time. Action by GTP was excluded since the hydrolysis resistant GTP analog, GMP-PNP did not stimulate glutamate uptake. Addition of an ecto-5'-nucleotidase inhibitor abolished GMP-stimulatory effect on glutamate uptake, without affecting GUO action. Taken together, these results suggest that GUO is the guanine-based purines responsible for glutamate uptake activation. In addition, the stimulatory effect on glutamate uptake was not observed with adenine-based purines. Moreover, GABA uptake was not activated by GUO. These results point to specificity in the interaction between GUO and the astrocyte glutamate uptake system.

  14. Drosophila melanogaster Prat, a Purine de Novo Synthesis Gene, Has a Pleiotropic Maternal-Effect Phenotype

    PubMed Central

    Malmanche, Nicolas; Clark, Denise V.

    2004-01-01

    In Drosophila melanogaster, two genes, Prat and Prat2, encode the enzyme, amidophosphoribosyltransferase, that performs the first and limiting step in purine de novo synthesis. Only Prat mRNA is present in the female germline and 0- to 2-hr embryos prior to the onset of zygotic transcription. We studied the maternal-effect phenotype caused by Prat loss-of-function mutations, allowing us to examine the effects of decreased purine de novo synthesis during oogenesis and the early stages of embryonic development. In addition to the purine syndrome previously characterized, we found that Prat mutant adult females have a significantly shorter life span and are conditionally semisterile. The semisterility is associated with a pleiotropic phenotype, including egg chamber defects and later effects on embryonic and larval viability. Embryos show mitotic synchrony and/or nuclear content defects at the syncytial blastoderm stages and segmentation defects at later stages. The semisterility is partially rescued by providing Prat mutant females with an RNA-enriched diet as a source of purines. Our results suggest that purine de novo synthesis is a limiting factor during the processes of cellular or nuclear proliferation that take place during egg chamber and embryonic development. PMID:15611171

  15. Coordinate developmental regulation of purine catabolic enzyme expression in gastrointestinal and postimplantation reproductive tracts

    PubMed Central

    1991-01-01

    Using histochemical detection, we have visualized in situ the complete metabolic pathway for the degradation of purine nucleotides. From the tongue to the ileum, diverse epithelial cell types lining the lumen of the mouse gastrointestinal (GI) tract strongly coexpress each of the five key purine catabolic enzymes. Dramatic increases in the expression of each enzyme occurred during postnatal maturation of the GI tract. Using in situ hybridization, an intense accumulation of adenosine deaminase (ADA) mRNA was detected only within GI epithelial cells undergoing postmitotic differentiation. In a similar manner, at the developing maternal-fetal interface, high level expression of the purine catabolic pathway also occurred in a unique subset of maternal decidual cells previously known to express high levels of alkaline phosphatase and ADA. This induction occurred almost immediately after implantation in the periembryonic maternal decidual cells, shortly thereafter in antimesometrial decidual cells, and later in cells of the placental decidua basalis: all of which contain cell types thought to be undergoing programmed cell death. The expression of the pathway at the site of embryo implantation appears to be critical because its pharmacologic inhibition during pregnancy has been found to be embryolethal or teratogenic. Purine destruction at these nutritional interfaces (placenta and gastrointestinal tract) seem to override any potential economy of purine salvage, and may represent biochemical adaptation to nucleic acid breakdown occurring in the context of dietary digestion or extensive programmed cell death. PMID:1918135

  16. Metabolic Engineering of the Purine Pathway for Riboflavin Production in Ashbya gossypii†

    PubMed Central

    Jiménez, Alberto; Santos, María A.; Pompejus, Markus; Revuelta, José L.

    2005-01-01

    Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid310, lysine333, and alanine417) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter). PMID:16204483

  17. Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative.

    PubMed Central

    Lacoste, J; François, J C; Hélène, C

    1997-01-01

    Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation. PMID:9115367

  18. Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases

    PubMed Central

    Hill, F.; Loakes, D.; Brown, D. M.

    1998-01-01

    A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base. PMID:9539724

  19. The effect of purine and pyrimidine analogues and virazole on adenovirus replication.

    PubMed

    Scheffler, P; Haghchenas, D; Wigand, R

    1975-04-01

    The multiplication of adenovirus 19 in HeLa cells was inhibited by various purine and pyrimidine analogues and by virazole. The formation of infectious virus and of capsid proteins (haemagglutin, group-specific complement-fixing antigen) was inhibited to the same degree, while the viral cytopathic effect (CPE) was not inhibited. The reversibility of the inhibition after removal of the substances was more complete for purine than for pyrimidine analogues. The inhibition was counteracted by simulataneous addition of the corresponding nucleosides. Adenosine was more effected than guanosine against purine analogues; both were partially effective against virazole, but none of them against arabinofuranosyladenine. The time-dependence of inhibition, the ensuing eclipse period after removal of the inhibitors, and the successive application of two inhibitors led to the conclusion that most of them affect the viral multiplication mainly by inhibition of DNA synthesis. Azacytidine inhibits the synthesis of structural proteins as well.

  20. Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

    PubMed Central

    Sharma, Vimal Kumar; Jelen, Frantisek; Trnkova, Libuse

    2015-01-01

    Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications. PMID:25594595

  1. Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

    NASA Astrophysics Data System (ADS)

    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-03-01

    Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB_ID: 4RJ2).

  2. Inhibitors of the purine salvage pathway: a valuable approach for antiprotozoal chemotherapy?

    PubMed

    Berg, M; Van der Veken, P; Goeminne, A; Haemers, A; Augustyns, K

    2010-01-01

    For many years, the purine salvage pathway of parasitic protozoa has been regarded as an attractive chemotherapeutic target. Parasitic protozoa lack de novo synthesis and rely entirely on the purine salvage pathway to meet their purine demands. Because of the great phylogenetic difference between parasite and host, there are often sufficient distinctions that can be exploited to design specific inhibitors for the parasitic enzymes. As a result, this pathway has been thoroughly investigated over the last twenty years. It is only quite recently that the genome studies of Trypanosoma, Leishmania and Plasmodium have been published. Based on these genomic data however, the existence of by-pass mechanisms by other enzymes and transporter systems could be suggested. Taking into account such proposition, the question might arise as to whether inhibition of a single salvage enzyme will be able or not to cause parasite death or growth arrest. In this paper, the key enzymes in the purine salvage pathways of relevant pathogenic species from the genera Trypanosoma, Leishmania and Plasmodium are reviewed. Their potential as drug targets is critically evaluated and where possible, correlated to literature data on antiparasitic activity of their inhibitors. While many studies over the past ten years have yielded contradictory results, this review attempts to clarify these findings by discussing the latest elements of progress in the field. Additionally, as part of a broader discussion on substrate analogue types of inhibitors, special attention is paid to iminoribitol derivatives, serving as transition state analogues of nucleoside-processing enzymes and comprising the most potent inhibitors reported for purine salvage enzymes. More specifically, the development of three generations of immucillins and a newer series of N-(arylmethyl-) substituted iminoribitol derivatives will be discussed. Finally, this review also covers subversive substrates of salvage enzymes: compounds that

  3. Purine enzyme activities in recent onset rheumatoid arthritis: are there differences between patients and healthy controls?

    PubMed Central

    Stolk, J N; Boerbooms, A M; De Abreu, R A; Kerstens, P J; de Koning, D G; de Graaf, R; Mulder, J; van de Putte, L B

    1996-01-01

    OBJECTIVE: Purine enzyme activities may predict the effectiveness of azathioprine treatment and be associated with increased deaths from infectious diseases. In rheumatoid arthritis, patients show variable responses to azathioprine and a higher percentage of death is caused by infections. The aim of the study was to investigate possible rheumatoid arthritis associated abnormalities of purine enzyme activities by measuring several of these enzymes in patients with recent onset rheumatoid arthritis before treatment with disease modifying antirheumatic drugs or prednisone. METHODS: 23 patients with recent onset rheumatoid arthritis and 28 healthy controls were studied. Activities of the enzymes 5'-nucleotidase, purine nucleoside phosphorylase (PNP), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and thiopurine methyltransferase (TPMT) were measured. Assessment of disease activity and blood sampling for routine measurements and HLA typing were done simultaneously. RESULTS: Purine enzyme activities did not differ between patients and healthy controls. Enzyme activities had no significant relations with indices of disease activity or rheumatoid factor titre or with the rheumatoid arthritis associated HLA types. Activity of 5'nucleotidase decreased with age (P < or = 0.05) and was lower by about 27% (P = 0.007) in males than in females. CONCLUSIONS: In rheumatoid arthritis patients, neither the variability in azathioprine effectiveness nor the increased death rate from infections can be explained by pre-existing abnormalities in the activities of the purine enzymes 5'-nucleotidase, PNP, HGPRT, or TPMT at an early stage of the disease, before disease modifying antirheumatic drugs or prednisone treatment. Besides adjustment for age, results of studies involving purine 5' nucleotidase activity should also be adjusted for sex. PMID:8984938

  4. Enhancement of the therapeutic effectiveness of methotrexate and protection of normal proliferating tissues with purines and pyrimidines.

    PubMed

    Harrap, K R; Taylor, G A; Browman, G P

    1977-07-01

    Mice can be protected against the toxicity arising from lethal doses of methotrexate with purine and pyrimidine combinations, but not with pyrimidine alone. Furthermore, methotrexate treatment of tumour-bearing mice, conjunction with purine/pyrimidine protection, can be more effective than conventional metotrexate/folinic acid treatment.

  5. Genetic and proteomic analyses of a Xanthomonas campestris pv. campestris purC mutant deficient in purine biosynthesis and virulence.

    PubMed

    Yuan, Zhihui; Wang, Li; Sun, Shutao; Wu, Yao; Qian, Wei

    2013-09-20

    Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. campestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.

  6. Selective DNA purine base photooxidation by bis-terdentate iridium(III) polypyridyl and cyclometalated complexes.

    PubMed

    Jacques, Alexandre; Kirsch-De Mesmaeker, Andrée; Elias, Benjamin

    2014-02-03

    Two bis-terdentate iridium(III) complexes with polypyridyl and cyclometalated ligands have been prepared and characterized. Their spectroscopic and electrochemical properties have been studied, and a photophysical scheme addressing their properties is proposed. Different types of excited states have been considered to account for the deactivation processes in each complex. Interestingly, in the presence of mono- or polynucleotides, a photoinduced electron-transfer process from a DNA purine base (i.e., guanine or adenine) to the excited complex is shown through luminescence quenching experiments. For the first time, this work reports evidence for selective DNA purine bases oxidation by excited iridium(III) bis-terdentate complexes.

  7. The role of purine degradation in methane biosynthesis and energy production in Methanococcus vannielii. Progress report

    SciTech Connect

    DeMoll, E.

    1998-11-01

    Firstly, characterization of a purine degrading pathway in Methanococcus vannielii was determined. The pathway is similar to that described for Clostridia. The M. vannielli pathway differs in a few respects from the Clostridial pathway. The pathway of Clostridia uses tetrahydrofolic acid (THF), whereas the pathway of M. vannielii uses tetrahydromethanopterin (H{sub 4}MPt) as a cofactor in the transfer of both the formimino moiety of formiminoglycine and apparently in the cleavage of glycine by a glycin decarboxylase type mechanism that is dependent upon at least H{sub 4}MPt and either NAD{sup +} or NADP{sup +}. Secondly, the relationship of purine degradation to methanogenesis was investigated.

  8. Synthesis of cycloalkyl substituted purine nucleosides via a metal-free radical route.

    PubMed

    Wang, Dong-Chao; Xia, Ran; Xie, Ming-Sheng; Qu, Gui-Rong; Guo, Hai-Ming

    2016-05-04

    An efficient route to synthesize cycloalkyl substituted purine nucleosides was developed. This metal-free C-H activation was accomplished by a tBuOOtBu initiated radical reaction. By adjusting the amount of tBuOOtBu and reaction time, the selective synthesis of C6-monocycloalkyl or C6,C8-dicycloalkyl substituted purine nucleosides could be realized. Furthermore, uracil and related nucleosides were also suitable substrates, giving the C5-cyclohexyl substituted uracil derivatives in good yields with excellent regioselectivities.

  9. meta-C-H Bromination on Purine Bases by Heterogeneous Ruthenium Catalysis.

    PubMed

    Warratz, Svenja; Burns, David J; Zhu, Cuiju; Korvorapun, Korkit; Rogge, Torben; Scholz, Julius; Jooss, Christian; Gelman, Dmitri; Ackermann, Lutz

    2017-02-01

    Methods for positionally selective remote C-H functionalizations are in high demand. Herein, we disclose the first heterogeneous ruthenium catalyst for meta-selective C-H functionalizations, which enabled remote halogenations with excellent site selectivity and ample scope. The versatile heterogeneous Ru@SiO2 catalyst was broadly applicable and could be easily recovered and reused, which set the stage for the direct fluorescent labeling of purines. In contrast to palladium, rhodium, iridium, or cobalt complexes, solely the ruthenium catalysis manifold provided access to meta-halogenated purine derivatives, illustrating the unique power of ruthenium C-H activation catalysis.

  10. Purine salvage pathways of Bacillus subtilis and effect of guanine on growth of GMP reductase mutants.

    PubMed Central

    Endo, T; Uratani, B; Freese, E

    1983-01-01

    We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic chart of the purine salvage pathway of Bacillus subtilis. PMID:6408059

  11. Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.

    PubMed Central

    Newman, J D; Diebold, R J; Schultz, B W; Noel, K D

    1994-01-01

    Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth. Images PMID:8195084

  12. Screening and Characterization of Purine Nucleoside Degrading Lactic Acid Bacteria Isolated from Chinese Sauerkraut and Evaluation of the Serum Uric Acid Lowering Effect in Hyperuricemic Rats

    PubMed Central

    Mei, Lu; Yuan, Lin; Xie, Ao; Yuan, Jieli

    2014-01-01

    Hyperuricemia is well known as the cause of gout. In recent years, it has also been recognized as a risk factor for arteriosclerosis, cerebrovascular and cardiovascular diseases, and nephropathy in diabetic patients. Foods high in purine compounds are more potent in exacerbating hyperuricemia. Therefore, the development of probiotics that efficiently degrade purine compounds is a promising potential therapy for the prevention of hyperuricemia. In this study, fifty-five lactic acid bacteria isolated from Chinese sauerkraut were evaluated for the ability to degrade inosine and guanosine, the two key intermediates in purine metabolism. After a preliminary screening based on HPLC, three candidate strains with the highest nucleoside degrading rates were selected for further characterization. The tested biological characteristics of candidate strains included acid tolerance, bile tolerance, anti-pathogenic bacteria activity, cell adhesion ability, resistance to antibiotics and the ability to produce hydrogen peroxide. Among the selected strains, DM9218 showed the best probiotic potential compared with other strains despite its poor bile resistance. Analysis of 16S rRNA sequences showed that DM9218 has the highest similarity (99%) to Lactobacillus plantarum WCFS1. The acclimated strain DM9218-A showed better resistance to 0.3% bile salt, and its survival in gastrointestinal tract of rats was proven by PCR-DGGE. Furthermore, the effects of DM9218-A in a hyperuricemia rat model were evaluated. The level of serum uric acid in hyperuricemic rat can be efficiently reduced by the intragastric administration of DM9218-A (P<0.05). The preventive treatment of DM9218-A caused a greater reduction in serum uric acid concentration in hyperuricemic rats than the later treatment (P<0.05). Our results suggest that DM9218-A may be a promising candidate as an adjunctive treatment in patients with hyperuricemia during the onset period of disease. DM9218-A also has potential as a probiotic

  13. Screening and characterization of purine nucleoside degrading lactic acid bacteria isolated from Chinese sauerkraut and evaluation of the serum uric acid lowering effect in hyperuricemic rats.

    PubMed

    Li, Ming; Yang, Dianbin; Mei, Lu; Yuan, Lin; Xie, Ao; Yuan, Jieli

    2014-01-01

    Hyperuricemia is well known as the cause of gout. In recent years, it has also been recognized as a risk factor for arteriosclerosis, cerebrovascular and cardiovascular diseases, and nephropathy in diabetic patients. Foods high in purine compounds are more potent in exacerbating hyperuricemia. Therefore, the development of probiotics that efficiently degrade purine compounds is a promising potential therapy for the prevention of hyperuricemia. In this study, fifty-five lactic acid bacteria isolated from Chinese sauerkraut were evaluated for the ability to degrade inosine and guanosine, the two key intermediates in purine metabolism. After a preliminary screening based on HPLC, three candidate strains with the highest nucleoside degrading rates were selected for further characterization. The tested biological characteristics of candidate strains included acid tolerance, bile tolerance, anti-pathogenic bacteria activity, cell adhesion ability, resistance to antibiotics and the ability to produce hydrogen peroxide. Among the selected strains, DM9218 showed the best probiotic potential compared with other strains despite its poor bile resistance. Analysis of 16S rRNA sequences showed that DM9218 has the highest similarity (99%) to Lactobacillus plantarum WCFS1. The acclimated strain DM9218-A showed better resistance to 0.3% bile salt, and its survival in gastrointestinal tract of rats was proven by PCR-DGGE. Furthermore, the effects of DM9218-A in a hyperuricemia rat model were evaluated. The level of serum uric acid in hyperuricemic rat can be efficiently reduced by the intragastric administration of DM9218-A (P<0.05). The preventive treatment of DM9218-A caused a greater reduction in serum uric acid concentration in hyperuricemic rats than the later treatment (P<0.05). Our results suggest that DM9218-A may be a promising candidate as an adjunctive treatment in patients with hyperuricemia during the onset period of disease. DM9218-A also has potential as a probiotic

  14. Metabolism of purine alkaloids and xanthine in leaves of maté (Ilex paraguariensis).

    PubMed

    Yin, Yuling; Katahira, Riko; Ashihara, Hiroshi

    2015-05-01

    Accumulation and metabolism of purine alkaloids in leaves of maté (Ilex paraguariensis) were investigated. In winter, leaves accumulated caffeine but not theobromine, indicating that caffeine is the end product of purine alkaloid synthesis in maté. To elucidate the purine alkaloid metabolism in maté leaves, the metabolic fate of [8-(14)C]theobromine, [8-(14)C]theophylline, [8-(14)C]caffeine and [8-(14)C] xanthine was investigated in the leaf disks of young and mature leaves. In young maté leaves, significant amounts of theobromine and theophylline were utilized for caffeine biosynthesis, but the conversion was not observed in mature leaves. A small amount of theophylline was converted to theobromine. Practically no caffeine catabolism was detected in maté leaves during a 24 h-incubation. Catabolism of theobromine and theophylline via 3-methylxanthine was observed mainly in mature leaves. Xanthine was catabolised extensively via ureides in both young and mature leaves, but limited amounts are also utilized for the synthesis of theobromine, theophylline and caffeine. Possible pathways for the metabolism of purine alkaloids in maté leaves are discussed.

  15. Targeting DNA repair with aphidicolin sensitizes primary chronic lymphocytic leukemia cells to purine analogs

    PubMed Central

    Starczewska, Eliza; Beyaert, Maxime; Michaux, Lucienne; Vekemans, Marie-Christiane; Saussoy, Pascale; Bol, Vanesa; Echarri, Ainhoa Arana; Smal, Caroline; Van Den Neste, Eric; Bontemps, Françoise

    2016-01-01

    Purine analogs are among the most effective chemotherapeutic drugs for the treatment of chronic lymphocytic leukemia (CLL). However, chemoresistance and toxicity limit their clinical use. Here, we report that the DNA polymerase inhibitor aphidicolin, which displayed negligible cytotoxicity as a single agent in primary CLL cells, markedly synergizes with fludarabine and cladribine via enhanced apoptosis. Importantly, synergy was recorded regardless of CLL prognostic markers. At the molecular level, aphidicolin enhanced purine analog-induced phosphorylation of p53 and accumulation of γH2AX, consistent with increase in DNA damage. In addition, aphidicolin delayed γH2AX disappearance that arises after removal of purine analogs, suggesting that aphidicolin causes an increase in DNA damage by impeding DNA damage repair. Similarly, aphidicolin inhibited UV-induced DNA repair known to occur primarily through the nucleotide excision repair (NER) pathway. Finally, we showed that fludarabine induced nuclear import of XPA, an indispensable factor for NER, and that XPA silencing sensitized cell lines to undergo apoptosis in response to fludarabine. Together, our data indicate that aphidicolin potentiates the cytotoxicity of purine analogs by inhibiting a DNA repair pathway that involves DNA polymerases, most likely NER, and provide a rationale for manipulating it to therapeutic advantage. PMID:27223263

  16. From Purines to Basic Biochemical Concepts: Experiments for High School Students

    ERIC Educational Resources Information Center

    Marini, Isabella; Ipata, Piero Luigi

    2007-01-01

    Many high school biology courses address mainly the molecular and cellular basis of life. The complexity that underlies the most essential processes is often difficult for the students to understand; possibly, in part, because of the inability to see and explore them. Six simple practical experiments on purine catabolism as a part of a…

  17. Participation of purines in the modulation of inflammatory response in rats experimentally infected by Cryptococcus neoformans.

    PubMed

    de Azevedo, Maria Isabel; Ferreiro, Laerte; Da Silva, Aleksandro S; Tonin, Alexandre A; Monteiro, Danieli Urach; Casali, Emerson A; Moritz, Cesar E J; Schirmbeck, Gabriel H; Cardoso, Valesca V; Flores, Mariana M; Fighera, Rafael; Stefani, Lenita M; Santurio, Janio M

    2016-10-01

    The present study was carried out to assess the participation of purines in the activation and modulation of inflammatory response of rats experimentally infected by Cryptococcus neoformans. Twenty four Wistar rats were divided into two groups of 12 animals each: Group A - uninfected control group and Group B - infected by C. neoformans. Blood was collected 20 and 50 days post-infection (PI) from six animals of each group in order to verify purine levels (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (HYPO), xanthine (XAN) and uric acid (URIC)). ATP levels were significantly increased (P < 0.05) in serum from infected animals on days 20 and 50 PI, as well as adenosine levels after 20 days PI on rats. On day 50 PI, levels of adenosine and uric acid were also reduced, but the levels of inosine and xanthine increased in animals infected by the fungus (P < 0.05). Therefore, it was possible to conclude that the purine levels in serum were altered and that these changes may be able to influence the pathogenesis of the disease caused by C. neoformans due the participation of purines (ATP and adenosine main) in the activation and modulation of inflammatory response.

  18. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

    PubMed

    Balestri, Francesco; Giannecchini, Michela; Sgarrella, Francesco; Carta, Maria Caterina; Tozzi, Maria Grazia; Camici, Marcella

    2007-02-01

    The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

  19. Changes in Purines Concentration in the Cerebrospinal Fluid of Pregnant Women Experiencing Pain During Active Labor.

    PubMed

    Schmidt, André P; Böhmer, Ana E; Hansel, Gisele; Soares, Félix A; Oses, Jean P; Giordani, Alex T; Posso, Irimar P; Auler, José Otávio C; Mendes, Florentino F; Félix, Elaine A; Portela, Luís V; Souza, Diogo O

    2015-11-01

    Labor pain has been reported as a severe pain and can be considered as a model of acute visceral pain. It is well known that extracellular purines have an important role in pain signaling in the central nervous system. This study analyzes the relationship between extracellular purines and pain perception during active labor. A prospective observational study was performed. Cerebrospinal fluid (CSF) levels of the purines and their metabolites were compared between women at term pregnancy with labor pain (n = 49) and without labor pain (Caesarian section; n = 47). Control groups (healthy men and women without chronic or acute pain-n = 40 and 32, respectively) were also investigated. The CSF levels of adenosine were significantly lower in the labor pain group (P = 0.026) and negatively correlated with pain intensity measured by a visual analogue scale (r = -0.48, P = 0.0005). Interestingly, CSF levels of uric acid were significantly higher in healthy men as compared to women. Additionally, pregnant women showed increased CSF levels of ADP, GDP, adenosine and guanosine and reduced CSF levels of AMP, GTP, and uric acid as compared to non-pregnant women (P < 0.05). These findings suggest that purines, in special the nucleoside adenosine, are associated with pregnancy and labor pain.

  20. Pronounced minimum of the thermodynamic Casimir forces of O(n) symmetric film systems: analytic theory.

    PubMed

    Dohm, Volker

    2014-09-01

    Thermodynamic Casimir forces of film systems in the O(n) universality classes with Dirichlet boundary conditions are studied below bulk criticality. Substantial progress is achieved in resolving the long-standing problem of describing analytically the pronounced minimum of the scaling function observed experimentally in ^{4}He films (n=2) by Garcia and Chan [Phys. Rev. Lett. 83, 1187 (1999)] and in Monte Carlo simulations for the three-dimensional Ising model (n=1) by O. Vasilyev et al. [Europhys. Lett. 80, 60009 (2007)]. Our finite-size renormalization-group approach describes the film systems as the limit of finite-slab systems with vanishing aspect ratio. This yields excellent agreement with the depth and the position of the minimum for n=1 and semiquantitative agreement with the minimum for n=2. Our theory also predicts a pronounced minimum for the n=3 Heisenberg universality class.

  1. Pronounced gender and age differences are evident in personal health care spending per person.

    PubMed

    Cylus, Jonathan; Hartman, Micah; Washington, Benjamin; Andrews, Kimberly; Catlin, Aaron

    2011-01-01

    This paper examines differences in national health care spending by gender and age. Our research found significant variations in per person spending by gender across age groups, health services, and types of payers. For example, in 2004 per capita health care spending for females was 32 percent more than for males. Per capita differences were most pronounced among the working-age population, largely because of spending for maternity care. Except for children, total spending for and by females was greater than that for and by males, for most services and payers. The gender difference in total spending was most pronounced in the elderly, as a result of the longer life expectancy of women.

  2. Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay.

    PubMed

    Domin, B A; Mahony, W B; Zimmerman, T P

    1988-07-05

    A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.

  3. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    PubMed Central

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  4. Synthesis and antimycobacterial activity of N-(2-aminopurin-6-yl) and N-(purin-6-yl) amino acids and dipeptides.

    PubMed

    Krasnov, Victor P; Vigorov, Alexey Yu; Musiyak, Vera V; Nizova, Irina A; Gruzdev, Dmitry A; Matveeva, Tatyana V; Levit, Galina L; Kravchenko, Marionella A; Skornyakov, Sergey N; Bekker, Olga B; Danilenko, Valery N; Charushin, Valery N

    2016-06-01

    Synthetic routes to novel N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates with amino acids and glycine-containing dipeptides were developed. In vitro testing of 42 new and known compounds made it possible to reveal a series of N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates exhibiting significant antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium, Mycobacterium terrae, and multidrug-resistant M. tuberculosis strain isolated from tuberculosis patients in the Ural region (Russia). N-(2-Aminopurin-6-yl)- and N-(purin-6-yl)-glycyl-(S)-glutamic acids were the most active compounds.

  5. Pronouncing the names of the moons of Saturn, or pulling teeth from Tethys

    NASA Astrophysics Data System (ADS)

    Consolmagno, G.; Reiche, H.

    The moons of Saturn, so Woody Allen recently observed [Allen, 1979], do not come up in conversation very often. This may be one reason why most people, planetary scientists included, have trouble pronouncing their names.Our troubles, however, stem not merely from the fact that these names are unfamiliar, or even that they are of non-English origin. After all, the same is true of many terms in technology and medicine [cf. Nybakken, 1959; Reiche, 1959]. They stem from the fact that they are drawn from Greco-Roman mythology and so possess the three chief difficulties troubling those who strive for ‘correct’ pronunciation anywhere in this area.

  6. Periodontal status of mandibular incisors after pronounced orthodontic advancement during adolescence: a follow-up evaluation.

    PubMed

    Artun, J; Grobéty, D

    2001-01-01

    The purpose of this study was to analyze whether pronounced orthodontic advancement of the mandibular incisors during Class II correction in the mixed dentition results in gingival recession. Through mandibular superimposition of the pretreatment and posttreatment cephalograms of 67 Class II patients who were treated with reverse headgear to the mandibular dentition, 45 patients with a minimum of a 1-mm advancement of the cementoenamel junction (CEJ; mean, 2.18 +/- 0.87) and a minimum of a 2-mm advancement of the incisal edge (mean, 3.87 +/- 1.34) were identified. Using the same protocol in Class II patients, 30 individuals who finished treatment at a similar time and age, but without reverse headgear and with no advancement of the CEJ (mean -0.43, SD 0.53) and a maximum of 1-mm advancement of the incisal edge (mean -0.26, SD 1.15) were identified. Before treatment, the mandibular incisors were more retruded, relative to the line from point A to pogonion and relative to the mandibular plane in the patients with pronounced advancement than in those with no advancement of the mandibular incisors; no differences were found at the time of appliance removal. A total of 30 patients with pronounced advancement and 21 patients with no advancement could meet for a follow-up examination a mean period of 7.83 years (SD, 4.44) and 9.38 years (SD, 4.39) after treatment, respectively. Clinical examinations at the time of follow-up revealed no differences in the amount of recession, the width of attached gingiva, the length of supracrestal connective tissue attachment, the probing pocket depth, and gingival bleeding index or visible plaque index of the mandibular incisors between the patients in the 2 groups. An examination of color slides demonstrated no differences in the number of mandibular incisors that developed recession from before treatment to after treatment and from after treatment to follow-up. Measurement of mandibular incisor crown height on the study models

  7. A Dual-Route Model that Learns to Pronounce English Words

    NASA Technical Reports Server (NTRS)

    Remington, Roger W.; Miller, Craig S.; Null, Cynthia H. (Technical Monitor)

    1995-01-01

    This paper describes a model that learns to pronounce English words. Learning occurs in two modules: 1) a rule-based module that constructs pronunciations by phonetic analysis of the letter string, and 2) a whole-word module that learns to associate subsets of letters to the pronunciation, without phonetic analysis. In a simulation on a corpus of over 300 words the model produced pronunciation latencies consistent with the effects of word frequency and orthographic regularity observed in human data. Implications of the model for theories of visual word processing and reading instruction are discussed.

  8. Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain.

    PubMed

    Gilbert, Sunny D; Stoddard, Colby D; Wise, Sarah J; Batey, Robert T

    2006-06-09

    Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.

  9. SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement.

    PubMed

    Nauš, Jan; Prokopová, Jitka; Rebíček, Jiří; Spundová, Martina

    2010-09-01

    Non-destructive assessment of chlorophyll content has recently been widely done by chlorophyll meters based on measurement of leaf transmittance (e.g. the SPAD-502 chlorophyll meter measures the leaf transmittance at 650 and 940 nm). However, the leaf transmittance depends not only on the content of chlorophylls but also on their distribution in leaves. The chlorophyll distribution within leaves is co-determined by chloroplast arrangement in cells that depends on light conditions. When tobacco leaves were exposed to a strong blue light (about 340 μmol of photons m⁻² s⁻¹), a very pronounced increase in the leaf transmittance was observed as chloroplasts migrated from face position (along cell walls perpendicular to the incident light) to side position (along cell walls parallel to the incoming light) and the SPAD reading decreased markedly. This effect was more pronounced in the leaves of young tobacco plants compared with old ones; the difference between SPAD values in face and side position reached even about 35%. It is shown how the chloroplast movement changes a relationship between the SPAD readings and real chlorophyll content. For an elimination of the chloroplast movement effect, it can be recommended to measure the SPAD values in leaves with a defined chloroplasts arrangement.

  10. Nonrandom distribution of cryptic repeating triplets of purines and pyrimidines (RNY)(n) in gp120 of HIV Type1.

    PubMed

    De Crignis, Elisa; Guglietta, Silvia; Foley, Brian T; Negroni, Matteo; Di Narzo, Antonio Fabio; Waelti Da Costa, Vreneli; Cavassini, Matthias; Bart, Pierre-Alexandre; Pantaleo, Giuseppe; Graziosi, Cecilia

    2012-05-01

    We have analyzed purine (R) and pyrimidine (Y) codon patterns in variable and constant regions of HIV-1 gp120 in seven patients infected with different HIV-1 subtypes and naive to antiretroviral therapy. We have calculated the relative frequency of each in-frame codon RNY, YNR, RNR, and YNY (N=any nucleotide) in variable and constant regions of gp120, in the sequence within indels and at indels' flanking sites. Our data show that hypervariable regions V1, V2, V4, and V5 are characterized by the presence of long stretches of RNY codons constituting the majority of the sequence portion within insertions/deletions. In full-length gp120 and within inserted/deleted fragments the number of AVT (V=A, C, G) codons did not exceed 50% of the total RNY codons. RNY strings in variable regions spanned up to 21 codons and were always in frame. In contrast, RNY strings in constant regions were mostly out of frame and their length was limited to five codons. The frequency of the codon RNY was found to be significantly higher in variable regions (p<0.0001; t-test), within indels, and at indels' flanking sites (p<0.0001; χ(2) test). Analysis of the distribution of RNY strings equal to or longer than five codons in the full genome of HXB2 also shows that these sequences are mostly out of frame, unless they contain a potential N-glycosylation site or an asparagine. These data suggest that cryptic repeats of RNY may play a role in the genesis of multiple base insertions and deletions in hypervariable regions of gp120.

  11. Amide-controlled, one-pot synthesis of tri-substituted purines generates structural diversity and analogues with trypanocidal activity.

    PubMed

    Pineda de las Infantas y Villatoro, Maria J; Unciti-Broceta, Juan D; Contreras-Montoya, Rafael; Garcia-Salcedo, Jose A; Gallo Mezo, Miguel A; Unciti-Broceta, Asier; Diaz-Mochon, Juan J

    2015-03-16

    A novel one-pot synthesis of tri-substituted purines and the discovery of purine analogues with trypanocidal activity are reported. The reaction is initiated by a metal-free oxidative coupling of primary alkoxides and diaminopyrimidines with Schiff base formation and subsequent annulation in the presence of large N,N-dimethylamides (e.g. N,N-dimethylpropanamide or larger). This synthetic route is in competition with a reaction previously-reported by our group, allowing the generation of a combinatorial library of tri-substituted purines by the simple modification of the amide and the alkoxide employed. Among the variety of structures generated, two purine analogues displayed trypanocidal activity against the protozoan parasite Trypanosoma brucei with IC50 < 5 μM, being each of those compounds obtained through each of the synthetic pathways.

  12. Akt Phosphorylation and Regulation of Transketolase Is a Nodal Point for Amino Acid Control of Purine Synthesis

    PubMed Central

    Saha, Arindam; Connelly, Stephen; Jiang, Jingjing; Zhuang, Shunhui; Amador, Deron T.; Phan, Tony; Pilz, Renate B.; Boss, Gerry R.

    2014-01-01

    SUMMARY The phosphatidylinositol 3-kinase (PI3K)/Akt pathway integrates environmental clues to regulate cell growth and survival. We showed previously that depriving cells of a single essential amino acid rapidly and reversibly arrests purine synthesis. Here we demonstrate that amino acids via mTORC2 and IκB kinase regulate Akt activity, and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the non-oxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the non-oxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for two days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a new mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  13. Characterization of a purine permease family gene OsPUP7 involved in growth and development control in rice.

    PubMed

    Qi, Zhuyun; Xiong, Lizhong

    2013-11-01

    In this study, PUP-type cytokinin transporter genes were identified in rice (Oryza sativa L.). The Oryza sativa purine permease (OsPUP) family has 12 members that show similar predicted protein sequences with AtPUPs. To reveal the functions of OsPUP genes, we searched the T-DNA mutant library of rice and found one mutant for the member OsPUP7. The T-DNA insertion caused a new transcript that encodes a protein with 26 amino acids different from the native OsPUP7 at the C-terminus. The mutant showed multiple phenotypic changes including increased plant height, big seeds, and delayed flowering. The mutant also showed increased sensitivity to drought and salt stresses and treatments with kinetin and abscisic acid. OsPUP7 is expressed mainly in the vascular bundle, pistil, and stamens. The measurement of cytokinins (CKs) showed that CK content in the mutant spikelets accumulated higher than that in the wild type. Moreover, uptake experiment in the yeast fcy2 mutant suggested that OsPUP7 has the ability to transport caffeine, a CK derivative. Our results indicate that the PUP transport system also exists in rice, and OsPUP7 has an important role in the transport of CK, thus affecting developmental process and stress responses.

  14. Structural characterization of purine nucleoside phosphorylase from human pathogen Helicobacter pylori.

    PubMed

    Štefanić, Zoran; Mikleušević, Goran; Luić, Marija; Bzowska, Agnieszka; Ašler, Ivana Leščić

    2017-03-20

    Microaerophilic bacterium Helicobacer pylori is a well known human pathogen involved in the development of many diseases. Due to the evergrowing infection rate and increase of H. pylori antibiotic resistence, it is of utmost importance to find a new way to attack and eradicate H. pylori. The purine metabolism in H. pylori is solely dependant on the salvage pathway and one of the key enzymes in this pathway is purine nucleoside phosphorylase (PNP). In this timely context, we report here the basic biochemical and structural characterization of recombinant PNP from the H. pylori clinical isolate expressed in Escherichia coli. Structure of H. pylori PNP is typical for high molecular mass PNPs. However, its activity towards adenosine is very low, thus resembling more that of low molecular mass PNPs. Understanding the molecular mechanism of this key enzyme may lead to the development of new drug strategies and help in the eradication of H. pylori.

  15. The prebiotic synthesis of modified purines and their potential role in the RNA world

    NASA Technical Reports Server (NTRS)

    Levy, M.; Miller, S. L.; Bada, J. L. (Principal Investigator)

    1999-01-01

    Modified purines are found in all organisms in the tRNA, rRNA, and even DNA, raising the possibility of an early role for these compounds in the evolution of life. These include N6-methyladenine, 1-methyladenine, N6,N6-dimethyladenine, 1-methylhypoxanthine, 1-methylguanine, and N2-methylguanine. We find that these bases as well as a number of nonbiological modified purines can be synthesized from adenine and guanine by the simple reaction of an amine or an amino group with adenine and guanine under the concentrated conditions of the drying-lagoon or drying-beach model of prebiotic synthesis with yields as high as 50%. These compounds are therefore as prebiotic as adenine and guanine and could have played an important role in the RNA world by providing additional functional groups in ribozymes, especially for the construction of hydrophobic binding pockets.

  16. 9H-Purine Scaffold Reveals Induced-Fit Pocket Plasticity of the BRD9 Bromodomain

    PubMed Central

    2015-01-01

    The 2-amine-9H-purine scaffold was identified as a weak bromodomain template and was developed via iterative structure based design into a potent nanomolar ligand for the bromodomain of human BRD9 with small residual micromolar affinity toward the bromodomain of BRD4. Binding of the lead compound 11 to the bromodomain of BRD9 results in an unprecedented rearrangement of residues forming the acetyllysine recognition site, affecting plasticity of the protein in an induced-fit pocket. The compound does not exhibit any cytotoxic effect in HEK293 cells and displaces the BRD9 bromodomain from chromatin in bioluminescence proximity assays without affecting the BRD4/histone complex. The 2-amine-9H-purine scaffold represents a novel template that can be further modified to yield highly potent and selective tool compounds to interrogate the biological role of BRD9 in diverse cellular systems. PMID:25703523

  17. Coexpression of two closely linked avian genes for purine nucleotide synthesis from a bidirectional promoter.

    PubMed Central

    Gavalas, A; Dixon, J E; Brayton, K A; Zalkin, H

    1993-01-01

    Two avian genes encoding essential steps in the purine nucleotide biosynthetic pathway are transcribed divergently from a bidirectional promoter element. The bidirectional promoter, embedded in a CpG island, directs coexpression of GPAT and AIRC genes from distinct transcriptional start sites 229 bp apart. The bidirectional promoter can be divided in half, with each half retaining partial activity towards the cognate gene. GPAT and AIRC genes encode the enzymes that catalyze step 1 and steps 6 plus 7, respectively, in the de novo purine biosynthetic pathway. This is the first report of genes coding for structurally unrelated enzymes of the same pathway that are tightly linked and transcribed divergently from a bidirectional promoter. This arrangement has the potential to provide for regulated coexpression comparable to that in a prokaryotic operon. Images PMID:8336716

  18. Purine derivatives as potent Bruton’s tyrosine kinase (BTK) inhibitors for autoimmune diseases

    SciTech Connect

    Shi, Qing; Tebben, Andrew; Dyckman, Alaric J.; Li, Hedy; Liu, Chunjian; Lin, James; Spergel, Steve; Burke, James R.; McIntyre, Kim W.; Olini, Gilbert C.; Strnad, Joann; Surti, Neha; Muckelbauer, Jodi K.; Chang, Chiehying; An, Yongmi; Cheng, Lin; Ruan, Qian; Leftheris, Katerina; Carter, Percy H.; Tino, Joseph; De Lucca, George V.

    2014-05-01

    Investigation of various heterocyclic core isosteres of imidazopyrazines 1 & 2 yielded purine derivatives 3 & 8 as potent and selective BTK inhibitors. Subsequent SAR studies of the purine series led to the discovery of 20 as a leading compound. Compound 20 is very selective when screened against a panel of 400 kinases and is a potent inhibitor in cellular assays of human B cell function including B-Cell proliferation and CD86 cell surface expression and exhibited in vivo efficacy in a mouse PCA model. Its X-ray co-crystal structure with BTK shows that the high selectivity is gained from filling a BTK specific lipophilic pocket. However, physical and ADME properties leading to low oral exposure hindered further development.

  19. Synthesis of Novel N9-Substituted Purine Derivatives from Polymer Supported α-Amino Acids.

    PubMed

    Vanda, David; Jorda, Radek; Lemrová, Barbora; Volná, Tereza; Kryštof, Vladimír; McMaster, Claire; Soural, Miroslav

    2015-07-13

    Solid-phase synthesis of purine derivatives bearing an α-amino acid motif in position 9 is described herein. Polymer supported amines were acylated with various Fmoc-α-amino acids and, after cleavage of the protecting group, arylation with 4,6-dichloro-5-nitropyrimidine or 2,4-dichloro-5-nitropyrimidine was performed. The second chlorine atom was replaced with various amines. Subsequent reduction of the nitro group, followed by reaction with aldehydes, afforded the purine scaffold. After cleavage from the polymer support, the target compounds were obtained in very good crude purity, good overall yields, and excellent enantiomeric purity. The anticancer activity of prepared compounds was tested in vitro against human cancer cell lines MCF7 and K562, and they were found to have mild, but clear dose-dependent effects.

  20. Direct Isolation of Purines and Pyrimidines from Nucleic Acids Using Sublimation

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Schubert, Michael; Bada, Jeffrey L.

    2003-01-01

    A sublimation technique was developed to isolate purines and pyrimidines directly from lambda-deoxyribonucleic acid (lambda-DNA) and Escherichia coli cells. The sublimation of adenine, cytosine, guanine, and thymine from lambda-DNA was tested under reduced pressure (approx. 0.5 Torr) at temperatures of >150 C. With the exception of guanine, approximately 60 -75% of each base was sublimed directly from the lambda-DNA and recovered on a coldfinger of the sublimation apparatus after heating to 450 C. Several nucleobases including adenine, cytosine, thymine, and uracil were also recovered from E. coli bacteria after heating the cells to the same temperature, although some thermal decomposition of the bases also occurred. These results demonstrate the feasibility of using sublimation to isolate purines and pyrimidines from native E. coli DNA and RNA without any chemical treatment of the cells.

  1. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

    SciTech Connect

    Abramchik, Yu. A. Timofeev, V. I. Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2015-07-15

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  2. Determination of Caffeine and Other Purine Compounds in Food and Pharmaceuitcals by Micellar Electrokinetic Chrmoatography

    NASA Astrophysics Data System (ADS)

    Vogt, Carla; Contradi, S.; Rohde, E.

    1997-09-01

    Capillary elctrophoresis is a modern separation technique, especially the extremely high efficiencies and minimal requirements with regard to buffers, samples and solvents lead to a dramatic increase of applications in the last few years. This paper offers an introduction to the technique of micellar elektrokinetic chromatography as a special kind of capillary electrophoresis. Caffeine and other purine compounds have been determined in foodstuff (tea, coffee, cocoa) as well as in pharmaceutical formulations. Different sample preparation procedures which have been developed with regard to the special properties of the sample matrices are discussed in the paper.This preparation facilitates the separation in many cases. So students have to solve a relatively simple separation problem by variation of buffer pH, buffer components and separation parameters. By doing a calibration for the analyzed purine compounds they will learn about reproducibility in capillary electrophoresis.

  3. Abiotic synthesis of purines and other heterocyclic compounds by the action of electrical discharges

    NASA Technical Reports Server (NTRS)

    Yuasa, S.; Flory, D.; Basile, B.; Oro, J.

    1984-01-01

    The synthesis of purines and pyrimidines using Oparin-Urey-type primitive earth atmospheres has been demonstrated by reacting methane, ethane, and ammonia in electrical discharges. Adenine, guaine, 4-aminoimidazole-5-carboxamide (AICA), and isocytosine have been identified by UV spectrometry and paper chromatography as the products of the reaction. The total yields of the identified heterocyclic compounds are 0.0023 percent. It is concluded that adenine synthesis occurs at a much lower concentration of hydrogen cyanide than has been shown by earlier studies. Pathways for the synthesis of purines from hydrogen cyanide are discussed, and a comparison of the heterocyclic compounds that have been identified in meteorites and in prebiotic reactions is presented.

  4. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

    NASA Astrophysics Data System (ADS)

    Abramchik, Yu. A.; Timofeev, V. I.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2015-07-01

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P21 and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  5. Stacking of purines in water: the role of dipolar interactions in caffeine.

    PubMed

    Tavagnacco, L; Di Fonzo, S; D'Amico, F; Masciovecchio, C; Brady, J W; Cesàro, A

    2016-05-11

    During the last few decades it has been ascertained that base stacking is one of the major contributions stabilizing nucleic acid conformations. However, the understanding of the nature of the interactions involved in the stacking process remains under debate and it is a subject of theoretical and experimental studies. Structural similarity between purine bases (guanine and adenine) in DNA and the caffeine molecule makes caffeine an excellent model for the purine bases. The present study clearly shows that dipolar interactions play a fundamental role in determining stacking of purine molecules in solution. In order to reach this achievement, polarized ultraviolet Raman resonant scattering experiments have been carried out on caffeine aqueous solutions as a function of concentration and temperature. The investigation pointed out at the aggregation and solvation properties, particularly at elevated temperatures. Kubo-Anderson theory was used as a framework to investigate the non-coincidence effect (NCE) occurring in the totally symmetric breathing modes of the purine rings, and in the bending modes of the methyl groups of caffeine. The NCE concentration dependence shows that caffeine aggregation at 80 °C occurs by planar stacking of the hydrophobic faces. The data clearly indicate that dipolar interactions determine the reorientational motion of the molecules in solution and are the driving force for the stacking of caffeine. In parallel, the observed dephasing times imply a change in caffeine interactions as a function of temperature and concentration. A decrease, at low water content, of the dephasing time for the ring breathing vibration mode indicates that self-association alters the solvation structure that is detectable at low concentration. These results are in agreement with simulation predictions and serve as an important validation of the models used in those calculations.

  6. L-Enantiomers of Transition State Analogue Inhibitors Bound to Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Rinaldo-Matthis,A.; Murkin, A.; Ramagopal, U.; Clinch, K.; Mee, S.; Evans, G.; Tyler, P.; Furneaux, R.; Almo, S.; Schramm, v.

    2008-01-01

    Human purine nucleoside phosphorylase (PNP) was crystallized with transition-state analogue inhibitors Immucillin-H and DADMe-Immucillin-H synthesized with ribosyl mimics of l-stereochemistry. The inhibitors demonstrate that major driving forces for tight binding of these analogues are the leaving group interaction and the cationic mimicry of the transition state, even though large geometric changes occur with d-Immucillins and l-Immucillins bound to human PNP.

  7. Electronic and structural elements that regulate the excited-state dynamics in purine nucleobase derivatives.

    PubMed

    Crespo-Hernández, Carlos E; Martínez-Fernández, Lara; Rauer, Clemens; Reichardt, Christian; Mai, Sebastian; Pollum, Marvin; Marquetand, Philipp; González, Leticia; Corral, Inés

    2015-04-08

    The excited-state dynamics of the purine free base and 9-methylpurine are investigated using experimental and theoretical methods. Femtosecond broadband transient absorption experiments reveal that excitation of these purine derivatives in aqueous solution at 266 nm results primarily in ultrafast conversion of the S2(ππ*) state to the vibrationally excited (1)nπ* state. Following vibrational and conformational relaxation, the (1)nπ* state acts as a doorway state in the efficient population of the triplet manifold with an intersystem crossing lifetime of hundreds of picoseconds. Experiments show an almost 2-fold increase in the intersystem crossing rate on going from polar aprotic to nonpolar solvents, suggesting that a solvent-dependent energy barrier must be surmounted to access the singlet-to-triplet crossing region. Ab initio static and surface-hopping dynamics simulations lend strong support to the proposed relaxation mechanism. Collectively, the experimental and computational results demonstrate that the accessibility of the nπ* states and the topology of the potential energy surfaces in the vicinity of conical intersections are key elements in controlling the excited-state dynamics of the purine derivatives. From a structural perspective, it is shown that the purine chromophore is not responsible for the ultrafast internal conversion in the adenine and guanine monomers. Instead, C6 functionalization plays an important role in regulating the rates of radiative and nonradiative relaxation. C6 functionalization inhibits access to the (1)nπ* state while simultaneously facilitating access to the (1)ππ*(La)/S0 conical intersection, such that population of the (1)nπ* state cannot compete with the relaxation pathways to the ground state involving ring puckering at the C2 position.

  8. Electronic and Structural Elements That Regulate the Excited-State Dynamics in Purine Nucleobase Derivatives

    PubMed Central

    2015-01-01

    The excited-state dynamics of the purine free base and 9-methylpurine are investigated using experimental and theoretical methods. Femtosecond broadband transient absorption experiments reveal that excitation of these purine derivatives in aqueous solution at 266 nm results primarily in ultrafast conversion of the S2(ππ*) state to the vibrationally excited 1nπ* state. Following vibrational and conformational relaxation, the 1nπ* state acts as a doorway state in the efficient population of the triplet manifold with an intersystem crossing lifetime of hundreds of picoseconds. Experiments show an almost 2-fold increase in the intersystem crossing rate on going from polar aprotic to nonpolar solvents, suggesting that a solvent-dependent energy barrier must be surmounted to access the singlet-to-triplet crossing region. Ab initio static and surface-hopping dynamics simulations lend strong support to the proposed relaxation mechanism. Collectively, the experimental and computational results demonstrate that the accessibility of the nπ* states and the topology of the potential energy surfaces in the vicinity of conical intersections are key elements in controlling the excited-state dynamics of the purine derivatives. From a structural perspective, it is shown that the purine chromophore is not responsible for the ultrafast internal conversion in the adenine and guanine monomers. Instead, C6 functionalization plays an important role in regulating the rates of radiative and nonradiative relaxation. C6 functionalization inhibits access to the 1nπ* state while simultaneously facilitating access to the 1ππ*(La)/S0 conical intersection, such that population of the 1nπ* state cannot compete with the relaxation pathways to the ground state involving ring puckering at the C2 position. PMID:25763596

  9. Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

    PubMed Central

    Kohl, D H; Schubert, K R; Carter, M B; Hagedorn, C H; Shearer, G

    1988-01-01

    N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides). The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vmax with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but less than 2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont. PMID:3353366

  10. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

    PubMed

    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas.

  11. The Formation of Nucleobases from the Irradiation of Purine in Astophysical Ices and Comparisons with Meteorites.

    NASA Technical Reports Server (NTRS)

    Sandford, S. A.; Materese, C. K.; Nuevo, M.

    2016-01-01

    N-heterocycles have been identified in meteorites and their extraterrestrial origins are suggested by isotopic ratio measurements. Although small N- heterocycles have not been detected in the interstellar medium (ISM), recent experiments in our lab have shown that the irradiation of the aromatic molecules like benzene (C6H6) and naphthalene (C10H8) in mixed molecular ices leads to the formation of O- and N-heterocyclic molecules. Among the class of N-heterocycles are the nucleobases, which are of astrobiological interest because they are the information bearing units of DNA and RNA. Nucleobases have been detected in meteorites [3-5], with isotopic signatures that are also consistent with an extraterrestrial origin. Three of the biologically relevant nucleobases (uracil, cytosine, and guanine) have a pyrimidine core structure while the remaining two (adenine and guanine) possess a purine core. Previous experiments in our lab have demonstrated that all of the bio-logical nucleobases (and numerous other molecules) with a pyrimidine core structure can be produced by irradiating pyrimidine in mixed molecular ices of several compositions [6-8]. In this work, we study the formation of purine-based molecules, including the nucleobases adenine, and guanine, from the ultraviolet (UV) irradiation of purine in ices consisting mixtures of H2O and NH3 at low temperature. The experiments are designed to simulate the astrophysical conditions under which these species may be formed in dense molecular clouds, protoplanetary disks, or on the surfaces of icy bodies in planetary systems.

  12. Hypercrosslinked strong cation-exchange polymers for selective extraction of serum purine metabolites associated with gout.

    PubMed

    Xu, Yating; Liu, Ju; Zhang, Hongyang; Jiang, Min; Cao, Lingling; Zhang, Min; Sun, Wei; Ruan, Shengli; Hu, Ping

    2016-05-01

    In this study, hypercrosslinked strong cation-exchange polymer resins (HXLPP-SCX) were synthesized and employed as selective sorbents for the solid-phase extraction (SPE) of basic purine metabolites associated with gout. The HXLPP-SCX material was prepared based on hypercrosslinking reactions and sulfonated with concentrated H2SO4. This synthetic procedure is facile and efficient without using highly toxic reagent. The resulting resins were characterized in the form of monodisperse microspheres (mean diameters of 3‒5μm) with narrow pore size (2.1nm) and relatively high specific surface areas (801m(2)/g). The polymers also possess high ion-exchange capacity (IEC, 2.22mmol/g) and good adsorption and selectivity performances for basic compounds. The resins used as SPE sorbents permit the selective enrichment of three pivotal purine metabolites (hypoxanthine, xanthine and inosine) in human serum followed by HPLC analysis. Method validation including linearity range, sensitivity, accuracy and reproducibility were evaluated. This method was exemplarily applied in the analysis of serum purines in gout patients and healthy controls. The present results demonstrate a promising potential of this HXLPP-SCX material for the clinical sample pretreatment.

  13. Isolation of Purines and Pyrimidines from the Murchison Meteorite Using Sublimation

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Bada, J. L.

    2004-01-01

    The origin of life on Earth, and possibly on other planets such as Mars, would have required the presence of liquid water and a continuous supply of prebiotic organic compounds. The exogenous delivery of organic matter by asteroids, comets, and carbonaceous meteorites could have contributed to the early Earth s prebiotic inventory by seeding the planet with biologically important organic compounds. A wide variety of prebiotic organic compounds have previously been detected in the Murchison CM type carbonaceous chondrite including amino acids, purines and pyrimidines. These compounds dominate terrestrial biochemistry and are integral components of proteins, DNA and RNA. Several purines including adenine, guanine, hypoxanthine, and xanthine, as well as the pyrimidine uracil, have previously been detected in water or formic acid extracts of Murchison using ion-exclusion chromatography and ultraviolet spectroscopy. However, even after purification of these extracts, the accurate identification and quantification of nucleobases is difficult due to interfering UV absorbing compounds. In order to reduce these effects, we have developed an extraction technique using sublimation to isolate purines and pyrimidines from other non-volatile organic compounds in Murchison acid extracts.

  14. Effects of allopurinol and oxipurinol on purine synthesis in cultured human cells

    PubMed Central

    Kelley, William N.; Wyngaarden, James B.

    1970-01-01

    In the present study we have examined the effects of allopurinol and oxipurinol on the de novo synthesis of purines in cultured human fibroblasts. Allopurinol inhibits de novo purine synthesis in the absence of xanthine oxidase. Inhibition at lower concentrations of the drug requires the presence of hypoxanthine-guanine phosphoribosyltransferase as it does in vivo. Although this suggests that the inhibitory effect of allopurinol at least at the lower concentrations tested is a consequence of its conversion to the ribonucleotide form in human cells, the nucleotide derivative could not be demonstrated. Several possible indirect consequences of such a conversion were also sought. There was no evidence that allopurinol was further utilized in the synthesis of nucleic acids in these cultured human cells and no effect of either allopurinol or oxipurinol on the long-term survival of human cells in vitro could be demonstrated. At higher concentrations, both allopurinol and oxipurinol inhibit the early steps of de novo purine synthesis in the absence of either xanthine oxidase or hypoxanthine-guanine phosphoribosyltransferase. This indicates that at higher drug concentrations, inhibition is occurring by some mechanism other than those previously postulated. PMID:5415686

  15. Chemical modification of the third strand: differential effects on purine and pyrimidine triple helix formation.

    PubMed

    Mills, Martin; Arimondo, Paola B; Lacroix, Laurent; Garestier, Thérèse; Klump, Horst; Mergny, Jean-Louis

    2002-01-08

    DNA triple helices offer exciting perspectives toward oligonucleotide-directed control of gene expression. Oligonucleotide analogues are routinely used with modifications in either the backbone or the bases to form more stable triple-helical structures or to prevent their degradation in cells. In this article, different chemical modifications are tested in a model system, which sets up a competition between the purine and pyrimidine motifs. For most modifications, the DeltaH degrees of purine triplex formation is close to zero, implying a nearly temperature-independent affinity constant. In contrast, the pyrimidine triplex is strongly favored at lower temperatures. The stabilization induced by modifications previously known to be favorable to the pyrimidine motif was quantified. Interestingly, modifications favorable to the GT motif (propynyl-U and dU replacing T) were also discovered. In a system where two third strands compete for triplex formation, replacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This purine-to-pyrimidine triplex conversion depends on the chemical nature of the triplex-forming strands and the stability of the corresponding triplexes.

  16. Purine and pyrimidine metabolism: Convergent evidence on chronic antidepressant treatment response in mice and humans

    PubMed Central

    Park, Dong Ik; Dournes, Carine; Sillaber, Inge; Uhr, Manfred; Asara, John M.; Gassen, Nils C.; Rein, Theo; Ising, Marcus; Webhofer, Christian; Filiou, Michaela D.; Müller, Marianne B.; Turck, Christoph W.

    2016-01-01

    Selective Serotonin Reuptake Inhibitors (SSRIs) are commonly used drugs for the treatment of psychiatric diseases including major depressive disorder (MDD). For unknown reasons a substantial number of patients do not show any improvement during or after SSRI treatment. We treated DBA/2J mice for 28 days with paroxetine and assessed their behavioral response with the forced swim test (FST). Paroxetine-treated long-time floating (PLF) and paroxetine-treated short-time floating (PSF) groups were stratified as proxies for drug non-responder and responder mice, respectively. Proteomics and metabolomics profiles of PLF and PSF groups were acquired for the hippocampus and plasma to identify molecular pathways and biosignatures that stratify paroxetine-treated mouse sub-groups. The critical role of purine and pyrimidine metabolisms for chronic paroxetine treatment response in the mouse was further corroborated by pathway protein expression differences in both mice and patients that underwent chronic antidepressant treatment. The integrated -omics data indicate purine and pyrimidine metabolism pathway activity differences between PLF and PSF mice. Furthermore, the pathway protein levels in peripheral specimens strongly correlated with the antidepressant treatment response in patients. Our results suggest that chronic SSRI treatment differentially affects purine and pyrimidine metabolisms, which may explain the heterogeneous antidepressant treatment response and represents a potential biosignature. PMID:27731396

  17. [Social inequalities in health less pronounced in women than in men: A question of measurements?].

    PubMed

    Cambois, E

    2016-04-01

    Social inequalities in mortality are generally less pronounced for women than for men. Are women's health risks and behaviours more homogeneous, or does this pattern arise from a measurement issue inducing an under-estimation of these inequalities? This article reviews a number of studies covering different dimensions of health and different dimensions of social status. Their findings show that there are large social inequalities in health among women. The focus on the working careers, family histories and conciliation of multiple activities provides evidence of major social determinants of health to which women are widely exposed. This article highlights the need to broaden the notion of social inequality and to redefine the social categories, notably by considering the distinct trajectories of men and women and their different spheres of activity. It highlights that gender differences in health are themselves partly socially constructed, as suggested by the gender approaches in the social sciences.

  18. LMTK3 deficiency causes pronounced locomotor hyperactivity and impairs endocytic trafficking.

    PubMed

    Inoue, Takeshi; Hoshina, Naosuke; Nakazawa, Takanobu; Kiyama, Yuji; Kobayashi, Shizuka; Abe, Takaya; Yamamoto, Toshifumi; Manabe, Toshiya; Yamamoto, Tadashi

    2014-04-23

    LMTK3 belongs to the LMTK family of protein kinases that are predominantly expressed in the brain. Physiological functions of LMTK3 and other members of the LMTK family in the CNS remain unknown. In this study, we performed a battery of behavioral analyses using Lmtk3(-/-) mice and showed that these mice exhibit abnormal behaviors, including pronounced locomotor hyperactivity, reduced anxiety behavior, and decreased depression-like behavior. Concurrently, the dopamine metabolite levels and dopamine turnover rate are increased in the striata of Lmtk3(-/-) mice compared with wild-type controls. In addition, using cultured primary neurons from Lmtk3(-/-) mice, we found that LMTK3 is involved in the endocytic trafficking of N-methyl-d-aspartate receptors, a type of ionotropic glutamate receptor. Altered membrane traffic of the receptor in Lmtk3(-/-) neurons may underlie behavioral abnormalities in the mutant animals. Together, our data suggest that LMTK3 plays an important role in regulating locomotor behavior in mice.

  19. Muscle carnosine loading by beta-alanine supplementation is more pronounced in trained vs. untrained muscles.

    PubMed

    Bex, T; Chung, W; Baguet, A; Stegen, S; Stautemas, J; Achten, E; Derave, W

    2014-01-15

    Carnosine occurs in high concentrations in human skeletal muscle and assists working capacity during high-intensity exercise. Chronic beta-alanine (BA) supplementation has consistently been shown to augment muscle carnosine concentration, but the effect of training on the carnosine loading efficiency is poorly understood. The aim of the present study was to compare muscle carnosine loading between trained and untrained arm and leg muscles. In a first study (n = 17), reliability of carnosine quantification by proton magnetic resonance spectroscopy ((1)H-MRS) was evaluated in deltoid and triceps brachii muscles. In a second study, participants (n = 35; 10 nonathletes, 10 cyclists, 10 swimmers, and 5 kayakers) were supplemented with 6.4 g/day of slow-release BA for 23 days. Carnosine content was evaluated in soleus, gastrocnemius medialis, and deltoid muscles by (1)H-MRS. All the results are reported as arbitrary units. In the nonathletes, BA supplementation increased carnosine content by 47% in the arm and 33% in the leg muscles (not significant). In kayakers, the increase was more pronounced in arm (deltoid) vs. leg (soleus + gastrocnemius) muscles (0.089 vs. 0.049), whereas the reverse pattern was observed in cyclists (0.065 vs. 0.084). Swimmers had significantly higher increase in carnosine in both deltoid (0.107 vs. 0.065) and gastrocnemius muscle (0.082 vs. 0.051) compared with nonathletes. We showed that 1) carnosine content can be reliably measured by (1)H-MRS in deltoid muscle, 2) carnosine loading is equally effective in arm vs. leg muscles of nonathletes, and 3) carnosine loading is more pronounced in trained vs. untrained muscles.

  20. A Source Area Approach Demonstrates Moderate Predictive Ability but Pronounced Variability of Invasive Species Traits.

    PubMed

    Klonner, Günther; Fischer, Stefan; Essl, Franz; Dullinger, Stefan

    2016-01-01

    The search for traits that make alien species invasive has mostly concentrated on comparing successful invaders and different comparison groups with respect to average trait values. By contrast, little attention has been paid to trait variability among invaders. Here, we combine an analysis of trait differences between invasive and non-invasive species with a comparison of multidimensional trait variability within these two species groups. We collected data on biological and distributional traits for 1402 species of the native, non-woody vascular plant flora of Austria. We then compared the subsets of species recorded and not recorded as invasive aliens anywhere in the world, respectively, first, with respect to the sampled traits using univariate and multiple regression models; and, second, with respect to their multidimensional trait diversity by calculating functional richness and dispersion metrics. Attributes related to competitiveness (strategy type, nitrogen indicator value), habitat use (agricultural and ruderal habitats, occurrence under the montane belt), and propagule pressure (frequency) were most closely associated with invasiveness. However, even the best multiple model, including interactions, only explained a moderate fraction of the differences in invasive success. In addition, multidimensional variability in trait space was even larger among invasive than among non-invasive species. This pronounced variability suggests that invasive success has a considerable idiosyncratic component and is probably highly context specific. We conclude that basing risk assessment protocols on species trait profiles will probably face hardly reducible uncertainties.

  1. A Source Area Approach Demonstrates Moderate Predictive Ability but Pronounced Variability of Invasive Species Traits

    PubMed Central

    Essl, Franz; Dullinger, Stefan

    2016-01-01

    The search for traits that make alien species invasive has mostly concentrated on comparing successful invaders and different comparison groups with respect to average trait values. By contrast, little attention has been paid to trait variability among invaders. Here, we combine an analysis of trait differences between invasive and non-invasive species with a comparison of multidimensional trait variability within these two species groups. We collected data on biological and distributional traits for 1402 species of the native, non-woody vascular plant flora of Austria. We then compared the subsets of species recorded and not recorded as invasive aliens anywhere in the world, respectively, first, with respect to the sampled traits using univariate and multiple regression models; and, second, with respect to their multidimensional trait diversity by calculating functional richness and dispersion metrics. Attributes related to competitiveness (strategy type, nitrogen indicator value), habitat use (agricultural and ruderal habitats, occurrence under the montane belt), and propagule pressure (frequency) were most closely associated with invasiveness. However, even the best multiple model, including interactions, only explained a moderate fraction of the differences in invasive success. In addition, multidimensional variability in trait space was even larger among invasive than among non-invasive species. This pronounced variability suggests that invasive success has a considerable idiosyncratic component and is probably highly context specific. We conclude that basing risk assessment protocols on species trait profiles will probably face hardly reducible uncertainties. PMID:27187616

  2. Pronounced subsurface cooling of North Atlantic waters off Northwest Africa during Dansgaard-Oeschger interstadials

    NASA Astrophysics Data System (ADS)

    Kim, Jung-Hyun; Romero, Oscar E.; Lohmann, Gerrit; Donner, Barbara; Laepple, Thomas; Haam, Eddie; Sinninghe Damsté, Jaap S.

    2012-07-01

    Millennial-scale Atlantic meridional overturning circulation (AMOC) variability has often been invoked to explain the Dansgaard-Oeschger (DO) events. However, the underlying causes responsible for millennial-scale AMOC variability are still debated. High-resolution U37K' and TEX86H temperature records for the last 50 kyr obtained from the tropical Northeast (NE) Atlantic (core GeoB7926-2, 20°13'N, 18°27'W, 2500 m water depth) show that distinctive DO-type subsurface (i.e. below the mixed layer: >20 m water depth) temperature oscillations occurred with amplitudes of up to 8 °C in the tropical NE Atlantic during Marine Isotope Stage 3 (MIS3). Statistical analyses reveal a positive relationship between the reconstructed substantial cooling of subsurface waters and prominent surface warming over Greenland during DO interstadials. General circulation model (GCM) simulations without external freshwater forcing, the mechanism often invoked in explaining DO events, demonstrate similar anti-phase correlations between AMOC and pronounced NE Atlantic subsurface temperatures under glacial climate conditions. Together with our paleoproxy dataset, this suggests that the vertical temperature structure and associated changes in AMOC were key elements governing DO events during the last glacial.

  3. Delay of Postnatal Maturation Sensitizes the Mouse Prostate to Testosterone-Induced Pronounced Hyperplasia

    PubMed Central

    Savolainen, Saija; Pakarainen, Tomi; Huhtaniemi, Ilpo; Poutanen, Matti; Mäkelä, Sari

    2007-01-01

    The role of estrogens in the etiology of prostate cancer is controversial. To demonstrate the specific effects of estrogens and androgens on the development of the prostatic epithelial hyperplasia, we used luteinizing hormone receptor knockout mice (LuRKO), which are resistant to pituitary regulation mediated by luteinizing hormone, lack postnatal androgen production, and have rudimentary accessory sex glands, the growth of which can be induced with exogenous androgen replacement. This model is thus ideal for the investigation of direct hormonal effects on the prostate. Testosterone, but not 5α-dihydrotestosterone, replacement from 21 days of life for 8 weeks induced pronounced hyperplasia and inflammation in the prostates of LuRKO mice. Interestingly, 5α-dihydrotestosterone combined with 17β-estradiol did not induce hyperplasia or inflammation, and treatments with inhibitors of estrogen action, aromatase inhibitor, and ICI 182780 further exacerbated testosterone-induced hyperplastic growth. However, the activation of estrogen receptor (ER)-β with a specific agonist, DPN [2,3-bis(4-hydroxyphenol)-propionitrile], prevented the development of prostatic hyperplasia and inflammation in testosterone-treated LuRKO mice. Thus, it seems that in the presence of sufficient androgenic stimulation, it is the balance between ER-α- and ER-β-mediated signaling that determines whether estrogens promote hyperplasia or protect the prostate against hyperplastic changes. PMID:17640960

  4. Pronounced fixation, strong population differentiation and complex population history in the Canary Islands blue tit subspecies complex.

    PubMed

    Hansson, Bengt; Ljungqvist, Marcus; Illera, Juan-Carlos; Kvist, Laura

    2014-01-01

    Evolutionary molecular studies of island radiations may lead to insights in the role of vicariance, founder events, population size and drift in the processes of population differentiation. We evaluate the degree of population genetic differentiation and fixation of the Canary Islands blue tit subspecies complex using microsatellite markers and aim to get insights in the population history using coalescence based methods. The Canary Island populations were strongly genetically differentiated and had reduced diversity with pronounced fixation including many private alleles. In population structure models, the relationship between the central island populations (La Gomera, Tenerife and Gran Canaria) and El Hierro was difficult to disentangle whereas the two European populations showed consistent clustering, the two eastern islands (Fuerteventura and Lanzarote) and Morocco weak clustering, and La Palma a consistent unique lineage. Coalescence based models suggested that the European mainland forms an outgroup to the Afrocanarian population, a split between the western island group (La Palma and El Hierro) and the central island group, and recent splits between the three central islands, and between the two eastern islands and Morocco, respectively. It is clear that strong genetic drift and low level of concurrent gene flow among populations have shaped complex allelic patterns of fixation and skewed frequencies over the archipelago. However, understanding the population history remains challenging; in particular, the pattern of extreme divergence with low genetic diversity and yet unique genetic material in the Canary Island system requires an explanation. A potential scenario is population contractions of a historically large and genetically variable Afrocanarian population, with vicariance and drift following in the wake. The suggestion from sequence-based analyses of a Pleistocene extinction of a substantial part of North Africa and a Pleistocene/Holocene eastward

  5. Structure and function of nucleoside hydrolases from Physcomitrella patens and maize catalyzing the hydrolysis of purine, pyrimidine, and cytokinin ribosides.

    PubMed

    Kopecná, Martina; Blaschke, Hanna; Kopecny, David; Vigouroux, Armelle; Koncitíková, Radka; Novák, Ondrej; Kotland, Ondrej; Strnad, Miroslav; Moréra, Solange; von Schwartzenberg, Klaus

    2013-12-01

    We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.

  6. [High-sensitivity analysis of purines in alcoholic beverages using hydrophilic interaction chromatography coupled with tandem mass spectrometry].

    PubMed

    Kakigi, Yasuhiro; Yoshioka, Toshiaki; Nagatomi, Yasushi; Uyama, Atsuo; Mochizuki, Naoki

    2014-01-01

    In this study, we established a high-sensitivity analytical method for purines in alcoholic beverages using hydrophilic interaction chromatography coupled with tandem mass spectrometry. The alcoholic beverages were hydrolyzed with perchloric acid (60%) and subjected to strong cation exchange solid-phase extraction (Bond Elut SCX). The four purine bases (hypoxanthine, adenine, xanthine, guanine) in the extracted solution were separated by hydrophilic interaction chromatography with TSKgel Amide-80 as a separation column, 10 mM ammonium formate (pH 2.0) as mobile phase A, and acetonitrile/100 mM ammonium formate (pH 2.0) (90/10) as mobile phase B. The detection of purine bases was performed by tandem mass spectrometry with ESI. The linearity of this analytical method was not less than 0.996, and the repeatability was not more than 8.4% for each purine base. The recovery was in the range of 60-105%, and the detection limit was not more than 0.005 mg/100 mL. This established method is expected to be useful for quality control and surveillance of purines in alcoholic beverages.

  7. 1,3,5-Triazine-based analogues of purine: from isosteres to privileged scaffolds in medicinal chemistry.

    PubMed

    Lim, Felicia Phei Lin; Dolzhenko, Anton V

    2014-10-06

    Purines can be considered as the most ubiquitous and functional N-heterocyclic compounds in nature. Structural modifications of natural purines, particularly using isosteric ring systems, have been in the focus of many drug discovery programs. Fusion of 1,3,5-triazine ring with pyrrole, pyrazole, imidazole, 1,2,3-triazole or 1,2,4-triazole results in seven bicyclic heterocyclic systems isosteric to purine. Application of the isosterism concept for the development of new compounds with therapeutic potential in areas involving purinergic regulation or purine metabolism led to significant advances in medicinal chemistry of the azolo[1,3,5]triazines. These 1,3,5-triazine-based purine-like scaffolds significantly increase level of molecular diversity and allow covering chemical space in the important areas of medicinal chemistry. Some of these azolo[1,3,5]triazine systems have become privileged scaffolds in the development of inhibitors of various kinases, phosphodiesterase, xanthine oxidase, and thymidine phosphorylase, antagonists of adenosine and corticotropin-releasing hormone receptors, anticancer and antiviral agents.

  8. GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway

    PubMed Central

    Li, Qiong; Leija, Christopher; Rijo-Ferreira, Filipa; Chen, Jun; Cestari, Igor; Stuart, Kenneth; Tu, Benjamin P.; Phillips, Margaret A.

    2015-01-01

    Summary The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine-5′-monophosphate (GMP) formation: conversion from xanthosine-5′-monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of HAT. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche. PMID:26043892

  9. On the origin of pronounced O3 gradients in the thunderstorm outflow region during DC3

    NASA Astrophysics Data System (ADS)

    Huntrieser, H.; Lichtenstern, M.; Scheibe, M.; Aufmhoff, H.; Schlager, H.; Pucik, T.; Minikin, A.; Weinzierl, B.; Heimerl, K.; Fütterer, D.; Rappenglück, B.; Ackermann, L.; Pickering, K. E.; Cummings, K. A.; Biggerstaff, M. I.; Betten, D. P.; Honomichl, S.; Barth, M. C.

    2016-06-01

    Unique in situ measurements of CO, O3, SO2, CH4, NO, NOx, NOy, VOC, CN, and rBC were carried out with the German Deutsches Zentrum für Luft- und Raumfahrt (DLR)-Falcon aircraft in the central U.S. thunderstorms during the Deep Convective Clouds and Chemistry experiment in summer 2012. Fresh and aged anvil outflow (9-12 km) from supercells, mesoscale convective systems, mesoscale convective complexes, and squall lines were probed over Oklahoma, Texas, Colorado, and Kansas. For three case studies (30 May and 8 and 12 June) a combination of trace species, radar, lightning, and satellite information, as well as model results, were used to analyze and design schematics of major trace gas transport pathways within and in the vicinity of the probed thunderstorms. The impact of thunderstorms on the O3 composition in the upper troposphere/lower stratosphere (LS) region was analyzed. Overshooting cloud tops injected high amounts of biomass burning and lightning-produced NOx emissions into the LS, in addition to low O3 mixing ratios from the lower troposphere. As a dynamical response, O3-rich air from the LS was transported downward into the anvil and also surrounded the outflow. The ΔO3/ΔCO ratio was determined in the anvil outflow region. A pronounced in-mixing of O3-rich stratospheric air masses was observed in the outflow indicated by highly positive or even negative ΔO3/ΔCO ratios (+1.4 down to -3.9). Photochemical O3 production (ΔO3/ΔCO = +0.1) was found to be minor in the recently lofted pollution plumes. O3 mixing ratios within the aged anvil outflow were mainly enhanced due to dynamical processes.

  10. Effects of riparian plant diversity loss on aquatic microbial decomposers become more pronounced with increasing time.

    PubMed

    Fernandes, Isabel; Duarte, Sofia; Cássio, Fernanda; Pascoal, Cláudia

    2013-11-01

    We examined the potential long-term impacts of riparian plant diversity loss on diversity and activity of aquatic microbial decomposers. Microbial assemblages were obtained in a mixed-forest stream by immersion of mesh bags containing three leaf species (alder, oak and eucalyptus), commonly found in riparian corridors of Iberian streams. Simulation of species loss was done in microcosms by including a set of all leaf species, retrieved from the stream, and non-colonized leaves of three, two or one leaf species. Leaves were renewed every month throughout six months, and microbial inoculum was ensured by a set of colonized leaves from the previous month. Microbial diversity, leaf mass loss and fungal biomass were assessed at the second and sixth months after plant species loss. Molecular diversity of fungi and bacteria, as the total number of operational taxonomic units per leaf diversity treatment, decreased with leaf diversity loss. Fungal biomass tended to decrease linearly with leaf species loss on oak and eucalyptus, suggesting more pronounced effects of leaf diversity on lower quality leaves. Decomposition of alder and eucalyptus leaves was affected by leaf species identity, mainly after longer times following diversity loss. Leaf decomposition of alder decreased when mixed with eucalyptus, while decomposition of eucalyptus decreased in mixtures with oak. Results suggest that the effects of leaf diversity on microbial decomposers depended on leaf species number and also on which species were lost from the system, especially after longer times. This may have implications for the management of riparian forests to maintain stream ecosystem functioning.

  11. Pronounced zonal heterogeneity in Eocene southern high-latitude sea surface temperatures

    PubMed Central

    Douglas, Peter M. J.; Affek, Hagit P.; Ivany, Linda C.; Houben, Alexander J. P.; Sijp, Willem P.; Sluijs, Appy; Schouten, Stefan; Pagani, Mark

    2014-01-01

    Paleoclimate studies suggest that increased global warmth during the Eocene epoch was greatly amplified at high latitudes, a state that climate models cannot fully reproduce. However, proxy estimates of Eocene near-Antarctic sea surface temperatures (SSTs) have produced widely divergent results at similar latitudes, with SSTs above 20 °C in the southwest Pacific contrasting with SSTs between 5 and 15 °C in the South Atlantic. Validation of this zonal temperature difference has been impeded by uncertainties inherent to the individual paleotemperature proxies applied at these sites. Here, we present multiproxy data from Seymour Island, near the Antarctic Peninsula, that provides well-constrained evidence for annual SSTs of 10–17 °C (1σ SD) during the middle and late Eocene. Comparison of the same paleotemperature proxy at Seymour Island and at the East Tasman Plateau indicate the presence of a large and consistent middle-to-late Eocene SST gradient of ∼7 °C between these two sites located at similar paleolatitudes. Intermediate-complexity climate model simulations suggest that enhanced oceanic heat transport in the South Pacific, driven by deep-water formation in the Ross Sea, was largely responsible for the observed SST gradient. These results indicate that very warm SSTs, in excess of 18 °C, did not extend uniformly across the Eocene southern high latitudes, and suggest that thermohaline circulation may partially control the distribution of high-latitude ocean temperatures in greenhouse climates. The pronounced zonal SST heterogeneity evident in the Eocene cautions against inferring past meridional temperature gradients using spatially limited data within given latitudinal bands. PMID:24753570

  12. Pronounced zonal heterogeneity in Eocene southern high-latitude sea surface temperatures.

    PubMed

    Douglas, Peter M J; Affek, Hagit P; Ivany, Linda C; Houben, Alexander J P; Sijp, Willem P; Sluijs, Appy; Schouten, Stefan; Pagani, Mark

    2014-05-06

    Paleoclimate studies suggest that increased global warmth during the Eocene epoch was greatly amplified at high latitudes, a state that climate models cannot fully reproduce. However, proxy estimates of Eocene near-Antarctic sea surface temperatures (SSTs) have produced widely divergent results at similar latitudes, with SSTs above 20 °C in the southwest Pacific contrasting with SSTs between 5 and 15 °C in the South Atlantic. Validation of this zonal temperature difference has been impeded by uncertainties inherent to the individual paleotemperature proxies applied at these sites. Here, we present multiproxy data from Seymour Island, near the Antarctic Peninsula, that provides well-constrained evidence for annual SSTs of 10-17 °C (1σ SD) during the middle and late Eocene. Comparison of the same paleotemperature proxy at Seymour Island and at the East Tasman Plateau indicate the presence of a large and consistent middle-to-late Eocene SST gradient of ∼7 °C between these two sites located at similar paleolatitudes. Intermediate-complexity climate model simulations suggest that enhanced oceanic heat transport in the South Pacific, driven by deep-water formation in the Ross Sea, was largely responsible for the observed SST gradient. These results indicate that very warm SSTs, in excess of 18 °C, did not extend uniformly across the Eocene southern high latitudes, and suggest that thermohaline circulation may partially control the distribution of high-latitude ocean temperatures in greenhouse climates. The pronounced zonal SST heterogeneity evident in the Eocene cautions against inferring past meridional temperature gradients using spatially limited data within given latitudinal bands.

  13. Pronounced and prevalent intersexuality does not impede the ‘Demon Shrimp’ invasion

    PubMed Central

    Green Etxabe, Amaia; Short, Stephen; Flood, Tim; Johns, Tim

    2015-01-01

    Crustacean intersexuality is widespread and often linked to infection by sex-distorting parasites. However, unlike vertebrate intersexuality, its association with sexual dysfunction is unclear and remains a matter of debate. The ‘Demon Shrimp,’ Dikerogammarus haemobaphes, an amphipod that has invaded continental waterways, has recently become widespread in Britain. Intersexuality has been noted in D. haemobaphes but not investigated further. We hypothesise that a successful invasive population should not display a high prevalence of intersexuality if this condition represents a truly dysfunctional phenotype. In addition, experiments have indicated that particular parasite burdens in amphipods may facilitate invasions. The rapid and ongoing invasion of British waterways represents an opportunity to determine whether these hypotheses are consistent with field observations. This study investigates the parasites and sexual phenotypes of D. haemobaphes in British waterways, characterising parasite burdens using molecular screening, and makes comparisons with the threatened Gammarus pulex natives. We reveal that invasive and native populations have distinct parasitic profiles, suggesting the loss of G. pulex may have parasite-mediated eco-system impacts. Furthermore, the parasite burdens are consistent with those previously proposed to facilitate biological invasions. Our study also indicates that while no intersexuality occurs in the native G. pulex, approximately 50% of D. haemobaphes males present pronounced intersexuality associated with infection by the microsporidian Dictyocoela berillonum. This unambiguously successful invasive population presents, to our knowledge, the highest reported prevalence of male intersexuality. This is the clearest evidence to date that such intersexuality does not represent a form of debilitating sexual dysfunction that negatively impacts amphipod populations. PMID:25699206

  14. [open quotes]Cryptic[close quotes] repeating triplets of purines and pyrimidines (cRRY(i)) are frequent and polymorphic: Analysis of coding cRRY(i) in the proopiomelanocortin (POMC) and TATA-binding protein (TBP) genes

    SciTech Connect

    Gostout, B.; Qiang Liu; Sommer, S.S. )

    1993-06-01

    Triplets of the form of purine, purine, pyrimidine (RRY(i)) are enhanced in frequency in the genomes of primates, rodents, and bacteria. Some RRY(i) are [open quotes]cryptic[close quotes] repeats (cRRY(i)) in which no one tandem run of a trinucleotide predominates. A search of human GenBank sequence revealed that the sequences of cRRY(i) are highly nonrandom. Three randomly chosen human cRRY(i) were sequenced in search of polymorphic alleles. Multiple polymorphic alleles were found in cRRY(i) in the coding regions of the genes for proopiomelanocortin (POMC) and TATA-binding protein (TBP). The highly polymorphic TBP cRRY(i) was characterized in detail. Direct sequencing of 157 unrelated human alleles demonstrated the presence of 20 different alleles which resulted in 29--40 consecutive glutamines in the amino-terminal region of TBP. These alleles are differently distributed among the races. PCR was used to screen 1,846 additional alleles in order to characterize more fully the range of variation in the population. Three additional alleles were discovered, but there was no example of a substantial sequence amplification as is seen in the repeat sequences associated with X-linked spinal and bulbar muscular atrophy, myotonic dystrophy, or the fragile-X syndrome. The structure of the TBP cRRY(i) is conserved in the five monkey species examined. In the chimpanzee, examination of four individuals revealed that the cRRY(i) was highly polymorphic, but the pattern of polymorphism differed from that in humans. The TBP cRRY(i) displays both similarities with and differences from the previously described RRY(i) in the coding sequence of the androgen receptor. The data suggest how simple tandem repeats could evolve from cryptic repeats. 18 refs., 3 figs., 6 tabs.

  15. Urinary excretion of purine derivatives, microbial protein synthesis, nitrogen use, and ruminal fermentation in sheep and goats fed diets of different quality.

    PubMed

    Carro, M D; Cantalapiedra-Hijar, G; Ranilla, M J; Molina-Alcaide, E

    2012-11-01

    The objective of this study was to compare N balance, microbial N flow (MNF) estimated from purine derivatives (PD) urinary excretion, and its variation when estimated using purine bases:N ratios in liquid associated bacteria (LAB) from models reported in the literature (MNF - response models) or measured ratios in liquid and solid-associated bacterial (SAB) pellets (MNF-LAB+SAB), diet digestibility, and rumen fermentation variables in sheep and goats fed 3 different practical, quality diets to study interspecies differences concerning N use as accurately as possible. Four mature female Merino sheep and 4 mature female Granadina goats, each fitted with a ruminal cannula, were used in 3 × 3 Latin square design with an extra animal. Two experimental diets had a forage-to-concentrate ratio of 70:30 (DM basis) with alfalfa hay (ALC) or grass hay (GRC) as forage, and the third diet contained 70% concentrate and 30% alfalfa hay (CAL). All animals were fed the diets at a daily rate of 56 g/kg BW(0.75) to minimize feed selection. Digestibility of nutrients was similar (P = 0.16 to 0.88) in the 2 species, but some animal species × diet interactions (P = 0.01 to 0.04) were detected. There were small differences between the fermentation patterns of both animal species. Goats showed decreased VFA concentrations (P = 0.005) and butyrate proportions (P = 0.04), and greater acetate proportions (P = 0.02) compared with sheep, whereas N intake and percentage of N intake excreted in feces were similar in both species (P = 0.58 and 0.15, respectively), the percentage excreted via the urine was greater in goats compared with sheep (P < 0.001). As a consequence, sheep had greater (P < 0.001) N retention than goats (averaged across diets, 32.6% and 16.1% of N intake, respectively). There were no differences (P = 0.95) between animal species in total PD excretion, but goats showed a greater excretion of allantoin (P = 0.01) and decreased excretion of xanthine (P = 0.008) and

  16. ATP and related purines stimulate motility, spatial congregation, and coalescence in red algal spores.

    PubMed

    Huidobro-Toro, Juan P; Donoso, Verónica; Flores, Verónica; Santelices, Bernabé

    2015-04-01

    Adenosine 5'-triphosphate (ATP) is a versatile extracellular signal along the tree of life, whereas cAMP plays a major role in vertebrates as an intracellular messenger for hormones, transmitters, tastants, and odorants. Since red algal spore coalescence may be considered analogous to the congregation process of social amoeba, which is stimulated by cAMP, we ascertained whether exogenous applications of ATP, cAMP, adenine, or adenosine modified spore survival and motility, spore settlement and coalescence. Concentration-response studies were performed with carpospores of Mazzaella laminarioides (Gigartinales), incubated with and without added purines. Stirring of algal blades released ADP/ATP to the cell media in a time-dependent manner. 10-300 μM ATP significantly increased spore survival; however, 1,500 μM ATP, cAMP or adenine induced 100% mortality within less than 24 h; the exception was adenosine, which up to 3,000 μM, did not alter spore survival. ATP exposure elicited spore movement with speeds of 2.2-2.5 μm · s(-1) . 14 d after 1,000 μM ATP addition, spore abundance in the central zone of the plaques was increased 2.7-fold as compared with parallel controls. Likewise, 1-10 μM cAMP or 30-100 μM adenine also increased central zone spore abundance, albeit these purines were less efficacious than ATP; adenosine up to 3,000 μM did not influence settlement. Moreover, 1,000 μM ATP markedly accelerated coalescence, the other purines caused a variable effect. We conclude that exogenous cAMP, adenine, but particularly ATP, markedly influence red algal spore physiology; effects are compatible with the expression of one or more membrane purinoceptor(s), discarding adenosine receptor participation.

  17. Enhancement of Peripheral Nerve Regrowth by the Purine Nucleoside Analog and Cell Cycle Inhibitor, Roscovitine

    PubMed Central

    Law, Vincent; Dong, Sophie; Rosales, Jesusa L.; Jeong, Myung-Yung; Zochodne, Douglas; Lee, Ki-Young

    2016-01-01

    Peripheral nerve regeneration is a slow process that can be associated with limited outcomes and thus a search for novel and effective therapy for peripheral nerve injury and disease is crucial. Here, we found that roscovitine, a synthetic purine nucleoside analog, enhances neurite outgrowth in neuronal-like PC12 cells. Furthermore, ex vivo analysis of pre-injured adult rat dorsal root ganglion (DRG) neurons showed that roscovitine enhances neurite regrowth in these cells. Likewise, in vivo transected sciatic nerves in rats locally perfused with roscovitine had augmented repopulation of new myelinated axons beyond the transection zone. By mass spectrometry, we found that roscovitine interacts with tubulin and actin. It interacts directly with tubulin and causes a dose-dependent induction of tubulin polymerization as well as enhances Guanosine-5′-triphosphate (GTP)-dependent tubulin polymerization. Conversely, roscovitine interacts indirectly with actin and counteracts the inhibitory effect of cyclin-dependent kinases 5 (Cdk5) on Actin-Related Proteins 2/3 (Arp2/3)-dependent actin polymerization, and thus, causes actin polymerization. Moreover, in the presence of neurotrophic factors such as nerve growth factor (NGF), roscovitine-enhanced neurite outgrowth is mediated by increased activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways. Since microtubule and F-actin dynamics are critical for axonal regrowth, the ability of roscovitine to activate the ERK1/2 and p38 MAPK pathways and support polymerization of tubulin and actin indicate a major role for this purine nucleoside analog in the promotion of axonal regeneration. Together, our findings demonstrate a therapeutic potential for the purine nucleoside analog, roscovitine, in peripheral nerve injury. PMID:27799897

  18. Measurement of small scalar and dipolar couplings in purine and pyrimidine bases.

    PubMed

    Zídek, L; Wu, H; Feigon, J; Sklenár, V

    2001-10-01

    A suite of spin-state-selective excitation (S3E) NMR experiments for the measurements of small one-bond (13C-13C, 15N-13C) and two-bond (1H-13C, 1H-15N) coupling constants in 13C,15N labeled purine and pyrimidine bases is presented. The incorporation of band-selective shaped pulses, elimination of the cross talk between alpha and beta sub-spectra, and accuracy and precision of the proposed approach are discussed. Merits of using S3E rather than alpha/beta-half-filter are demonstrated using results obtained on isotopically labeled DNA oligonucleotides.

  19. HCN - A plausible source of purines, pyrimidines and amino acids on the primitive earth

    NASA Technical Reports Server (NTRS)

    Ferris, J.-P.; Joshi, P. C.; Edelson, E. H.; Lawless, J. G.

    1978-01-01

    Dilute (0.1 M) solutions of HCN condense to oligomers at pH 9.2, and hydrolysis of these oligomers yields 4,5-dihydroxypyrimidine, orotic acid, 5-hydroxyuracil, adenine, 4-aminoimidazole-5-carboxamide, and amino acids. It is suggested that the three main classes of nitrogen-containing biomolecules - purines, pyrimidines, and amino acids may have originated from HCN on the primitive earth. It is also suggested that the presence of orotic acid and 4-aminoimidazole-5-carboxamide might indicate that contemporary biosynthetic pathways for nucleotides evolved from the compounds released on hydrolysis of HCN oligomers.

  20. Validation and steady-state analysis of a power-law model of purine metabolism in man.

    PubMed Central

    Curto, R; Voit, E O; Sorribas, A; Cascante, M

    1997-01-01

    The paper introduces a model of human purine metabolism in situ. Chosen from among several alternative system descriptions, the model is formulated as a Generalized Mass Action system within Biochemical Systems Theory and validated with analyses of steady-state and dynamic characteristics. Eigenvalue and sensitivity analyses indicate that the model has a stable and robust steady-state. The model quite accurately reproduces numerous biochemical and clinical observations in healthy subjects as well as in patients with disorders of purine metabolism. These results suggest that the model can be used to assess biochemical and clinical aspects of human purine metabolism. It provides a means of exploring effects of enzyme deficiencies and is a potential tool for identifying steps of the pathway that could be the target of therapeutical intervention. Numerous quantitative comparisons with data are given. The model can be used for biomathematical exploration of relationships between enzymic deficiencies and clinically manifested diseases. PMID:9210399

  1. Pronounced microheterogeneity in a sorbitol-water mixture observed through variable temperature neutron scattering.

    PubMed

    Chou, Shin G; Soper, Alan K; Khodadadi, Sheila; Curtis, Joseph E; Krueger, Susan; Cicerone, Marcus T; Fitch, Andrew N; Shalaev, Evgenyi Y

    2012-04-19

    In this study, the structure of concentrated d-sorbitol-water mixtures is studied by wide- and small-angle neutron scattering (WANS and SANS) as a function of temperature. The mixtures are prepared using both deuterated and regular sorbitol and water at a molar fraction of sorbitol of 0.19 (equivalent to 70% by weight of regular sorbitol in water). Retention of an amorphous structure (i.e., absence of crystallinity) is confirmed for this system over the entire temperature range, 100-298 K. The glass transition temperature, Tg, is found from differential scanning calorimetry to be approximately 200 K. WANS data are analyzed using empirical potential structure refinement, to obtain the site-site radial distribution functions (RDFs) and coordination numbers. This analysis reveals the presence of nanoscaled water clusters surrounded by (and interacting with) sorbitol molecules. The water clusters appear more structured compared to bulk water and, especially at the lowest temperatures, resemble the structure of low-density amorphous ice (LDA). Upon cooling to 100 K the peaks in the water RDFs become markedly sharper, with increased coordination number, indicating enhanced local (nanometer-scale) ordering, with changes taking place both above and well below the Tg. On the mesoscopic (submicrometer) scale, although there are no changes between 298 and 213 K, cooling the sample to 100 K results in a significant increase in the SANS signal, which is indicative of pronounced inhomogeneities. This increase in the scattering is partly reversed during heating, although some hysteresis is observed. Furthermore, a power law analysis of the SANS data indicates the existence of domains with well-defined interfaces on the submicrometer length scale, probably as a result of the appearance and growth of microscopic voids in the glassy matrix. Because of the unusual combination of small and wide scattering data used here, the present results provide new physical insight into the

  2. Fertility and Pregnancy Outcome after Myoma Enucleation by Minilaparotomy under Microsurgical Conditions in Pronounced Uterus Myomatosus.

    PubMed

    Floss, K; Garcia-Rocha, G-J; Kundu, S; von Kaisenberg, C S; Hillemanns, P; Schippert, C

    2015-01-01

    Introduction: Besides the typical complaints and symptoms, myomas can cause sterility, infertility and complications during pregnancy. Laparoscopic interventions reach their limits with regard to organ preservation and the simultaneous desire to have children in the removal of multiple and larger intramural myoma nodes. The aim of this study is to examine fertility status and pregnancy outcome after myoma removal by minilaparotomy (skin incision maximal 8 cm) in women with pronounced uterus myomatosus. Materials and Methods: This retrospective study makes use of the data from 160 patients with an average age of 34.6 years. Factors analysed include number, size and localisation of the myomas, complaints due to the myoma, pre- and postoperative gravidity, mode of delivery, and complications of birth. Results: Indications for organ-sparing myoma enucleation were the desire to have children (72.5 %), bleeding disorders (60 %) and pressure discomfort (36.5 %). On average 4.95 (SD ± 0.41), maximally 46 myomas were removed. The largest myoma had a diameter of 6.64 cm (SD ± 2.74). 82.5 % of the patients had transmural myomas, in 17.5 % the uterine cavity was inadvertently opened. On average the operating time was 163 minutes (SD ± 45.47), the blood loss 1.59 g/dL (SD ± 0.955). 60.3 % of the patients with the desire to have children became pregnant postoperatively. 75.3 % of the pregnancies were on average carried through to the 38th week (28.4 % vaginal deliveries, 71.6 % Caesarean sections). In the postoperative period there was one case of uterine rupture in the vicinity of a previous scar. Discussion: By means of the microsurgical "mini-laparotomy" even extensive myomatous uterine changes can, in the majority of cases, be operated in an organ-sparing manner with retention of the ability to conceive and to carry a pregnancy through to maturity of the infant. The risk for a postoperative uterine rupture in a subsequent pregnancy and

  3. Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer

    PubMed Central

    Barfeld, Stefan J.; Fazli, Ladan; Persson, Margareta; Marjavaara, Lisette; Urbanucci, Alfonso; Kaukoniemi, Kirsi M.; Rennie, Paul S.; Ceder, Yvonne; Chabes, Andrei; Visakorpi, Tapio; Mills, Ian G.

    2015-01-01

    The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses. PMID:25869206

  4. An Investigation of Prebiotic Purine Synthesis from the Hydrolysis of HCN Polymers

    NASA Astrophysics Data System (ADS)

    Borquez, Eduardo; Cleaves, H. James; Lazcano, Antonio; Miller, Stanley L.

    2005-04-01

    The polymerization of concentrated NH4CN solutions has been studied at various temperatures and ammonia concentrations. The products of the oligomerization of ammonium cyanide include adenine and guanine, as well as trace amounts of 2,6-diaminopurine. Our results indicate that the adenine yield is not strongly dependent on temperature. Guanine is produced in lower yield. The original studies by Oró and Kimball (1961) showed that the 6 N HCl hydrolysis of the NH4CN polymerization supernatant greatly increased the adenine yield. However, this hydrolysis also decomposes adenine and other purines. Therefore, we have measured the yields from an NH4CN polymerization as a function of hydrolysis time, and found that shorter hydrolytic periods give higher yields of adenine.We have also investigated the hydrolysis of the supernatant at pH 8, which is a more reasonable model of primitive oceanic conditions, and found that the adenine yield is comparable to that obtained with acid hydrolysis (approximately 0.1%). The yield of adenine does not decline at longer hydrolysis times because of the greater stability of adenine at pH 8. The insoluble black polymer formed from NH4CN has been analyzed by both acid and neutral hydrolysis. In both cases adenine yields of approximately 0.05% were obtained. This suggests that the polymer may have been as important a prebiotic source of purines as the usually analyzed supernatant.

  5. Sustainable synthesis and automated deposition: an accessible discovery screening library of fragment-like purines.

    PubMed

    Kamper, Christoph; Korpis, Katharina; Specker, Edgar; Anger, Lennart; Neuenschwander, Martin; Bednarski, Patrick J; Link, Andreas

    2012-08-01

    A sub-library of 88 information-rich lead-like purine derivatives were prepared and deposited in an open access academic screening facility. The rationale for the synthesis of these rigid low complexity structures was the privileged character of the purine heterocycle associated with its inherent probability of interactions with multiple adenine-related targets. Although generally expected to be weak binders in many assays, such fragment-like compounds are estimated to match diverse binding sites. It is suggested that heterocycles with many anchor points for hydrogen bonds can be anticipated to undergo very specific interactions to produce more negative enthalpies and thus provide superior starting points for lead optimization than compounds that owe their activity to entropic effects. The in vitro cytotoxicity of the small compounds on a panel of human cancer cell lines has been investigated and some of them showed marked unselective or selective toxicity. This data may be useful if these fragments are to be incorporated into drug-like structures via metabolically cleavable connections. The sub-library will be implemented as part of the ChemBioNet ( www.chembionet.info ) library, and it is open to screening campaigns of academic research groups striving for a fragment-based approach in their biological assays.

  6. Purine-benzimidazole hybrids: synthesis, single crystal determination and in vitro evaluation of antitumor activities.

    PubMed

    Sharma, Alka; Luxami, Vijay; Paul, Kamaldeep

    2015-03-26

    In an effort to identify novel compounds for the treatment of cancer, a diverse array of potential bioactive hybrid, purine-benzimidazole was synthesized in good yields through nucleophilic substitution at C6 position of purine ring with versatile cyclic amines at C2 position. The structures of newly prepared compounds were confirmed by IR, (1)H, (13)C NMR, mass spectroscopy and, in case of 19, by single crystal X-ray diffraction analysis. The newly synthesized compounds were evaluated against 60 human tumour cell lines at one dose concentration level. Compound 6 exhibited significant growth inhibition and was evaluated as 60 cell panel at five dose concentration levels. Compound 6 proved to be 1.25 fold more active than the positive control 5-FU, with GI50 value of 18.12 μM (MG-MID). Interaction of the compounds with Aurora-A enzyme involved in the process of propagation of cancer, has also been investigated. Compound 6 showed selectivity towards Aurora-A kinase inhibition with IC50 value of 0.0l μM. Molecular docking studies in the active binding site provided theoretical support for the experimental biological data acquired.

  7. Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin

    PubMed Central

    Yee, Rebecca; Cui, Peng; Shi, Wanliang; Feng, Jie; Zhang, Ying

    2015-01-01

    Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus. PMID:27025643

  8. Single tryptophan of disordered loop from Plasmodium falciparum purine nucleoside phosphorylase: involvement in catalysis and microenvironment.

    PubMed

    Suthar, Manish Kumar; Verma, Anita; Doharey, Pawan Kumar; Singh, Shiv Vardan; Saxena, Jitendra Kumar

    2013-06-01

    Among various tropical diseases, malaria is a major life-threatening disease caused by Plasmodium parasite. Plasmodium falciparum is responsible for the deadliest form of malaria, so-called cerebral malaria. Purine nucleoside phosphorylase from P. falciparum is a homohexamer containing single tryptophan residue per subunit that accepts inosine and guanosine but not adenosine for its activity. This enzyme has been exploited as drug target against malaria disease. It is important to draw together significant knowledge about inherent properties of this enzyme which will be helpful in better understanding of this drug target. The enzyme shows disorder to order transition during catalysis. The single tryptophan residue residing in conserved region of transition loop is present in purine nucleoside phosphorylases throughout the Plasmodium genus. This active site loop motif is conserved among nucleoside phosphorylases from apicomplexan parasites. Modification of tryptophan residue by N-bromosuccinamide resulted in complete loss of activity showing its importance in catalysis. Inosine was not able to protect enzyme against N-bromosuccinamide modification. Extrinsic fluorescence studies revealed that tryptophan might not be involved in substrate binding. The tryptophan residue localised in electronegative environment showed collisional and static quenching in the presence of quenchers of different polarities.

  9. Purine 5‧,8-cyclo-2‧-deoxynucleoside lesions in irradiated DNA

    NASA Astrophysics Data System (ADS)

    Chatgilialoglu, Chryssostomos; Krokidis, Marios G.; Papadopoulos, Kyriakos; Terzidis, Michael A.

    2016-11-01

    Having their position gained among the smallest bulky DNA lesions recognized by the nucleotide excision repair (NER) enzyme, purine 5‧,8-cyclo-2‧-deoxynucleosides (5‧,8-cPu) are increasingly attracting the interest in the field of genome integrity in health and diseases. Exclusively generated by one of the most harmful of the reactive oxygen species, the hydroxyl radical, 5‧,8-cPu can be utilized also for highly valuable information regarding the oxidative status nearby the area where the genetic information is stored. Herein, we have collected the most recently reported biological studies, focusing on the repair mechanism of these lesions and their biological significance particularly in transcription. The LC-MS/MS quantification protocols that appeared in the literature are discussed in details, along with the reported values for the four 5‧,8-cPu produced by in vitro γ-radiolysis experiments with calf thymus DNA. Mechanistic insights in the formation of the purine 5‧,8-cyclo-2‧-deoxynucleosides and their chemical stability are also given in the light of their potential to be utilized as DNA biomarkers of oxidative stress.

  10. New bis-N9-(methylphenylmethyl)purine derivatives: synthesis and antitumor activity.

    PubMed

    Kode, Nageswara; Chen, Liying; Murthy, Devangachinta; Adewumi, Dare; Phadtare, Shashikant

    2007-03-01

    A series of ortho-, meta- and para-bis-N9-(methylphenylmethyl)purine derivatives 4-15 were obtained by two-step synthesis from various substituted chloropurines with alpha,alpha'-dichloroxylenes. These bis-N9-(methylphenylmethyl)purines 4-15 were evaluated for the primary cytotoxic activity against a panel of NCI-H460 (lung), MCF-7 (breast) and SF-268 (CNS) cancer cell lines. The 'active' compounds which reduced growth of cancer cells to ca. 32% or less, have been evaluated in a full panel of 60 human cancer cell lines over a 5-log dose range at the National Cancer Institute, Bethesda, MD. In this series, the most activity is correlated to the compounds derived from the 2,6-dichloropurines such as bis-9-[o-(methylphenylmethyl)]2,6-dichloropurine (5), bis-9-[m-(methylphenylmethyl)]2,6-dichloropurine (8), and bis-9-[p-(methylphenylmethyl)]2,6-dichloropurine (11). In particular compound 8 exhibited high sensitivity in leukemia cell lines and compounds 5, 8 and 11 exhibited consistent high sensitivity in many breast cancer cell lines. Compound 11 was the most potent in this series and exhibited GI(50)<0.01 microM sensitivity against non-small lung cancer EKVX, colon cancer HT-29, melanoma SK-MEL-28, renal cancer RXF 393, prostate cancer DU-145 and several breast cancer HS 578T and BT-549 cell lines.

  11. An Ancient Riboswitch Class in Bacteria Regulates Purine Biosynthesis and One-carbon Metabolism

    PubMed Central

    Kim, Peter B.; Nelson, James W.; Breaker, Ronald R.

    2015-01-01

    SUMMARY Over thirty years ago, ZTP (5-amino-4-imidazole carboxamide riboside 5'-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one carbon metabolism. Biochemical data confirms that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity, while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches in vivo. The wide distribution of this riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages. PMID:25616067

  12. Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography.

    PubMed

    Marrubini, Giorgio; Mendoza, Bolivar Enrique Castillo; Massolini, Gabriella

    2010-03-01

    The separation of 12 model compounds chosen among purine and pyrimidine bases and nucleosides was studied by using hydrophilic interaction chromatography (HILIC). The compounds investigated were small molecules with relevant properties for biomedical and pharmaceutical studies. The mixture of pyrimidines and purines was applied on a ZIC-HILIC 150 x 2.1 mm, 5 microm, and two TSKgel Amide-80 150 x 2.0 mm, 5 microm and 3 microm particle size columns. The retention of the analytes was studied by varying ACN%, ammonium formate concentration, pH, and column temperature. The results obtained confirmed the elution order of nucleobases, nucleosides, and nucleotides based on their hydrophobicity. The retention mechanism of the columns was studied considering the models used for describing partitioning and surface adsorption. The influence on retention of chromatographic conditions (ACN%, salt concentration, pH, and temperature) was described and discussed for both columns. The optimization of the conditions studied allowed to assess a gradient method for the separation of the 12 analytes. The developed method is a valuable alternative to existing methods for the separation of the compounds concerned.

  13. An expanding role for purine uptake permease-like transporters in plant secondary metabolism

    PubMed Central

    Jelesko, John G.

    2012-01-01

    For the past decade, our understanding of the plant purine uptake permease (PUP) transporter family was primarily oriented on purine nucleobase substrates and their tissue-specific expression patterns in Arabidopsis. However, a tobacco PUP-like homolog demonstrating nicotine uptake permease activity was recently shown to affect both nicotine metabolism and root cell growth. These new findings expand the physiological role for PUP-like transporters to include plant secondary metabolism. Molecular evolution analyses of PUP-like transporters indicate they are distinct group within an ancient super family of drug and metabolite transporters (DMTs). The PUP-like family originated during terrestrial plant evolution sometime between the bryophytes and the lycophytes. A phylogenetic analysis indicates that the PUP-like transporters were likely derived from a pre-existing nucleotide-sugar transporter family within the DMT super family. Within the lycophyte Selaginella, there are three paralogous groups of PUP-like transporters. One of the three PUP-like paralogous groups showed an extensive pattern of gene duplication and diversification within the angiosperm lineage, whereas the more ancestral PUP-like paralogous groups did not. Biochemical characterization of four closely related PUP-like paralogs together with model-based phylogenetic analyses indicate both subfunctionalization and neofunctionalization during the molecular evolution of angiosperm PUP-like transporters. These findings suggest that members of the PUP-like family of DMT transporters are likely involved in diverse primary and secondary plant metabolic pathways. PMID:22639664

  14. Does pronounceability modulate the letter string deficit of children with dyslexia? A study with the rate and amount model

    PubMed Central

    Marinelli, Chiara V.; Traficante, Daniela; Zoccolotti, Pierluigi

    2014-01-01

    The locus of the deficit of children with dyslexia in dealing with strings of letters may be a deficit at a pre-lexical graphemic level or an inability to bind orthographic and phonological information. We evaluate these alternative hypotheses in two experiments by examining the role of stimulus pronounceability in a lexical decision task (LDT) and in a forced-choice letter discrimination task (Reicher–Wheeler paradigm). Seventeen fourth grade children with dyslexia and 24 peer control readers participated to two experiments. In the LDT children were presented with high-, low-frequency words, pronounceable pseudowords (such as DASU) and unpronounceable non-words (such as RNGM) of 4-, 5-, or 6- letters. No sign of group by pronounceability interaction was found when over-additivity was taken into account. Children with dyslexia were impaired when they had to process strings, not only of pronounceable stimuli but also of unpronounceable stimuli, a deficit well accounted for by a single global factor. Complementary results were obtained with the Reicher–Wheeler paradigm: both groups of children gained in accuracy in letter discrimination in the context of pronounceable primes (words and pseudowords) compared to unpronounceable primes (non-words). No global factor was detected in this task which requires the discrimination between a target letter and a competitor but does not involve simultaneous letter string processing. Overall, children with dyslexia show a selective difficulty in simultaneously processing a letter string as a whole, independent of its pronounceability; however, when the task involves isolated letter processing, also these children can make use of the ortho-phono-tactic information derived from a previously seen letter string. This pattern of findings is in keeping with the idea that an impairment in pre-lexical graphemic analysis may be a core deficit in developmental dyslexia. PMID:25520680

  15. Osmylated DNA, a novel concept for sequencing DNA using nanopores

    NASA Astrophysics Data System (ADS)

    Kanavarioti, Anastassia

    2015-03-01

    Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. ‘Base calling’ becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.

  16. Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

    PubMed

    Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

    2013-07-01

    Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

  17. TD-DFT investigation of the magnetic circular dichroism spectra of some purine and pyrimidine bases of nucleic acids.

    PubMed

    Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick; Santoro, Fabrizio; Improta, Roberto; Coriani, Sonia

    2015-05-28

    We present a computational study of the magnetic circular dichroism (MCD) spectra in the 200-300 nm wavelength region of purine and its derivative hypoxanthine, as well as of the pyrimidine bases of nucleic acids uracil, thymine, and cytosine, using the B3LYP and CAM-B3LYP functionals. Solvent effects are investigated within the polarizable continuum model and by inclusion of explicit water molecules. In general, the computed spectra are found to be in good agreement with the experimental ones, apart from some overall blue shifts. Both the pseudo-A term shape of the MCD spectra of the purines and the B term shape of the spectra of pyrimidine bases are reproduced. Our calculations also correctly reproduce the reversed phase of the MCD bands in purine compared to that of its derivatives present in nucleic acids. Solvent effects are sizable and system specific, but they do not in general alter the qualitative shape of the spectra. The bands are dominated by the bright π → π* transitions, and our calculations in solution nicely reproduce their energy differences, improving the estimates obtained in the gas phase. Shoulders are predicted for purine and uracil due to n → π* excitations, but they are too weak to be observed in the experiment.

  18. Plasmodium falciparum Parasites Are Killed by a Transition State Analogue of Purine Nucleoside Phosphorylase in a Primate Animal Model

    PubMed Central

    Cassera, María B.; Hazleton, Keith Z.; Merino, Emilio F.; Obaldia, Nicanor; Ho, Meng-Chiao; Murkin, Andrew S.; DePinto, Richard; Gutierrez, Jemy A.; Almo, Steven C.; Evans, Gary B.; Babu, Yarlagadda S.; Schramm, Vern L.

    2011-01-01

    Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent. PMID:22096507

  19. Inhibition and Structure of Trichomonas vaginalis Purine Nucleoside Phosphorylase with Picomolar Transition State Analogues

    SciTech Connect

    Rinaldo-Matthis,A.; Wing, C.; Ghanem, M.; Deng, H.; Wu, P.; Gupta, A.; Tyler, P.; Evans, G.; Furneaux, R.; et al.

    2007-01-01

    Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition stte mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a K{sub m}/K{sub d} ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a K{sub m}/K{sub d} ratio of 203,300. The tight binding of DADMe-ImmA supports a late S{sub N}1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP-ImmA{center_dot}PO{sub 4} and TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4} ternary complexes differ from previous structures with substrate anologues. The tight binding with DADMe-ImmA is in part due to a 2.7 {angstrom} ionic interaction between a PO{sub 4} oxygen and the N1 cation of the hydroxypyrrolidine and is weaker in the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure at 3.5 {angstrom}. However, the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4}. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope

  20. Novel Ligands for a Purine Riboswitch Discovered by RNA-Ligand Docking

    PubMed Central

    Daldrop, Peter; Reyes, Francis E.; Robinson, David A.; Hammond, Colin M.; Lilley, David M.; Batey, Robert T.; Brenk, Ruth

    2011-01-01

    Summary The increasing number of RNA crystal structures enables a structure-based approach to the discovery of new RNA-binding ligands. To develop the poorly explored area of RNA-ligand docking, we have conducted a virtual screening exercise for a purine riboswitch to probe the strengths and weaknesses of RNA-ligand docking. Using a standard protein-ligand docking program with only minor modifications, four new ligands with binding affinities in the micromolar range were identified, including two compounds based on molecular scaffolds not resembling known ligands. RNA-ligand docking performed comparably to protein-ligand docking indicating that this approach is a promising option to explore the wealth of RNA structures for structure-based ligand design. PMID:21439477

  1. Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle.

    PubMed Central

    Sabina, R L; Swain, J L; Olanow, C W; Bradley, W G; Fishbein, W N; DiMauro, S; Holmes, E W

    1984-01-01

    To assess the role of the purine nucleotide cycle in human skeletal muscle function, we evaluated 10 patients with AMP deaminase deficiency (myoadenylate deaminase deficiency; MDD). 4 MDD and 19 non-MDD controls participated in an exercise protocol. The latter group was composed of a patient cohort (n = 8) exhibiting a constellation of symptoms similar to those of the MDD patients, i.e., postexertional aches, cramps, and pains; as well as a cohort of normal, unconditioned volunteers (n = 11). The individuals with MDD fatigued after performing only 28% as much work as their non-MDD counterparts. Muscle biopsies were obtained from the four MDD patients and the eight non-MDD patients at rest and following exercise to the point of fatigue. Creatine phosphate content fell to a comparable extent in the MDD (69%) and non-MDD (52%) patients at the onset of fatigue. Following exercise the 34% decrease in ATP content of muscle from the non-MDD subjects was significantly greater than the 6% decrease in ATP noted in muscle from the MDD patients (P = 0.048). Only one of four MDD patients had a measurable drop in ATP compared with seven of eight non-MDD patients. At end-exercise the muscle content of inosine 5'-monophosphate (IMP), a product of AMP deaminase, was 13-fold greater in the non-MDD patients than that observed in the MDD group (P = 0.008). Adenosine content of muscle from the MDD patients increased 16-fold following exercise, while there was only a twofold increase in adenosine content of muscle from the non-MDD patients (P = 0.028). Those non-MDD patients in whom the decrease in ATP content following exercise was measurable exhibited a stoichiometric increase in IMP, and total purine content of the muscle did not change significantly. The one MDD patient in whom the decrease in ATP was measurable, did not exhibit a stoichiometric increase in IMP. Although the adenosine content increased 13-fold in this patient, only 48% of the ATP catabolized could be accounted for

  2. Release of extracellular purines from plant roots and effect on ion fluxes.

    PubMed

    Dark, Adeeba; Demidchik, Vadim; Richards, Siân L; Shabala, Sergey; Davies, Julia M

    2011-11-01

    Extracellular purine nucleotides appear capable of regulating plant development, defence and stress responses by acting in part as agonists of plasma membrane calcium channels. Factors stimulating ATP release include wounding, osmotic stress and elicitors. Here we show that exogenous abscisic acid and L-glutamate can also cause ATP accumulation around Arabidopsis thaliana roots. Release of ADP from root epidermis would trigger ionotropic receptor-like activity in the plasma membrane, resulting in transient elevation of cytosolic free calcium. Root epidermal protoplasts (expressing aequorin as a cytosolic free calcium reporter) can support an extracellular ADP-induced cytosolic calcium elevation in the presence of an extracellular reductant. This confirms that ADP could elicit calcium-based responses distinct to those of ATP, which have been shown previously to involve production of extracellular reactive oxygen species.

  3. Theacrine, a purine alkaloid with anti-inflammatory and analgesic activities.

    PubMed

    Wang, Yuanyuan; Yang, Xiaorong; Zheng, Xinqiang; Li, Jing; Ye, Chuangxing; Song, Xiaohong

    2010-09-01

    The anti-inflammatory and analgesic effects of theacrine (1, 3, 7, 9-tetramethyluric acid), a purine alkaloid which is abundantly present in Camellia kucha, were investigated. Xylene-induced ear edema, acetic acid-induced vascular permeability and lambda-carrageenan-induced paw edema were used to investigate anti-inflammatory activity, and acetic acid-induced writhing and hot-plate tests were used to determine analgesic effect. Oral administration of theacrine (8-32 mg/kg) induced dose-related anti-inflammatory and analgesic effects. On the other hand, oral caffeine administration (8-32 mg/kg) did not show an inhibitory effect on the inhibition of inflammatory response or cause analgesia. Additionally, the result of the acute toxicity test showed that the LD(50) of theacrine was 810.6 mg/kg (769.5-858.0mg/kg). The data obtained suggest theacrine possessed analgesic and anti-inflammatory activities.

  4. De novo synthesis of purine nucleotides in different fiber types of rat skeletal muscle

    SciTech Connect

    Tullson, P.C.; John-Alder, H.; Hood, D.A.; Terjung, R.L.

    1986-03-01

    The contribution of de novo purine nucleotide synthesis to nucleotide metabolism in skeletal muscles is not known. The authors have determined rates of de novo synthesis in soleus (slow-twitch red), red gastrocnemius (fast-twitch red), and white gastrocnemius (fast-twitch white) using the perfused rat hindquarter. /sup 14/C glycine incorporation into ATP was linear after 1 and 2 hours of perfusion with 0.2 mM added glycine. The intracellular (I) and extracellular (E) specific activity of /sup 14/C glycine was determined by HPLC of phenylisothiocyanate derivatives of neutralized PCA extracts. The rates of de novo synthesis when expressed relative to muscle ATP content show slow and fast-twitch red muscles to be similar and about twice as great as fast-twitch white muscles. This could represent a greater turnover of the adenine nucleotide pool in more oxidative red muscle types.

  5. Identification of amino acid residues responsible for the pyrimidine and purine nucleoside specificities of human concentrative Na(+) nucleoside cotransporters hCNT1 and hCNT2.

    PubMed

    Loewen, S K; Ng, A M; Yao, S Y; Cass, C E; Baldwin, S A; Young, J D

    1999-08-27

    hCNT1 and hCNT2 mediate concentrative (Na(+)-linked) cellular uptake of nucleosides and nucleoside drugs by human cells and tissues. The two proteins (650 and 658 residues, 71 kDa) are 72% identical in sequence and contain 13 putative transmembrane helices (TMs). When produced in Xenopus oocytes, recombinant hCNT1 is selective for pyrimidine nucleosides (system cit), whereas hCNT2 is selective for purine nucleosides (system cif). Both transport uridine. We have used (i) chimeric constructs between hCNT1 and hCNT2, (ii) sequence comparisons with a newly identified broad specificity concentrative nucleoside transporter (system cib) from Eptatretus stouti, the Pacific hagfish (hfCNT), and (iii) site-directed mutagenesis of hCNT1 to identify two sets of adjacent residues in TMs 7 and 8 of hCNT1 (Ser(319)/Gln(320) and Ser(353)/Leu(354)) that, when converted to the corresponding residues in hCNT2 (Gly(313)/Met(314) and Thr(347)/Val(348)), changed the specificity of the transporter from cit to cif. Mutation of Ser(319) in TM 7 of hCNT1 to Gly enabled transport of purine nucleosides, whereas concurrent mutation of Gln(320) to Met (which had no effect on its own) augmented this transport. The additional mutation of Ser(353) to Thr in TM 8 converted hCNT1/S319G/Q320M, from cib to cif, but with relatively low adenosine transport activity. Additional mutation of Leu(354) to Val (which had no effect on its own) increased the adenosine transport capability of hCNT1/S319G/Q320M/S353T, producing a full cif-type transporter phenotype. On its own, the S353T mutation converted hCNT1 into a transporter with novel uridine-selective transport properties. Helix modeling of hCNT1 placed Ser(319) (TM 7) and Ser(353) (TM 8) within the putative substrate translocation channel, whereas Gln(320) (TM 7) and Leu(354) (TM 8) may exert their effects through altered helix packing.

  6. On the accessibility to conical intersections in purines: hypoxanthine and its singly protonated and deprotonated forms.

    PubMed

    Villabona-Monsalve, Juan P; Noria, Raquel; Matsika, Spiridoula; Peón, Jorge

    2012-05-09

    The dynamics following electronic excitation of hypoxanthine and its nucleoside inosine were studied by femtosecond fluorescence up-conversion. Our objective was to explore variants of the purinic DNA bases in order to determine the molecular parameters that increase or reduce the accessibility to ground state conical intersections. From experiments in water and methanol solution we conclude that both dominant neutral tautomers of hypoxanthine exhibit ultrashort excited state lifetimes (τ < 0.2 ps), which are significantly shorter than in the related nucleobase guanine. This points to a more accessible conical intersection for the fluorescent state upon removal of the amino group, present in guanine but absent in hypoxanthine. The excited state dynamics of singly protonated hypoxanthine were also studied, showing biexponential decays with a 1.1 ps component (5%) besides a sub-0.2 ps ultrafast component. On the other hand, the S(1) lifetimes of the singly deprotonated forms of hypoxanthine and inosine show drastic differences, where the latter remains ultrafast but the singly deprotonated hypoxanthine shows a much longer lifetime of 19 ps. This significant variation is related to the different deprotonation sites in hypoxanthine versus inosine, which gives rise to significantly different resonance structures. In our study we also include multireference perturbation theory (MRMP2) excited state calculations in order to determine the nature of the initial electronic excitation in our experiments and clarify the ordering of the states in the singlet manifold at the ground state geometry. In addition, we performed multireference configuration interaction calculations (MR-CIS) that identify the presence of low-lying conical intersections for both prominent neutral tautomers of hypoxanthine. In both cases, the surface crossings occur at geometries reached by out of plane opposite motions of C2 and N3. The study of this simpler purine gives several insights into how small

  7. Effects of dairy cow diet forage proportion on duodenal nutrient supply and urinary purine derivative excretion.

    PubMed

    Moorby, J M; Dewhurst, R J; Evans, R T; Danelón, J L

    2006-09-01

    Four mature Holstein-Friesian dairy cows were used in a 4 x 4 Latin square change-over design experiment made up of four 4-wk periods to investigate the relationship between microbial protein flow to the duodenum and excretion of purine derivatives (PD) in the urine. Four dietary treatments based on ad libitum access to ryegrass silage were offered, with a standard dairy concentrate included at different forage:concentrate (F:C) ratios, calculated on a dry matter basis: 80:20, 65:35, 50:50, and 35:65. Feed intakes increased as the proportion of concentrate in the diet increased, despite a concurrent decrease in silage intake. Increased feed intake led to increased nutrient flow to the duodenum. Milk yields increased as the diet F:C ratio decreased, with cows offered the 35:65 diet yielding nearly 8 kg/d more milk than cows offered the 80:20 diet; the concentrations of milk fat decreased and milk protein increased with a decreasing F:C ratio. Purine derivative excretion in the urine increased with an increasing proportion of concentrate in the diet, and there was a strong linear relationship between total PD excretion (allantoin and uric acid) and microbial N flow to the duodenum: microbial N (g/d) = 19.9 + 0.689 x total PD (mmol/d); R = 0.887. This strengthens the case for using PD excretion as a noninvasive marker of microbial protein flow from the rumen in dairy cows.

  8. Purine metabolism in response to hypoxic conditions associated with breath-hold diving and exercise in erythrocytes and plasma from bottlenose dolphins (Tursiops truncatus).

    PubMed

    del Castillo Velasco-Martínez, Iris; Hernández-Camacho, Claudia J; Méndez-Rodríguez, Lía C; Zenteno-Savín, Tania

    2016-01-01

    In mammalian tissues under hypoxic conditions, ATP degradation results in accumulation of purine metabolites. During exercise, muscle energetic demand increases and oxygen consumption can exceed its supply. During breath-hold diving, oxygen supply is reduced and, although oxygen utilization is regulated by bradycardia (low heart rate) and peripheral vasoconstriction, tissues with low blood flow (ischemia) may become hypoxic. The goal of this study was to evaluate potential differences in the circulating levels of purine metabolism components between diving and exercise in bottlenose dolphins (Tursiops truncatus). Blood samples were taken from captive dolphins following a swimming routine (n=8) and after a 2min dive (n=8). Activity of enzymes involved in purine metabolism (hypoxanthine guanine phosphoribosyl transferase (HGPRT), inosine monophosphate deshydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP)), and purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD(+)), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, guanosine diphosphate (GDP), guanosine triphosphate (GTP)) concentrations were quantified in erythrocyte and plasma samples. Enzymatic activity and purine metabolite concentrations involved in purine synthesis and degradation, were not significantly different between diving and exercise. Plasma adenosine concentration was higher after diving than exercise (p=0.03); this may be related to dive-induced ischemia. In erythrocytes, HGPRT activity was higher after diving than exercise (p=0.007), suggesting an increased capacity for purine recycling and ATP synthesis from IMP in ischemic tissues of bottlenose dolphins during diving. Purine recycling and physiological adaptations may maintain the ATP concentrations in bottlenose dolphins after diving and exercise.

  9. Identification of a purine-rich intronic enhancer element in the mouse eosinophil-associated ribonuclease 2 (mEar 2) gene.

    PubMed

    Dyer, Kimberly D; Nitto, Takeaki; Moreau, Joanne M; McDevitt, Amanda L; Rosenberg, Helene F

    2004-02-01

    The Mus musculus eosinophil-associated ribonuclease (mEar) gene cluster includes multiple distinct coding sequences that are highly divergent orthologs of the human eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3). We present a transcriptional analysis of the gene encoding mEar 2, the only member of this cluster with a well-defined expression profile. In this work, we demonstrate that the presence of non-coding exon 1 and the intron in tandem with a 361-bp 5' promoter of mEar 2 results in enhanced reporter gene expression, as much as 6-to 10-fold over the activity observed with the 5' promoter alone. We have identified a conserved purine-rich element in the intron of the mEar 2 gene that is necessary for maximum transcription and that interacts specifically with NFAT-binding proteins in nuclear extracts derived from the mouse LA4 epithelial cell line. Similar intronic enhancers have been described as regulating transcription of the human EDN gene, suggesting an overall conservation of an important regulatory strategy.

  10. Synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, an impurity of acyclovir.

    PubMed

    Suárez, Rosa M; Matía, Maria Paz; Novella, José Luis; Molina, Andres; Cosme, Antonio; Vaquero, Juan José; Alvarez-Builla, Julio

    2012-07-25

    The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial N-(9-acetyl-6-oxo-1H-purin-2-yl)acetamide, the process uses an acid catalysed phase transfer catalysis (PTC) process to produce the selective alkylation at the 9 position of the guanine ring.

  11. Theacrine, a special purine alkaloid with sedative and hypnotic properties from Cammelia assamica var. kucha in mice.

    PubMed

    Xu, Jie-Kun; Kurihara, Hiroshi; Zhao, Liang; Yao, Xin-Sheng

    2007-01-01

    The central nervous system activities of theacrine (1,3,7,9-tetramethyluric acid), a purine alkaloid which is abundantly present in Camellia assamica var. kucha, were investigated in ambulatory activity, pentobarbital-induced sleep and forced swimming test in mice, compared with two other purine alkaloids, caffeine and theobromine. Caffeine treatment led to a marked increase in the ambulatory activity accompanied with decreasing of the immobility time in forced swimming test at both 10 and 30 mg/kg. Under the same conditions, neither theacrine nor theobromine showed obvious excited efficacy. Both doses of theacrine could significantly prolong the sleeping time induced by pentobarbital, while caffeine and theobromine exhibited an inverted effect. These results indicated that theacrine possessed potent sedative and hypnotic properties and its central nervous system effects were different from those of caffeine and theobromine.

  12. Isolation and Characterization of a Slowly Milk-Coagulating Variant of Lactobacillus helveticus Deficient in Purine Biosynthesis

    PubMed Central

    Hebert, Elvira M.; De Giori, Graciela S.; Raya, Raul R.

    2001-01-01

    A slowly milk-coagulating variant (Fmc−) of Lactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc− strain was threefold lower than that of the wild-type strain. L. helveticus S1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc+ phenotype of L. helveticus S1. PMID:11282642

  13. Distribution coefficients of purine alkaloids in water-ammonium sulfate-alkyl acetate-dialkyl phthalate systems

    NASA Astrophysics Data System (ADS)

    Korenman, Ya. I.; Krivosheeva, O. A.; Mokshina, N. Ya.

    2012-12-01

    The distribution of purine alkaloids (caffeine, theobromine, theophylline) was studied in the systems: alkyl acetates-dialkyl phtalate-salting-out agent (ammonium sulfate). The quantitative characteristics of the extraction-distribution coefficients ( D) and the degree of extraction ( R, %) are calculated. The relationships between the distribution coefficients of alkaloids and the length of the hydrocarbon radical in the molecule of alkyl acetate (dialkyl phtalate) are determined. The possibility of predicting the distribution coefficients is demonstrated.

  14. Study of Copper and Purine-Copper Complexes on Modified Carbon Electrodes by Cyclic and Elimination Voltammetry

    PubMed Central

    Trnkova, Libuse; Zerzankova, Lenka; Dycka, Filip; Mikelova, Radka; Jelen, Frantisek

    2008-01-01

    Using a paraffin impregnated graphite electrode (PIGE) and mercury-modified pyrolytic graphite electrode with basal orientation (Hg-PGEb) copper(II) and Cu(II)-DNA purine base solutions have been studied by cyclic (CV) and linear sweep voltammetry (LSV) in connection with elimination voltammetry with linear scan (EVLS). In chloride and bromide solutions (pH 6), the redox process of Cu(II) proceeded on PIGE with two cathodic and two anodic potentially separated signals. According to the elimination function E4, the first cathodic peak corresponds to the reduction Cu(II) + e- → Cu(I) with the possibility of fast disproportionation 2Cu(I) → Cu(II)+ Cu(0). The E4 of the second cathodic peak signalized an electrode process controlled by a surface reaction. The electrode system of Cu(II) on Hg-PGEb in borate buffer (pH 9.2) was characterized by one cathodic and one anodic peak. Anodic stripping voltammetry (ASV) on PIGE and cathodic stripping voltammetry (CSV) on Hg-PGEb were carried out at potentials where the reduction of copper ions took place and Cu(I)-purine complexes were formed. By using ASV and CSV in combination with EVLS, the sensitivity of Cu(I)-purine complex detection was enhanced relative to either ASV or CSV alone, resulting in higher peak currents of more than one order of magnitude. The statistical treatment of CE data was used to determine the reproducibility of measurements. Our results show that EVLS in connection with the stripping procedure is useful for both qualitative and quantitative microanalysis of purine derivatives and can also reveal details of studied electrode processes. PMID:27879715

  15. Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

    PubMed

    Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

    2016-06-12

    Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD.

  16. Profiles of phenolic compounds and purine alkaloids during the development of seeds of Theobroma cacao cv. Trinitario.

    PubMed

    Pereira-Caro, Gema; Borges, Gina; Nagai, Chifumi; Jackson, Mel C; Yokota, Takao; Crozier, Alan; Ashihara, Hiroshi

    2013-01-16

    Changes occurring in phenolic compounds and purine alkaloids, during the growth of seeds of cacao (Theobroma cacao) cv. Trinitario, were investigated using HPLC-MS/MS. Extracts of seeds with a fresh weight of 125, 700, 1550, and 2050 mg (stages 1-4, respectively) were analyzed. The phenolic compounds present in highest concentrations in developing and mature seeds (stages 3 and 4) were flavonols and flavan-3-ols. Flavan-3-ols existed as monomers of epicatechin and catechin and as procyanidins. Type B procyanidins were major components and varied from dimers to pentadecamer. Two anthocyanins, cyanidin-3-O-arabinoside and cyanidin-3-O-galactoside, along with the N-phenylpropernoyl-l-amino acids, N-caffeoyl-l-aspartate, N-coumaroyl-l-aspartate, N-coumaroyl-3-hydroxytyrosine (clovamide), and N-coumaroyltyrosine (deoxyclovamide), and the purine alkaloids theobromine and caffeine, were present in stage 3 and 4 seeds. Other purine alkaloids, such as theophylline and additional methylxanthines, did not occur in detectable quantities. Flavan-3-ols were the only components to accumulate in detectable quantities in young seeds at developmental stages 1 and 2.

  17. [Genetic control of metabolism of mutagenic purine base analogs 6-hydroxylaminopurine and 2-amino-6-hydroxylaminopurine in yeast Saccharomyces cerevisiae].

    PubMed

    Stepchenkova, E I; Koz'min, S G; Alenin, V V; Pavlov, Iu I

    2009-04-01

    The influence of inactivation of genes, which control biosynthesis of inosine monophosphate (IMP) de novo and the purine utilization and interconversion pathway, on sensitivity of yeast Saccharomyces cerevisiae cells to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA) was studied. It was shown that the manifestation of HAP and AHA mutagenic properties involves the action of enzyme adenine phosphoribosyltransferase encoded in yeast by APT1 gene. A blockade of each stage of IMP biosynthesis, with the exception of the block mediated by inactivation of genes ADE16 and ADE17 leading to the accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), was shown to enhance yeast cell sensitivity to the HAP mutagenic effect; however, it does not affect the sensitivity to AHA. A blockade of conversion of IMP into adenosine monophosphate (AMP) causes hypersensitivity of yeast cells to the mutagenic action of HAP and to the toxic effect of HAP, AHA, and hypoxanthine. It is fully probable that this enhancement of sensitivity to HAP and AHA is conditioned by changes in the pool of purines. This indicates that genes ADE12, ADE13, AAH1, and HAM1 controlling processes of purine utilization and interconversion in yeast make the greatest contribution to the system of protection against the toxic and mutagenic action of the examined analogs. Possible mechanisms of HAP detoxication in bacteria, yeast, and humans are considered.

  18. An Unusual Protector-Protégé Strategy for the Biosynthesis of Purine Nucleoside Antibiotics.

    PubMed

    Wu, Pan; Wan, Dan; Xu, Gudan; Wang, Gui; Ma, Hongmin; Wang, Tingting; Gao, Yaojie; Qi, Jianzhao; Chen, Xiaoxia; Zhu, Jian; Li, Yong-Quan; Deng, Zixin; Chen, Wenqing

    2017-02-16

    Pentostatin (PTN, deoxycoformycin) and arabinofuranosyladenine (Ara-A, vidarabine) are purine nucleoside antibiotics used clinically to treat hematological cancers and human DNA virus infections, respectively. PTN has a 1,3-diazepine ring, and Ara-A is an adenosine analog with an intriguing epimerization at the C-2' hydroxyl group. However, the logic underlying the biosynthesis of these interesting molecules has long remained elusive. Here, we report that the biosynthesis of PTN and Ara-A employs an unusual protector-protégé strategy. To our surprise, we determined that a single gene cluster governs PTN and Ara-A biosynthesis via two independent pathways. Moreover, we verified that PenB functions as a reversible oxidoreductase for the final step of PTN. Remarkably, we provided the first direct biochemical evidence that PTN can protect Ara-A from deamination by selective inhibition of the host adenosine deaminase. These findings expand our knowledge of natural product biosynthesis and open the way for target-directed genome mining of Ara-A/PTN-related antibiotics.

  19. Activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) on undernourished and renourished rats' thymus.

    PubMed

    Feliu, M S.; Slobodianik, N H.

    2001-02-01

    We studied the effect of administration of a low quality dietary protein, from weaning onwards, on the thymus of undernourished rats and the posterior effect of refeeding with a high quality dietary protein. Changes in thymus weight and the activity of Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNP) on thymus, were determined. Wistar rats were suckled in groups of 14-16 per dam since birth to weaning (23 days) to obtain undernutrition. At weaning, a group of 14-16 rats received pre-cooked maize flour (Protein content: 6.5%) for 18 days. One group was sacrificed (M) and the other rats were refed with the casein diet (Protein content: 20%) during 20 days (R). The age-matched control groups were fed stock diet since 40 (C40) and 60 (C60) days of age, respectively. At the end of the experimental period, body (Bw) and thymus weight were determined. ADA and PNP activities were determined in thymocyte suspensions. Highly significant differences in thymus weight-expressed as mg or mg/Bw(0.75)-and the activity of ADA and PNP were observed in rats fed the experimental diet containing maize flour, when compared to the respective age-matched control. No statistical differences were observed between R and C60.The administration of a high quality dietary protein to undernourished weanling rats is capable to reverse the damage produced by the low quality dietary protein on thymus weight and ADA and PNP thymus activities.

  20. The role of a purine-specific nucleoside hydrolase in spore germination of Bacillus thuringiensis.

    PubMed

    Liang, Liang; He, Xihong; Liu, Gang; Tan, Huarong

    2008-05-01

    A homologous gene (iunH) of a putative nucleoside hydrolase (NH), which had been identified from the exosporia of Bacillus cereus and Bacillus anthracis spores, was cloned from Bacillus thuringiensis subsp. kurstaki. Disruption of iunH did not affect the vegetative growth and sporulation of Bacillus thuringiensis, but promoted both inosine- and adenosine-induced spore germination. The inosine- or adenosine-induced germination rate decreased when the wild-type iunH gene was overexpressed in Bacillus thuringiensis. The iunH gene product was characterized as a purine-specific NH. The kinetic parameters of IunH with inosine as substrate were K(m)=399+/-115 microM, k(cat)=48.9+/-8.5 s(-1) and k(cat)/K(m)=1.23 x 10(5) M(-1) s(-1). The optimal pH and temperature for IunH were found to be pH 6 and 80 degrees C. Meanwhile, the specific activity of inosine hydrolase in intact spores of the wild-type strain with inosine as substrate was 2.89+/-0.23x10(-2) micromol min(-1) (mg dry wt)(-1). These results indicate that IunH is important in moderating inosine- or adenosine-induced germination of Bacillus thuringiensis spores.

  1. A 90-Day Oral Toxicological Evaluation of the Methylurate Purine Alkaloid Theacrine

    PubMed Central

    Hirka, Gábor; Glávits, Róbert; Palmer, Philip A.; Endres, John R.; Pasics Szakonyiné, Ilona

    2016-01-01

    A 90-day repeated-dose oral toxicological evaluation was conducted according to GLP and OECD guidelines on the methylurate purine alkaloid theacrine, which is found naturally in certain plants. Four groups of Hsd.Brl.Han Wistar rats (ten/sex/group) were administered theacrine by gavage doses of 0 (vehicle only), 180, 300, and 375 mg/kg bw/day. Two females and one male in the 300 and 375 mg/kg bw/day groups, respectively, died during the study. Histological examination revealed centrilobular hepatocellular necrosis as the probable cause of death. In 375 mg/kg bw/day males, slight reductions in body weight development, food consumption, and feed efficiency, decreased weight of the testes and epididymides and decreased intensity of spermatogenesis in the testes, lack or decreased amount of mature spermatozoa in the epididymides, and decreased amount of prostatic secretions were detected at the end of the three months. At 300 mg/kg bw/day, slight decreases in the weights of the testes and epididymides, along with decreased intensity of spermatogenesis in the testes, and lack or decreased amount of mature spermatozoa in the epididymides were detected in male animals. The NOAEL was considered to be 180 mg/kg bw/day, as at this dose there were no toxicologically relevant treatment-related findings in male or female animals. PMID:27635133

  2. Effect of ethanol on metabolism of purine bases (hypoxanthine, xanthine, and uric acid).

    PubMed

    Yamamoto, Tetsuya; Moriwaki, Yuji; Takahashi, Sumio

    2005-06-01

    There are many factors that contribute to hyperuricemia, including obesity, insulin resistance, alcohol consumption, diuretic use, hypertension, renal insufficiency, genetic makeup, etc. Of these, alcohol (ethanol) is the most important. Ethanol enhances adenine nucleotide degradation and increases lactic acid level in blood, leading to hyperuricemia. In beer, purines also contribute to an increase in plasma uric acid. Although rare, dehydration and ketoacidosis (due to ethanol ingestion) are associated with the ethanol-induced increase in serum uric acid levels. Ethanol also increases the plasma concentrations and urinary excretion of hypoxanthine and xanthine via the acceleration of adenine nucleotide degradation and a possible weak inhibition of xanthine dehydrogenase activity. Since many factors such as the ALDH2*1 gene and ADH2*2 gene, daily drinking habits, exercise, and dehydration enhance the increase in plasma concentration of uric acid induced by ethanol, it is important to pay attention to these factors, as well as ingested ethanol volume, type of alcoholic beverage, and the administration of anti-hyperuricemic agents, to prevent and treat ethanol-induced hyperuricemia.

  3. The antibacterial activity and toxicity of enrofloxacin are decreased by nanocellulose conjugated with aminobenzyl purin.

    PubMed

    Yasini, Seyed Ali; Zadeh, Mohammad Hossein Balal; Shahdadi, Hossein

    2015-11-01

    The first aim of this study was to synthesize nanocellulose conjugated with aminobenzyl purin (NCABP), and the second aim was to evaluate the effect of NCABP on both toxicity and antibacterial activity of enrofloxacin. Here, the adsorption of enrofloxacin by NCABP was first modeled by molecular dynamic (MD) simulation. In the next step, NCABP was synthesized, and was exposed to enrofloxacin, 1000 μg mL(-1), at various conditions. Then, the quantity of adsorption and release was separately measured. Furthermore, both toxicity and antibacterial activity of NCABP, enrofloxacin, and (NCABP+enrofloxacin) were separately evaluated. In this study, MD simulation clearly showed the adsorption after 50 picoseconds. The adsorption tests revealed that the increase of incubation time and NCABP concentration, at range of 50-200 μg mL(-1), led to increase of adsorption. Moreover, the decrease of pH led to increase of adsorption. Interestingly, NCABP could adsorb enrofloxacin, up to 1000 μg mL(-1), in different types of meat. Moreover, the increase of incubation time and temperature did not release enrofloxacin, but the increase of pH increased release. This study showed that both toxicity and antibacterial activity of enrofloxacin were decreased when exposed together with NCABP.

  4. Influence of sprint training on human skeletal muscle purine nucleotide metabolism.

    PubMed

    Stathis, C G; Febbraio, M A; Carey, M F; Snow, R J

    1994-04-01

    To examine the effect of sprint training on human skeletal muscle purine nucleotide metabolism, eight active untrained subjects completed a maximal 30-s sprint bout on a cycle ergometer before and after 7 wk of sprint training. Resting muscle ATP and total adenine nucleotide content were reduced (P < 0.05) by 19 and 18%, respectively, after training. Training resulted in a 52% attenuation (P < 0.05) in the magnitude of ATP depletion after exercise and a similar reduction (P < 0.05) in the accumulation of inosine 5'-monophosphate and ammonia. During recovery, muscle inosine 5'-monophosphate (P < 0.05) and inosine (P < 0.01) content were reduced after training, as was the accumulation of inosine (P < 0.05). Plasma ammonia was higher (P < 0.05) after training early in recovery; in contrast, plasma hypoxanthine concentrations were reduced (P < 0.05) during the latter stages of recovery. The attenuated resting ATP and total adenine nucleotide contents after training probably result from the acute effects of prior training sessions. The reduction in the magnitude of ATP depletion during a 30-s sprint bout after training must reflect an improved balance between ATP hydrolysis and resynthesis. It is unclear which mechanism(s) is responsible for the reduction in the magnitude of ATP degradation after training.

  5. A 90-Day Oral Toxicological Evaluation of the Methylurate Purine Alkaloid Theacrine.

    PubMed

    Clewell, Amy; Hirka, Gábor; Glávits, Róbert; Palmer, Philip A; Endres, John R; Murbach, Timothy S; Marx, Tennille; Pasics Szakonyiné, Ilona

    2016-01-01

    A 90-day repeated-dose oral toxicological evaluation was conducted according to GLP and OECD guidelines on the methylurate purine alkaloid theacrine, which is found naturally in certain plants. Four groups of Hsd.Brl.Han Wistar rats (ten/sex/group) were administered theacrine by gavage doses of 0 (vehicle only), 180, 300, and 375 mg/kg bw/day. Two females and one male in the 300 and 375 mg/kg bw/day groups, respectively, died during the study. Histological examination revealed centrilobular hepatocellular necrosis as the probable cause of death. In 375 mg/kg bw/day males, slight reductions in body weight development, food consumption, and feed efficiency, decreased weight of the testes and epididymides and decreased intensity of spermatogenesis in the testes, lack or decreased amount of mature spermatozoa in the epididymides, and decreased amount of prostatic secretions were detected at the end of the three months. At 300 mg/kg bw/day, slight decreases in the weights of the testes and epididymides, along with decreased intensity of spermatogenesis in the testes, and lack or decreased amount of mature spermatozoa in the epididymides were detected in male animals. The NOAEL was considered to be 180 mg/kg bw/day, as at this dose there were no toxicologically relevant treatment-related findings in male or female animals.

  6. Analysis of abnormalities in purine metabolism leading to gout and to neurological dysfunctions in man.

    PubMed Central

    Curto, R; Voit, E O; Cascante, M

    1998-01-01

    A modelling approach is used to analyse diseases associated with purine metabolism in man. The specific focus is on deficiencies in two enzymes, hypoxanthine:guanine phosphoribosyltransferase and adenylosuccinate lyase. These deficiencies can lead to a number of symptoms, including neurological dysfunctions and mental retardation. Although the biochemical mechanisms of dysfunctions associated with adenylosuccinate lyase deficiency are not completely understood, there is at least general agreement in the literature about possible causes. Simulations with our model confirm that accumulation of the two substrates of the enzyme can lead to significant biochemical imbalance. In hypoxanthine:guanine phosphoribosyltransferase deficiency the biochemical mechanisms associated with neurological dysfunctions are less clear. Model analyses support some old hypotheses but also suggest new indicators for possible causes of neurological dysfunctions associated with this deficiency. Hypoxanthine:guanine phosphoribosyltransferase deficiency is known to cause hyperuricaemia and gout. We compare the relative importance of this deficiency with other known causes of gout in humans. The analysis suggests that defects in the excretion of uric acid are more consequential than defects in uric acid synthesis such as hypoxanthine:guanine phosphoribosyltransferase deficiency. PMID:9445373

  7. Solution-phase parallel synthesis of acyclic nucleoside libraries of purine, pyrimidine, and triazole acetamides.

    PubMed

    Pathak, Ashish K; Pathak, Vibha; Reynolds, Robert C

    2014-09-08

    Molecular diversity plays a pivotal role in modern drug discovery against phenotypic or enzyme-based targets using high throughput screening technology. Under the auspices of the Pilot Scale Library Program of the NIH Roadmap Initiative, we produced and report herein a diverse library of 181 purine, pyrimidine, and 1,2,4-triazole-N-acetamide analogues which were prepared in a parallel high throughput solution-phase reaction format. A set of assorted amines were reacted with several nucleic acid N-acetic acids utilizing HATU as the coupling reagent to produce diverse acyclic nucleoside N-acetamide analogues. These reactions were performed using 24 well reaction blocks and an automatic reagent-dispensing platform under inert atmosphere. The targeted compounds were purified on an automated purification system using solid sample loading prepacked cartridges and prepacked silica gel columns. All compounds were characterized by NMR and HRMS, and were analyzed for purity by HPLC before submission to the Molecular Libraries Small Molecule Repository (MLSMR) at NIH. Initial screening through the Molecular Libraries Probe Production Centers Network (MLPCN) program, indicates that several analogues showed diverse and interesting biological activities.

  8. Recognizing Sequences of Sequences

    PubMed Central

    Kiebel, Stefan J.; von Kriegstein, Katharina; Daunizeau, Jean; Friston, Karl J.

    2009-01-01

    The brain's decoding of fast sensory streams is currently impossible to emulate, even approximately, with artificial agents. For example, robust speech recognition is relatively easy for humans but exceptionally difficult for artificial speech recognition systems. In this paper, we propose that recognition can be simplified with an internal model of how sensory input is generated, when formulated in a Bayesian framework. We show that a plausible candidate for an internal or generative model is a hierarchy of ‘stable heteroclinic channels’. This model describes continuous dynamics in the environment as a hierarchy of sequences, where slower sequences cause faster sequences. Under this model, online recognition corresponds to the dynamic decoding of causal sequences, giving a representation of the environment with predictive power on several timescales. We illustrate the ensuing decoding or recognition scheme using synthetic sequences of syllables, where syllables are sequences of phonemes and phonemes are sequences of sound-wave modulations. By presenting anomalous stimuli, we find that the resulting recognition dynamics disclose inference at multiple time scales and are reminiscent of neuronal dynamics seen in the real brain. PMID:19680429

  9. Pronounced differences between the native K+ channels and KAT1 and KST1 alpha-subunit homomers of guard cells.

    PubMed

    Brüggemann, L; Dietrich, P; Dreyer, I; Hedrich, R

    1999-01-01

    Stomatal opening is the result of K(+)-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K(+)-selective ion channels. Here we compare the invitro properties of two guard-cell K(+)-channel alpha-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K(+)-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K(+)-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K+ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs+, 50% of the guard-cell K(+)-uptake channels (GCKClin) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to -90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as -155 mV were required. In contrast, compared to the K+ channels in vivo the functional alpha-subunit homomers almost lacked a voltage-dependent block by extracellular Ca2+. Similar to the block by Cs+ and Ca2+, the acid activation of the alpha-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCLin in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K(+)-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K(+)-uptake channels result either from the heterometric assembly of different alpha-subunits or evolve from cell-type specific posttranslational modification.

  10. Pronounced Climatic and Environmental Changes in the South West Pacific Ocean Following the End-Cretaceous Extinction Event

    NASA Astrophysics Data System (ADS)

    Crouch, E. M.; Taylor, K. W.; Willumsen, P. S.; Hollis, C. J.; Pancost, R. D.

    2014-12-01

    Dinoflagellate cyst assemblages from Cretaceous/Paleogene (K/Pg) boundary sections in eastern New Zealand record an alternating succession of pronounced abundance changes in two peridinioid (primarily heterotrophic) genera following the K/Pg boundary event. In Canterbury and East Coast Basin sections, two phases of abundant Trithyrodinium evittii, the first immediately following the K/Pg boundary, are interposed by two acme intervals of Palaeoperidinium pyrophorum. While several lines of evidence suggest T. evittii was a warm-water species and P. pyrophorum flourished in cooler oceanic conditions, robust temperature records have not been available from these K/Pg boundary sections. We have completed sea surface temperature (SST) reconstructions, based on glycerol dialkyl glycerol tetraether (GDGT) distributions, at mid-Waipara River, North Canterbury, from ~1 m below to 20 m above the K/Pg boundary. Changes in GDGT distribution across the K/Pg boundary indicates warming of 2-3°C, regardless of which TEX86-based proxy is used, coincident with the interval of abundant T. evittii. Detailed climatic records at the K/Pg boundary layer are hampered by intense bioturbation. Above an unconformity (at 23 cm) notable shifts in GDGT distribution indicates pronounced cooling, yielding SST estimates that are 7°C lower than the uppermost Cretaceous. The acme of P. pyrophorum corresponds with these cooler SSTs, and an unusual increase in the proportion of GDGT-2 in this interval can be attributed to cool water upwelling. The P. pyrophorum acme is also documented in distal diatom-rich siliceous sediments in Marlborough, where siliceous microfossils and element geochemistry indicate cool-water upwelling in the basal Paleocene. The second phase of abundant T. evittii, at ~2 m in Waipara, coincides with an interval of more stable SSTs that are comparable to the uppermost Cretaceous. Further discussion of the TEX86-based SST proxy and GDGT distributions will be provided in the

  11. Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases.

    PubMed

    Wielgus-Kutrowska, B; Kulikowska, E; Wierzchowski, J; Bzowska, A; Shugar, D

    1997-01-15

    Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP. Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with Ki values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Ki values similar to those for inhibition of other natural substrates. The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates. The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.

  12. Hydrogen bond donors accelerate vibrational cooling of hot purine derivatives in heavy water.

    PubMed

    Zhang, Yuyuan; Chen, Jinquan; Kohler, Bern

    2013-08-08

    Natural nucleobases and many of their derivatives have ultrashort excited state lifetimes that make them excellent model systems for studying intermolecular energy flow from a hot solute molecule to the solvent. UV-pump/broadband-mid-IR-probe transient absorption spectra of canonical purine nucleobases and several xanthine derivatives were acquired in D2O and acetonitrile in the probe frequency range of 1500-1750 cm(-1). The spectra reveal that vibrationally hot ground state molecules created by ultrafast internal conversion return to thermal equilibrium in several picoseconds by dissipating their excess energy to solvent molecules. In acetonitrile solution, where hydrogen bonding is minimal, vibrational cooling (VC) occurs with the same time constant of 10 ± 3 ps for paraxanthine, theophylline, and caffeine within experimental uncertainty. In D2O, VC by these molecules occurs more rapidly and at different rates that are correlated with the number of N-D bonds. Hypoxanthine has a VC time constant of 3 ± 1 ps, while similar lifetimes of 2.3 ± 0.8 ps and 3.1 ± 0.3 ps are seen for 5'-adenosine monophosphate and 5'-guanosine monophosphate, respectively. All three molecules have at least two N-D bonds. Slightly slower VC time constants are measured for paraxanthine (4 ± 1 ps) and theophylline (5.1 ± 0.8 ps), dimethylated xanthines that have only one N-D bond. Caffeine, a trimethylated xanthine with no N-D bonds, has a VC time constant of 7.7 ± 0.9 ps, the longest ever observed for any nucleobase in aqueous solution. Hydrogen bond donation by solute molecules is proposed to enable rapid energy disposal to water via direct coupling of high frequency solute-solvent modes.

  13. The Evolutionary Fate of the Genes Encoding the Purine Catabolic Enzymes in Hominoids, Birds, and Reptiles

    PubMed Central

    Keebaugh, Alaine C.; Thomas, James W.

    2010-01-01

    Gene loss has been proposed to play a major role in adaptive evolution, and recent studies are beginning to reveal its importance in human evolution. However, the potential consequence of a single gene-loss event upon the fates of functionally interrelated genes is poorly understood. Here, we use the purine metabolic pathway as a model system in which to explore this important question. The loss of urate oxidase (UOX) activity, a necessary step in this pathway, has occurred independently in the hominoid and bird/reptile lineages. Because the loss of UOX would have removed the functional constraint upon downstream genes in this pathway, these downstream genes are generally assumed to have subsequently deteriorated. In this study, we used a comparative genomics approach to empirically determine the fate of UOX itself and the downstream genes in five hominoids, two birds, and a reptile. Although we found that the loss of UOX likely triggered the genetic deterioration of the immediate downstream genes in the hominoids, surprisingly in the birds and reptiles, the UOX locus itself and some of the downstream genes were present in the genome and predicted to encode proteins. To account for the variable pattern of gene retention and loss after the inactivation of UOX, we hypothesize that although gene loss is a common fate for genes that have been rendered obsolete due to the upstream loss of an enzyme a metabolic pathway, it is also possible that same lack of constraint will foster the evolution of new functions or allow the optimization of preexisting alternative functions in the downstream genes, thereby resulting in gene retention. Thus, adaptive single-gene losses have the potential to influence the long-term evolutionary fate of functionally interrelated genes. PMID:20106906

  14. Thermodynamics of the Purine Nucleoside Phosphorylase Reaction Revealed by Computer Simulations.

    PubMed

    Isaksen, Geir Villy; Åqvist, Johan; Brandsdal, Bjørn Olav

    2017-01-10

    Enzymes are able to catalyze chemical reactions by reducing the activation free energy, yielding significant increases in the reaction rates. This can thermodynamically be accomplished by either reducing the activation enthalpy or increasing the activation entropy. The effect of remote mutations on the thermodynamic activation parameters of human purine nucleoside phosphorylase is examined using extensive molecular dynamics and free energy simulations. More than 2700 independent reaction free energy profiles for six different temperatures have been calculated to obtain high-precision computational Arrhenius plots. On the basis of these, the activation enthalpies and entropies were computed from linear regression of the plots with ΔG(⧧) as a function of 1/T, and the obtained thermodynamic activation parameters are in very good agreement with those from experiments. The Arrhenius plots immediately show that the 6-oxopurines (INO and GUO) have identical slopes, whereas the 6-aminopurine (ADO) has a significantly different slope, indicating that the substrate specificity is related to the difference in thermodynamic activation parameters. Furthermore, the calculations show that the human PNP specificity for 6-oxopurines over 6-aminopurines originates from significant differences in electrostatic preorganization. The effect of the remote double mutation, K22E and H104R (E:R), has also been examined, as it alters human PNP toward the bovine PNP. These residues are situated on the protein surface, 28-35 Å from the active site, and the mutation alters the enthalpy-entropy balance with little effect on the catalytic rates. It is thus quite remarkable that the empirical valence bond method can reproduce the enthalpies and entropies induced by these long-range mutations.

  15. Four Generations of Transition State Analogues for Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Ho, M.; Shi, W; Rinaldo-Mathis, A; Tyler, P; Evans, G; Almo, S; Schramm, V

    2010-01-01

    Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*{sub i} = 58 pM, first-generation) contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*{sub i} = 9 pM, second-generation), uses a methylene-bridged dihydroxypyrrolidine cation with two asymmetric centers. DATMe-Immucillin-H (K*{sub i} = 9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*{sub i} = 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; (1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, (2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, (3) interaction between phosphate and inhibitor hydroxyl groups, and (4) His257 interacting with the 5{prime}-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

  16. Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland.

    PubMed

    Sanderson, Julie; Dartt, Darlene A; Trinkaus-Randall, Vickery; Pintor, Jesus; Civan, Mortimer M; Delamere, Nicholas A; Fletcher, Erica L; Salt, Thomas E; Grosche, Antje; Mitchell, Claire H

    2014-10-01

    This review highlights recent findings that describ how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca(2+) concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target

  17. Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

    PubMed

    Maynes, J T; Yam, W; Jenuth, J P; Gang Yuan, R; Litster, S A; Phipps, B M; Snyder, F F

    1999-12-01

    Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.

  18. Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

    PubMed Central

    Maynes, J T; Yam, W; Jenuth, J P; Gang Yuan, R; Litster, S A; Phipps, B M; Snyder, F F

    1999-01-01

    Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively. PMID:10567244

  19. The effects of morphine and methionine-enkephalin on the release of purines from cerebral cortex slices of rats and mice.

    PubMed Central

    Stone, T. W.

    1981-01-01

    1 Slices of cerebral cortex from Wistar rats, TO mice or C57 mice were preincubated with [3H]-adenosine, and labelled purines were subsequently releases by electrical stimulation or by perfusing with ouabain, 100 micro M. 2 Electrically-evoked purine release was substantially reduced when the Ca2+ concentration in the medium was lowered from 2.4 to 0.1 mM. In both rats and mice, the electrically-evoked release was increased by morphine and methionine-enkephalin (Met-enkephalin), 10 micro M, and in rats and TO mice by morphine 1 micro M, both drug effects being prevented by naloxone. 3 Purine release evoked by ouabain was also increased by morphine 1 and 10 micro M, though not by Met-enkephalin, from slices of rat cortex. Ouabain-induced release from TO mice was reduced by morphine, and from C57 mice was unchanged. 4 The enhancement by morphine of electrically-evoked purine release may indicate that purines mediate some effects of morphine in the CNS. PMID:7272599

  20. Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine, Pyrimidine, and Cytokinin Ribosides1[W

    PubMed Central

    Kopečná, Martina; Blaschke, Hanna; Kopečný, David; Vigouroux, Armelle; Končitíková, Radka; Novák, Ondřej; Kotland, Ondřej; Strnad, Miroslav; Moréra, Solange; von Schwartzenberg, Klaus

    2013-01-01

    We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism. PMID:24170203

  1. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences

    NASA Technical Reports Server (NTRS)

    Nordheim, A.; Rich, A.

    1983-01-01

    Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50-80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

  2. A simple HPLC method for determining the purine content of beer and beer-like alcoholic beverages.

    PubMed

    Fukuuchi, Tomoko; Yasuda, Makoto; Inazawa, Katsunori; Ota, Tatsuhiro; Yamaoka, Noriko; Mawatari, Ken-ichi; Nakagomi, Kazuya; Kaneko, Kiyoko

    2013-01-01

    Several methods for quantifying the purine content in food and drink have been described using high-performance liquid chromatography (HPLC). We have developed an improved HPLC method that is based on a method reported by Kaneko et al. and that is more sensitive yet simple, and suitable for determining the purine content of beer and beer-like alcoholic beverages. Quantitative HPLC separation was performed on a Shodex Asahi Pak GS-320HQ column with an isocratic elution of 150 mmol/L sodium phosphate buffer (H(3)PO(4)/NaH(2)PO(4) = 20:100 (v/v)). The retention times for the four analytes, namely, adenine, guanine, hypoxanthine and xanthine, were 19.9, 25.0, 29.3 and 43.0 min, respectively. The resolution was good, and there was no excessive interference from the other compounds in the beverages at these retention times. Furthermore, the detection limit for all the analytes was improved to less than 0.0075 mg/L, and all the calibration curves showed good linearity (r(2) > 0.999) between 0.013 and 10 mg/L for adenine and guanine, and between 0.025 and 10 mg/L for hypoxanthine and xanthine. The pretreatment was simplified by removing some procedures and optimizing the perchloric acid hydrolysis and the enzymatic peak-shift assay. We reduced the sample dilution rate by almost 50%, and the time spent on pretreatment from 4 days to only 180 min. The recovery of the analytes from spiked samples was 94.8 - 103.8%. This method may be useful for evaluating quantitative and qualitative differences in the purine content of beer and beer-like alcoholic beverages.

  3. Study of Copper and Purine-Copper Complexes on Modified Carbon Electrodes by Cyclic and Elimination Voltammetry.

    PubMed

    Trnkova, Libuse; Zerzankova, Lenka; Dycka, Filip; Mikelova, Radka; Jelen, Frantisek

    2008-01-24

    Using a paraffin impregnated graphite electrode (PIGE) and mercury-modifiedpyrolytic graphite electrode with basal orientation (Hg-PGEb) copper(II) and Cu(II)-DNApurine base solutions have been studied by cyclic (CV) and linear sweep voltammetry(LSV) in connection with elimination voltammetry with linear scan (EVLS). In chlorideand bromide solutions (pH 6), the redox process of Cu(II) proceeded on PIGE with twocathodic and two anodic potentially separated signals. According to the eliminationfunction E4, the first cathodic peak corresponds to the reduction Cu(II) e⁻ → Cu(I) withthe possibility of fast disproportionation 2Cu(I) → Cu(II) Cu(0). The E4 of the secondcathodic peak signalized an electrode process controlled by a surface reaction. Theelectrode system of Cu(II) on Hg-PGEb in borate buffer (pH 9.2) was characterized by onecathodic and one anodic peak. Anodic stripping voltammetry (ASV) on PIGE and cathodicstripping voltammetry (CSV) on Hg-PGEb were carried out at potentials where thereduction of copper ions took place and Cu(I)-purine complexes were formed. By usingASV and CSV in combination with EVLS, the sensitivity of Cu(I)-purine complexdetection was enhanced relative to either ASV or CSV alone, resulting in higher peakcurrents of more than one order of magnitude. The statistical treatment of CE data wasused to determine the reproducibility of measurements. Our results show that EVLS inconnection with the stripping procedure is useful for both qualitative and quantitativemicroanalysis of purine derivatives and can also reveal details of studied electrodeprocesses.

  4. [Separation of purines, pyrimidines, pterins and flavonoids on magnolol-bonded silica gel stationary phase by high performance liquid chromatography].

    PubMed

    Chen, Hong; Li, Laishen; Zhang, Yang; Zhou, Rendan

    2012-10-01

    A new magnolol-bonded silica gel stationary phase (MSP) was used to separate the basic drugs including four purines, eight pyrimidines, four pterins and five flavonoids as polar representative samples by high performance liquid chromatography (HPLC). To clarify the separation mechanism, a commercial ODS column was also tested under the same chromatographic conditions. The high selectivities and fast baseline separations of the above drugs were achieved by using simple mobile phases on MSP. Although there is no end-caped treatment, the peak shapes of basic drugs containing nitrogen such as purines, pyrimidines and pterins were rather symmetrical on MSP, which indicated the the magnolol as ligand with multi-sites could shield the side effect of residual silanol groups on the surface of silica gel. Although somewhat different in the separation resolution, it was found that the elution orders of some drugs were generally similar on both MSP and ODS. The hydrophobic interaction should play a significant role in the separations of the above basic drugs, which was attributed to their reversed-phase property in the nature. However, MSP could provide the additional sites for many polar solutes, which was a rational explanation for the high selectivity of MSP. For example, in the separation of purines, pyrimidines and pterins on MSP, hydrogen-bonding and dipole-dipole interactions played leading roles besides hydrophobic interaction. Some solute molecules (such as mercaptopurine, vitexicarpin) and MSP can form the strong pi-pi stacking in the separation process. All enhanced the retention and improved the separation selectivity of MSP, which facilitated the separation of the basic drugs.

  5. Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib).

    PubMed

    Ritzel, M W; Ng, A M; Yao, S Y; Graham, K; Loewen, S K; Smith, K M; Ritzel, R G; Mowles, D A; Carpenter, P; Chen, X Z; Karpinski, E; Hyde, R J; Baldwin, S A; Cass, C E; Young, J D

    2001-01-26

    The human concentrative (Na(+)-linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na(+)-nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691- and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na(+)-dependent cib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na(+) concentration indicated a Na(+):uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetate-induced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element.

  6. Hetero-ring-expansion design for purine analogs: A theoretical study on the structural, electronic, and excited-state properties

    NASA Astrophysics Data System (ADS)

    Zhang, Laibin; Zhou, Liuzhu; Tian, Jianxiang; Li, Xiaoming

    2014-03-01

    A series of hetero-ring-expanded purine analogs are designed and their structural, electronic and excited-state properties are investigated by DFT calculations. The results indicate that the designed analogs can form stable base pairs with natural counterparts. Compared with natural ones, these size-expanded analogs and corresponding base pairs have smaller ionization potentials and HOMO-LUMO gaps. Furthermore, the A-analogs have ionization potentials even lower than natural G. Finally, the electronic absorption spectra are calculated and the nature of the low-lying excited states is discussed. These observations imply their promising applications as molecular wires and new DNA motifs.

  7. Methanocarba ring as a ribose modification in ligands of G protein-coupled purine and pyrimidine receptors: synthetic approaches

    PubMed Central

    Tosh, Dilip K.

    2015-01-01

    Adenosine receptors (ARs) and P2Y receptors for purine and pyrimidine nucleotides have widespread distribution and regulate countless physiological processes. Various synthetic ligands are in clinical trials for treatment of inflammatory diseases, pain, cancer, thrombosis, ischemia, and other conditions. The methanocarba (bicyclo[3.1.0]hexane) ring system as a rigid substitution for ribose, which maintains either a North (N) or South (S) conformation, tends to preserve or enhance the potency and/or selectivity for certain receptor subtypes. This review summarizes recent developments in the synthetic approaches to these biologically important nucleoside and nucleotide analogues. PMID:26161251

  8. Sequence dependence of isothermal DNA amplification via EXPAR

    PubMed Central

    Qian, Jifeng; Ferguson, Tanya M.; Shinde, Deepali N.; Ramírez-Borrero, Alissa J.; Hintze, Arend; Adami, Christoph; Niemz, Angelika

    2012-01-01

    Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions. PMID:22416064

  9. Unexpected pronounced heating in the uppermost layer of the Dead Sea after a sharp drop in noon surface solar radiation

    NASA Astrophysics Data System (ADS)

    Kishcha, Pavel; Starobinets, Boris; Gertman, Isaac; Ozer, Tal; Alpert, Pinhas

    2016-04-01

    A passage of frontal cloudiness accompanied by dust pollution over the Judean Mountains and the Dead Sea valley, which occurred on March 22, 2013, led to a sharp drop in noon solar radiation under weak winds (from 860 W m-2 to 50 W m-2). Solar radiation measurements showed that the transition from clear-sky to overcast conditions was sharper over the Dead Sea than over the Israel Mediterranean coast. The maximal rate of decrease in noon solar radiation at the Dead Sea almost doubled that near the Mediterranean coast (17 W m-2 min-1 vs. 10 W m-2 min-1). The temperature stratification was observed in the uppermost layer of the Dead Sea before the aforementioned drop in noon solar radiation. This temperature stratification was evidence that the weak winds were incapable of producing significant mixing in the Dead Sea. Buoy measurements showed that, unexpectedly, a sharp decrease in noon solar radiation caused pronounced heating in the uppermost layer of the Dead Sea. Evaporation from the Dead Sea surface leads to an increase in salinity in the surface layer. In the presence of significant solar radiation, this increased salinity in the surface layer did not lead to an increase in water density. The gravitational stability and temperature stratification in the uppermost layer were observed. By contrast, after the drop in solar radiation, the increased salinity in the surface layer led to an increase in water density and, consequently, to gravitational instability, because of higher density of surface seawater compared to the density in the layers below. The gravitational instability switched on a pronounced heating process in the 2-m uppermost layer of the Dead Sea. This temperature increase took place under weak winds, which were incapable of creating significant mechanical mixing in the Dead Sea. The heating of seawater in the 2-m uppermost layer was switched off later by the sharp influx of hot foehn winds up to 20 m/s from the lee side of the Judean Mts. into the

  10. Modeling, Substrate Docking, and Mutational Analysis Identify Residues Essential for the Function and Specificity of a Eukaryotic Purine-Cytosine NCS1 Transporter*

    PubMed Central

    Krypotou, Emilia; Kosti, Vasiliki; Amillis, Sotiris; Myrianthopoulos, Vassilios; Mikros, Emmanuel; Diallinas, George

    2012-01-01

    The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a three-dimensional model of the Aspergillus nidulans purine-cytosine/H+ FcyB symporter. The model consists of 12 transmembrane α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct core of 10 TMSs is made of two intertwined inverted repeats (TMS1–5 and TMS6–10) that are followed by two additional TMSs. TMS1, TMS3, TMS6, and TMS8 form an open cavity that is predicted to host the substrate binding site. Based on primary sequence alignment, three-dimensional topology, and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; Ser-85 (TMS1), Trp-159, Asn-163 (TMS3), Trp-259 (TMS6), and Asn-354 (TMS8). To validate the role of these and other putatively critical residues, we performed a systematic functional analysis of relevant mutants. We show that the proposed substrate binding residues, plus Asn-350, Asn-351, and Pro-353 are irreplaceable for FcyB function. Among these residues, Ser-85, Asn-163, Asn-350, Asn-351, and Asn-354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that Ser-85, Asn-163, and Asn-354 directly interact with substrates, Trp-159 and Trp-259 stabilize binding through π-π stacking interactions, and Pro-353 affects the local architecture of substrate binding site, whereas Asn-350 and Asn-351 probably affect substrate binding indirectly. Our work is the first systematic approach to address structure-function-specificity relationships in a eukaryotic member of NCS1 family by combining genetic and computational approaches. PMID:22969088

  11. Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution.

    PubMed

    Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi; Fujii, Kentaro; Yokoya, Akinari; Fukuda, Yoshihiro; Saitoh, Yuji

    2014-08-07

    The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5'-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5'-monophosphate, and adenosine 5'-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

  12. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose

    PubMed Central

    Hibbs, John B.; Vavrin, Zdenek; Cox, James E.

    2016-01-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces. PMID:26895212

  13. Functional traits variation explains the distribution of Aextoxicon punctatum (Aextoxicaceae) in pronounced moisture gradients within fog-dependent forest fragments

    PubMed Central

    Salgado-Negret, Beatriz; Canessa, Rafaella; Valladares, Fernando; Armesto, Juan J.; Pérez, Fernanda

    2015-01-01

    Climate change and fragmentation are major threats to world forests. Understanding how functional traits related to drought tolerance change across small-scale, pronounced moisture gradients in fragmented forests is important to predict species’ responses to these threats. In the case of Aextoxicon punctatum, a dominant canopy tree in fog-dependent rain forest patches in semiarid Chile, we explored how the magnitude, variability and correlation patterns of leaf and xylem vessel traits and hydraulic conductivity varied across soil moisture (SM) gradients established within and among forest patches of different size, which are associated with differences in tree establishment and mortality patterns. Leaf traits varied across soil-moisture gradients produced by fog interception. Trees growing at drier leeward edges showed higher leaf mass per area, trichome and stomatal density than trees from the wetter core and windward zones. In contrast, xylem vessel traits (vessels diameter and density) did not vary producing loss of hydraulic conductivity at drier leeward edges. We also detected higher levels of phenotypic integration and variability at leeward edges. The ability of A. punctatum to modify leaf traits in response to differences in SM availability established over short distances (<500 m) facilitates its persistence in contrasting microhabitats within forest patches. However, xylem anatomy showed limited plasticity, which increases cavitation risk at leeward edges. Greater patch fragmentation, together with fluctuations in irradiance and SM in small patches, could result in higher risk of drought-related tree mortality, with profound impacts on hydrological balances at the ecosystem scale. PMID:26257746

  14. Functional traits variation explains the distribution of Aextoxicon punctatum (Aextoxicaceae) in pronounced moisture gradients within fog-dependent forest fragments.

    PubMed

    Salgado-Negret, Beatriz; Canessa, Rafaella; Valladares, Fernando; Armesto, Juan J; Pérez, Fernanda

    2015-01-01

    Climate change and fragmentation are major threats to world forests. Understanding how functional traits related to drought tolerance change across small-scale, pronounced moisture gradients in fragmented forests is important to predict species' responses to these threats. In the case of Aextoxicon punctatum, a dominant canopy tree in fog-dependent rain forest patches in semiarid Chile, we explored how the magnitude, variability and correlation patterns of leaf and xylem vessel traits and hydraulic conductivity varied across soil moisture (SM) gradients established within and among forest patches of different size, which are associated with differences in tree establishment and mortality patterns. Leaf traits varied across soil-moisture gradients produced by fog interception. Trees growing at drier leeward edges showed higher leaf mass per area, trichome and stomatal density than trees from the wetter core and windward zones. In contrast, xylem vessel traits (vessels diameter and density) did not vary producing loss of hydraulic conductivity at drier leeward edges. We also detected higher levels of phenotypic integration and variability at leeward edges. The ability of A. punctatum to modify leaf traits in response to differences in SM availability established over short distances (<500 m) facilitates its persistence in contrasting microhabitats within forest patches. However, xylem anatomy showed limited plasticity, which increases cavitation risk at leeward edges. Greater patch fragmentation, together with fluctuations in irradiance and SM in small patches, could result in higher risk of drought-related tree mortality, with profound impacts on hydrological balances at the ecosystem scale.

  15. Pronounced genetic differentiation and recent secondary contact in the mangrove tree Lumnitzera racemosa revealed by population genomic analyses

    PubMed Central

    Li, Jianfang; Yang, Yuchen; Chen, Qipian; Fang, Lu; He, Ziwen; Guo, Wuxia; Qiao, Sitan; Wang, Zhengzhen; Guo, Miaomiao; Zhong, Cairong; Zhou, Renchao; Shi, Suhua

    2016-01-01

    Systematically investigating the impacts of Pleistocene sea-level fluctuations on mangrove plants may provide a better understanding of their demographic history and useful information for their conservation. Therefore, we conducted population genomic analyses of 88 nuclear genes to explore the population dynamics of a mangrove tree Lumnitzera racemosa across the Indo-West Pacific region. Our results revealed pronounced genetic differentiation in this species between the populations from the Indian Ocean and the Pacific Ocean, which may be attributable to the long-term isolation between the western and eastern coasts of the Malay Peninsula during sea-level drops in the Pleistocene glacial periods. The mixing of haplotypes from the two highly divergent groups was identified in a Cambodian population at almost all 88 nuclear genes, suggesting genetic admixture of the two lineages at the boundary region. Similar genetic admixture was also found in other populations from Southeast Asia based on the Bayesian clustering analysis of six nuclear genes, which suggests extensive and recent secondary contact of the two divergent lineages in Southeast Asia. Computer simulations indicated substantial migration from the Indian Ocean towards the South China Sea, which likely results in the genetic admixture in Southeast Asia. PMID:27380895

  16. Response of urinary purine derivatives excretion to different levels of ruminal glucose infusion in heifers.

    PubMed

    Dickhoefer, Uta; Ahnert, Sandra; Schoof, Hartwig; Moritz, Niels; Susenbeth, Andreas

    2015-01-01

    This study investigated the response of urinary purine derivatives (PD) excretion to increasing levels of intraruminal glucose infusion to evaluate how well this indicator reflects induced changes in microbial crude protein flow. Four rumen-cannulated heifers (482 ± 25 kg body weight) were fed at maintenance energy level with a basal diet (on fresh matter basis) of 4 kg/d hay, 1.5 kg/d concentrate and 60 g/d minerals in two equal meals. The trial comprised a control period (Control I) without glucose infusion followed by four consecutive periods in which all animals received 125 g, 250 g, 500 g or 1000 g/d of glucose, respectively. For this, daily dosages of glucose and urea (90 g/d during all periods) were divided into three portions that were dissolved in water and directly administered into the rumen during morning and afternoon feedings and once during noon. After the highest glucose dosage, a second control period was carried out (Control II). Urinary PD excretion increased with glucose infusion of 125 g/d (71.4 mmol/d) and 1000 g/d (74.2 mmol/d) over the level at Control I (53.9 mmol/d (standard error of the mean (SEM) 3.4; p = 0.012). After withdrawing glucose infusion, PD excretion (79.0 mmol/d) did not return to Control I level (p = 0.001). In contrast, faecal nitrogen (N) excretions linearly increased with incremental glucose infusion (p < 0.001) from 33.9 g/d at Control I to 39.7 g/d (SEM 0.5) at 1000 g/d of glucose and were similar in Control I and II (p = 0.086). The contradicting responses in the excretions of faecal N and urinary PD to increasing glucose infusions highlight the limited accuracy of the PD excretion as a non-invasive indicator when incremental dosages of rapidly fermentable carbohydrates are supplied.

  17. Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids

    PubMed Central

    Summers, Ryan M.; Louie, Tai Man; Yu, Chi-Li; Gakhar, Lokesh; Louie, Kailin C.

    2012-01-01

    The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with N1- and N3-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His6 fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His6 plus His6-NdmD catalyzed N1-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His6 plus His6-NdmD catalyzed N3-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N7-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste. PMID:22328667

  18. Nanopores and nucleic acids: prospects for ultrarapid sequencing

    NASA Technical Reports Server (NTRS)

    Deamer, D. W.; Akeson, M.

    2000-01-01

    DNA and RNA molecules can be detected as they are driven through a nanopore by an applied electric field at rates ranging from several hundred microseconds to a few milliseconds per molecule. The nanopore can rapidly discriminate between pyrimidine and purine segments along a single-stranded nucleic acid molecule. Nanopore detection and characterization of single molecules represents a new method for directly reading information encoded in linear polymers. If single-nucleotide resolution can be achieved, it is possible that nucleic acid sequences can be determined at rates exceeding a thousand bases per second.

  19. Simultaneous determination of purine metabolites, creatinine and pseudouridine in ruminant urine by reversed-phase high-performance liquid chromatography.

    PubMed

    Shingfield, K J; Offer, N W

    1999-02-19

    Determination of purine metabolites, pseudouridine and creatinine in both bovine and ovine urine using high-performance liquid chromatography (HPLC) is described. Following dilution and filtration, urine samples were analysed directly. Separation and quantification was achieved using a Spherisorb ODS II C18 column (250x4.6 mm I.D.) under isocratic conditions. The mobile phase contained 7.5 mM ammonium dihydrogen phosphate, 10 mM sodium 1-heptane sulphonic acid and 1.0 mM triethylamine at pH 3.0. Chromatography was achieved at a flow-rate of 1.0 ml/min and monitoring column effluent at 218 nm. Total analysis time was 60 min. Recovery of all compound standards added to urine was above 96%. In all cases, close spectral matches of compound standards and corresponding identified peaks in ovine and bovine urine were obtained. Lowest detectable concentrations of allantoin, uric acid, xanthine, hypoxanthine, creatinine and pseudouridine were 1.1, 1.0, 1.0, 3.0 and 0.4 micromol/l, respectively. Advantages of simultaneous determination of purine metabolites, creatinine and pseudouridine in ruminant urine collected from both sheep and cattle exist over current methods.

  20. Consortium analysis of gene and gene-folate interactions in purine and pyrimidine metabolism pathways with ovarian carcinoma risk

    PubMed Central

    Kelemen, Linda E.; Terry, Kathryn L.; Goodman, Marc T.; Webb, Penelope M.; Bandera, Elisa V.; McGuire, Valerie; Rossing, Mary Anne; Wang, Qinggang; Dicks, Ed; Tyrer, Jonathan P.; Song, Honglin; Kupryjanczyk, Jolanta; Dansonka-Mieszkowska, Agnieszka; Plisiecka-Halasa, Joanna; Timorek, Agnieszka; Menon, Usha; Gentry-Maharaj, Aleksandra; Gayther, Simon A.; Ramus, Susan J.; Narod, Steven A.; Risch, Harvey A.; McLaughlin, John R.; Siddiqui, Nadeem; Glasspool, Rosalind; Paul, James; Carty, Karen; Gronwald, Jacek; Lubiński, Jan; Jakubowska, Anna; Cybulski, Cezary; Kiemeney, Lambertus A.; Massuger, Leon F. A. G.; van Altena, Anne M.; Aben, Katja K. H.; Olson, Sara H.; Orlow, Irene; Cramer, Daniel W.; Levine, Douglas A.; Bisogna, Maria; Giles, Graham G.; Southey, Melissa C.; Bruinsma, Fiona; Kjær, Susanne Krüger; Høgdall, Estrid; Jensen, Allan; Høgdall, Claus K.; Lundvall, Lene; Engelholm, Svend-Aage; Heitz, Florian; du Bois, Andreas; Harter, Philipp; Schwaab, Ira; Butzow, Ralf; Nevanlinna, Heli; Pelttari, Liisa M.; Leminen, Arto; Thompson, Pamela J.; Lurie, Galina; Wilkens, Lynne R.; Lambrechts, Diether; Van Nieuwenhuysen, Els; Lambrechts, Sandrina; Vergote, Ignace; Beesley, Jonathan; Fasching, Peter A.; Beckmann, Matthias W.; Hein, Alexander; Ekici, Arif B.; Doherty, Jennifer A.; Wu, Anna H.; Pearce, Celeste L.; Pike, Malcolm C.; Stram, Daniel; Chang-Claude, Jenny; Rudolph, Anja; Dörk, Thilo; Dürst, Matthias; Hillemanns, Peter; Runnebaum, Ingo B.; Bogdanova, Natalia; Antonenkova, Natalia; Odunsi, Kunle; Edwards, Robert P.; Kelley, Joseph L.; Modugno, Francesmary; Ness, Roberta B.; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; Orsulic, Sandra; Fridley, Brooke L.; Vierkant, Robert A.; Cunningham, Julie M.; Wu, Xifeng; Lu, Karen; Liang, Dong; Hildebrandt, Michelle A.T.; Weber, Rachel Palmieri; Iversen, Edwin S.; Tworoger, Shelley S.; Poole, Elizabeth M.; Salvesen, Helga B.; Krakstad, Camilla; Bjorge, Line; Tangen, Ingvild L.; Pejovic, Tanja; Bean, Yukie; Kellar, Melissa; Wentzensen, Nicolas; Brinton, Louise A.; Lissowska, Jolanta; Garcia-Closas, Montserrat; Campbell, Ian G.; Eccles, Diana; Whittemore, Alice S.; Sieh, Weiva; Rothstein, Joseph H.; Anton-Culver, Hoda; Ziogas, Argyrios; Phelan, Catherine M.; Moysich, Kirsten B.; Goode, Ellen L.; Schildkraut, Joellen M.; Berchuck, Andrew; Pharoah, Paul D.P.; Sellers, Thomas A.; Brooks-Wilson, Angela; Cook, Linda S.; Le, Nhu D.

    2014-01-01

    Scope We re-evaluated previously reported associations between variants in pathways of one-carbon (folate) transfer genes and ovarian carcinoma (OC) risk, and in related pathways of purine and pyrimidine metabolism, and assessed interactions with folate intake. Methods and Results Odds ratios (OR) for 446 genetic variants were estimated among 13,410 OC cases and 22,635 controls and among 2,281 cases and 3,444 controls with folate information. Following multiple testing correction, the most significant main effect associations were for DPYD variants rs11587873 (OR=0.92, P=6x10−5) and rs828054 (OR=1.06, P=1x10−4). Thirteen variants in the pyrimidine metabolism genes, DPYD, DPYS, PPAT and TYMS, also interacted significantly with folate in a multi-variant analysis (corrected P=9.9x10−6) but collectively explained only 0.2% of OC risk. Although no other associations were significant after multiple testing correction, variants in SHMT1 in one-carbon transfer, previously reported with OC, suggested lower risk at higher folate (Pinteraction=0.03-0.006). Conclusions Variation in pyrimidine metabolism genes, particularly DPYD, which was previously reported to be associated with OC, may influence risk; however, stratification by folate intake is unlikely to modify disease risk appreciably in these women. SHMT1 SNP-byfolate interactions are plausible but require further validation. Polymorphisms in selected genes in purine metabolism were not associated with OC. PMID:25066213

  1. Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk.

    PubMed

    Ishikawa, Toshihisa; Aw, Wanping; Kaneko, Kiyoko

    2013-11-04

    In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid) in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs), i.e., 421C>A (major) and 376C>T (minor), in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

  2. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates.

    PubMed

    López-Zavala, Alonso A; Quintero-Reyes, Idania E; Carrasco-Miranda, Jesús S; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R

    2014-09-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.

  3. The pharmacological role of phosphatases (acid and alkaline phosphomonoesterases) in snake venoms related to release of purines - a multitoxin.

    PubMed

    Dhananjaya, Bhadrapura L; D'Souza, Cletus J M

    2011-02-01

    Snake venom components, acting in concert in the prey, cause their immobilization and initiate digestion. To achieve this, several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as phosphatases (acid and alkaline phosphomonoesterases) are less studied and their pharmacological role in venoms is not clearly defined. Also, they show overlapping substrate specificities and have other common biochemical properties causing uncertainty about their identity in venoms. The near-ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It appears that these enzymes may play a central role in liberating purines (mainly adenosine) - a multitoxin and through the action of purines help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities as venom enzymes have been evolved to interfere in diverse physiological processes. This has not been verified by pharmacological studies using purified enzymes. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established.

  4. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

    PubMed Central

    López-Zavala, Alonso A.; Quintero-Reyes, Idania E.; Carrasco-Miranda, Jesús S.; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.

    2014-01-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012 ▶), J. Bioenerg. Biomembr. 44, 325–331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2′-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

  5. Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress

    PubMed Central

    Yamauchi, Yukina; Izawa, Shingo

    2016-01-01

    Severe ethanol stress (>9% ethanol, v/v) as well as glucose deprivation rapidly induces a pronounced repression of overall protein synthesis in budding yeast Saccharomyces cerevisiae. Therefore, transcriptional activation in yeast cells under severe ethanol stress does not always indicate the production of expected protein levels. Messenger RNAs of genes containing heat shock elements can be intensively translated under glucose deprivation, suggesting that some mRNAs are preferentially translated even under severe ethanol stress. In the present study, we tried to identify the mRNA that can be preferentially translated under severe ethanol stress. BTN2 encodes a v-SNARE binding protein, and its null mutant shows hypersensitivity to ethanol. We found that BTN2 mRNA was efficiently translated under severe ethanol stress but not under mild ethanol stress. Moreover, the increased Btn2 protein levels caused by severe ethanol stress were smoothly decreased with the elimination of ethanol stress. These findings suggested that severe ethanol stress extensively induced BTN2 expression. Further, the BTN2 promoter induced protein synthesis of non-native genes such as CUR1, GIC2, and YUR1 in the presence of high ethanol concentrations, indicating that this promoter overcame severe ethanol stress-induced translation repression. Thus, our findings provide an important clue about yeast response to severe ethanol stress and suggest that the BTN2 promoter can be used to improve the efficiency of ethanol production and stress tolerance of yeast cells by modifying gene expression in the presence of high ethanol concentration. PMID:27602028

  6. Pronounced Minima in Tropospheric Ozone and OH above the Tropical West Pacific and their Role for Stratospheric Composition

    NASA Astrophysics Data System (ADS)

    Rex, M.; Wohltmann, I.; Lehmann, R.; Rosenlof, K. H.; Wennberg, P. O.; Weisenstein, D. K.; Notholt, J.; Krüger, K.; Mohr, V.; Tegtmeier, S.

    2014-12-01

    Hundreds of organic species are emitted into the atmosphere mostly from biogenic processes. The rapid breakdown by reactions with OH radicals prevents most of them from reaching the stratosphere. Hence, the omnipresent layer of OH in the troposphere shields the stratosphere from these emissions and is particularly relevant for those species that do not photolyse efficiently. Reactions involving ozone are a strong source of OH in clean tropical air. Hence the OH concentration is closely coupled to ozone abundances. The Western Pacific warm pool is key for troposphere to stratosphere exchange. We report measurements of 14 ozonesondes launched during the Transbrom ship cruise through the center of the warm pool. During a 2500km portion of the ship track between 10S and 15N we found ozone concentrations below the detection limit of the sondes throughout the troposphere. We will discuss the uncertainties of ozonesonde measurements at very low ozone concentrations, the robustness of our observations and the upper limit of the ozone concentration that would be consistent with our raw data. Based on modelling and measurements of OH on the ER-2 during the STRAT campaign we suggest that there also is a pronounced minimum in the tropospheric column of OH over the tropical West Pacific. We show that this increases the lifetime of chemical species and has the potential to amplify the impact of surface emissions on the stratospheric composition. Specifically, we discuss the role of emissions of biogenic halogenated species from this geographic region for stratospheric ozone depletion. Also, we discuss the potential role of increasing anthropogenic emissions of SO2 in South East Asia or from minor volcanic eruptions for the stratospheric aerosol budget.

  7. Near-surface mixing and pronounced deep-water stratification in a compartmentalised, human-disturbed atoll lagoon system

    NASA Astrophysics Data System (ADS)

    Gardner, J. P. A.; Garton, D. W.; Collen, J. D.

    2011-03-01

    Palmyra Atoll has four partially isolated lagoons up to 50 m in depth, each with complex and variable bottom topographies. Measurements of depth, temperature, salinity, turbidity and dissolved oxygen (DO) revealed a well-mixed shallow surface layer (0-10 m depth) and below that pronounced stratification of DO in the absence of a pycnocline. Turbidity increased in a step-like manner at ~25 m depth, at the oxycline. For all deep sections of the lagoon (>30 m), DO declined uniformly to 0% saturation. As determined from filtration, mass of particulates was independent of depth. Surface mixing and deep-water stratification are both stable at different temporal scales, including day versus night, daily, weekly and annually. We suggest that lagoon circulation is represented by a shallow, westward-moving surface layer of well-to-partially mixed water with high DO and low turbidity, underlain by a relatively static and temporally stable layer with low to zero DO and elevated turbidity. This is the first report of such conditions within a deep lagoon system, and only the second report of anoxic conditions in any such system. In deep-water, stable euxinic conditions reflect bottom topography, with dysoxic and anoxic water being constrained within silled basins. The occurrence and depth of large volumes of sediment-laden and dysoxic/anoxic water need to be considered in management proposals designed to increase water flow through the lagoon. These novel water column conditions most probably arose as a consequence of military construction work, consistent with published reports of profound changes to the atoll during 1940-1945. If so, they highlight the need to better understand the possible consequences of cutting channels and modification of lagoon flow at many atolls across the central Pacific Ocean.

  8. Metabolic physiology of the Humboldt squid, Dosidicus gigas: Implications for vertical migration in a pronounced oxygen minimum zone

    NASA Astrophysics Data System (ADS)

    Rosa, Rui; Seibel, Brad A.

    2010-07-01

    The Humboldt (or jumbo) squid, Dosidicus gigas, is an active predator endemic to the Eastern Pacific that undergoes diel vertical migrations into a pronounced oxygen minimum layer (OML). Here, we investigate the physiological mechanisms that facilitate these migrations and assess the associated costs and benefits. Exposure to hypoxic conditions equivalent to those found in the OML (∼10 μM O 2 at 10 °C) led to a significant reduction in the squid’s routine metabolic rate (RMR), from 8.9 to 1.6 μmol O 2 g -1 h -1 ( p < 0.05), and a concomitant increase in mantle muscle octopine levels (from 0.50 to 5.24 μmol g -1 tissue, p < 0.05). Enhanced glycolitic ATP production accounted for only 7.0% and 2.8% at 10 °C and 20 °C, respectively, of the energy deficit that resulted from the decline in aerobic respiration. The observed metabolic suppression presumably extends survival time in the OML by conserving the finite stores of fermentable substrate and avoiding the accumulation of the deleterious anaerobic end products in the tissues. RMR increased significantly with temperature ( p < 0.05), from 8.9 (at 10 °C) to 49.85 μmol O 2 g -1 h -1 (at 25 °C) which yielded a Q10 of 2.0 between 10 and 20 °C and 7.9 between 20 and 25 °C ( p < 0.05). These results suggest that 25 °C, although within the normal surface temperature range in the Gulf of California, is outside this species’ normal temperature range. By following the scattering layer into oxygen-enriched shallow water at night, D. gigas may repay any oxygen debt accumulated during the daytime. The dive to deeper water may minimize exposure to stressful surface temperatures when most prey have migrated to depth during the daytime. The physiological and ecological strategies demonstrated here may have facilitated the recent range expansion of this species into northern waters where expanding hypoxic zones prohibit competing top predators.

  9. The genome-wide DNA sequence specificity of the anti-tumour drug bleomycin in human cells.

    PubMed

    Murray, Vincent; Chen, Jon K; Tanaka, Mark M

    2016-07-01

    The cancer chemotherapeutic agent, bleomycin, cleaves DNA at specific sites. For the first time, the genome-wide DNA sequence specificity of bleomycin breakage was determined in human cells. Utilising Illumina next-generation DNA sequencing techniques, over 200 million bleomycin cleavage sites were examined to elucidate the bleomycin genome-wide DNA selectivity. The genome-wide bleomycin cleavage data were analysed by four different methods to determine the cellular DNA sequence specificity of bleomycin strand breakage. For the most highly cleaved DNA sequences, the preferred site of bleomycin breakage was at 5'-GT* dinucleotide sequences (where the asterisk indicates the bleomycin cleavage site), with lesser cleavage at 5'-GC* dinucleotides. This investigation also determined longer bleomycin cleavage sequences, with preferred cleavage at 5'-GT*A and 5'- TGT* trinucleotide sequences, and 5'-TGT*A tetranucleotides. For cellular DNA, the hexanucleotide DNA sequence 5'-RTGT*AY (where R is a purine and Y is a pyrimidine) was the most highly cleaved DNA sequence. It was striking that alternating purine-pyrimidine sequences were highly cleaved by bleomycin. The highest intensity cleavage sites in cellular and purified DNA were very similar although there were some minor differences. Statistical nucleotide frequency analysis indicated a G nucleotide was present at the -3 position (relative to the cleavage site) in cellular DNA but was absent in purified DNA.

  10. Separation and identification of purine nucleosides in the urine of patients with malignant cancer by reverse phase liquid chromatography/electrospray tandem mass spectrometry.

    PubMed

    Li, Hua-Yu; Wang, Shao-Min; Liu, Hong-Min; Bu, Shan-Shan; Li, Juan; Han, Dong; Zhang, Ming-zhi; Wu, Guang-Yin

    2009-05-01

    Urinary-modified nucleosides have a potential role as cancer biomarkers for a number of malignant diseases. High performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, MS/MS analysis and accurate mass measurements in order to identify purine nucleosides purified from urine. Potential purine nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the mass spectrometric techniques. In this manner, numerous modified purine nucleosides were identified in the urine samples from cancer patients including xanthine, adenosine, N1-methyladenosine, 5'-deoxy-5'-methylthioadenosine, 2-methyladenosine, N6-threonylcarbamoyladenosine, inosine, N1-methylinosine, guanosine, N1-methylguanosine, N7-methylguanine, N2-methylguanosine, N2,N2-dimethyguanosine, N2,N2,N7-trimethylguanosine. Furthermore, a number of novel purine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data including N3-methyladenosine, N7-methyladenine, 5'-dehydro-2'-deoxyinosine, N3-methylguanine, O6-methylguanosine, N1,N2,N7-trimethylguanosine, N1-methyl-N2-ethylguanosine and N7-methyl-N1-ethylguanosine.

  11. Integrative Analysis of Circadian Transcriptome and Metabolic Network Reveals the Role of De Novo Purine Synthesis in Circadian Control of Cell Cycle

    PubMed Central

    Li, Ying; Li, Guang; Görling, Benjamin; Luy, Burkhard; Du, Jiulin; Yan, Jun

    2015-01-01

    Metabolism is the major output of the circadian clock in many organisms. We developed a computational method to integrate both circadian gene expression and metabolic network. Applying this method to zebrafish circadian transcriptome, we have identified large clusters of metabolic genes containing mostly genes in purine and pyrimidine metabolism in the metabolic network showing similar circadian phases. Our metabolomics analysis found that the level of inosine 5'-monophosphate (IMP), an intermediate metabolite in de novo purine synthesis, showed significant circadian oscillation in larval zebrafish. We focused on IMP dehydrogenase (impdh), a rate-limiting enzyme in de novo purine synthesis, with three circadian oscillating gene homologs: impdh1a, impdh1b and impdh2. Functional analysis revealed that impdh2 contributes to the daily rhythm of S phase in the cell cycle while impdh1a contributes to ocular development and pigment synthesis. The three zebrafish homologs of impdh are likely regulated by different circadian transcription factors. We propose that the circadian regulation of de novo purine synthesis that supplies crucial building blocks for DNA replication is an important mechanism conferring circadian rhythmicity on the cell cycle. Our method is widely applicable to study the impact of circadian transcriptome on metabolism in complex organisms. PMID:25714999

  12. Complete assignment of the (1)H and (13)C NMR spectra of cis and trans isonucleoside derivatives of purine with a tetrahydropyran ring.

    PubMed

    Besada, Pedro; Costas, Tamara; Terán, Carmen

    2010-06-01

    (1)H and (13)C NMR chemical shifts of cis and trans isonucleoside analogues of purine in which the furanose moiety is substituted by a tetrahydropyran ring were completely assigned using one- and two-dimensional NMR experiments that include NOE, DEPT, COSY and HSQC. The significant (1)H and (13)C NMR signals differentiating between the cis and trans stereoisomers were compared.

  13. Wobble↔Watson-Crick tautomeric transitions in the homo-purine DNA mismatches: a key to the intimate mechanisms of the spontaneous transversions.

    PubMed

    Brovarets', Ol'ha O; Hovorun, Dmytro M

    2015-01-01

    The intrinsic capability of the homo-purine DNA base mispairs to perform wobble↔Watson-Crick/Topal-Fresco tautomeric transitions via the sequential intrapair double proton transfer was discovered for the first time using QM (MP2/DFT) and QTAIM methodologies that are crucial for understanding the microstructural mechanisms of the spontaneous transversions.

  14. Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

    PubMed

    Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

    2014-04-09

    As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use.

  15. Genome Sequencing.

    PubMed

    Verma, Mansi; Kulshrestha, Samarth; Puri, Ayush

    2017-01-01

    Genome sequencing is an important step toward correlating genotypes with phenotypic characters. Sequencing technologies are important in many fields in the life sciences, including functional genomics, transcriptomics, oncology, evolutionary biology, forensic sciences, and many more. The era of sequencing has been divided into three generations. First generation sequencing involved sequencing by synthesis (Sanger sequencing) and sequencing by cleavage (Maxam-Gilbert sequencing). Sanger sequencing led to the completion of various genome sequences (including human) and provided the foundation for development of other sequencing technologies. Since then, various techniques have been developed which can overcome some of the limitations of Sanger sequencing. These techniques are collectively known as "Next-generation sequencing" (NGS), and are further classified into second and third generation technologies. Although NGS methods have many advantages in terms of speed, cost, and parallelism, the accuracy and read length of Sanger sequencing is still superior and has confined the use of NGS mainly to resequencing genomes. Consequently, there is a continuing need to develop improved real time sequencing techniques. This chapter reviews some of the options currently available and provides a generic workflow for sequencing a genome.

  16. Role of Achiral Nucleobases in Multicomponent Chiral Self-Assembly: Purine-Triggered Helix and Chirality Transfer.

    PubMed

    Deng, Ming; Zhang, Li; Jiang, Yuqian; Liu, Minghua

    2016-11-21

    Chiral self-assembly is a basic process in biological systems, where many chiral biomolecules such as amino acids and sugars play important roles. Achiral nucleobases usually covalently bond to saccharides and play a significant role in the formation of the double helix structure. However, it remains unclear how the achiral nucleobases can function in chiral self-assembly without the sugar modification. Herein, we have clarified that purine nucleobases could trigger N-(9-fluorenylmethox-ycarbonyl) (Fmoc)-protected glutamic acid to self-assemble into helical nanostructures. Moreover, the helical nanostructure could serve as a matrix and transfer the chirality to an achiral fluorescence probe, thioflavin T (ThT). Upon chirality transfer, the ThT showed not only supramolecular chirality but also circular polarized fluorescence (CPL). Without the nucleobase, the self-assembly processes cannot happen, thus providing an example where achiral molecules played an essential role in the expression and transfer of the chirality.

  17. A Canonical Correlation Analysis of AIDS Restriction Genes and Metabolic Pathways Identifies Purine Metabolism as a Key Cooperator

    PubMed Central

    Ye, Hanhui; Yuan, Jinjin; Wang, Zhengwu; Huang, Aiqiong; Liu, Xiaolong; Han, Xiao; Chen, Yahong

    2016-01-01

    Human immunodeficiency virus causes a severe disease in humans, referred to as immune deficiency syndrome. Studies on the interaction between host genetic factors and the virus have revealed dozens of genes that impact diverse processes in the AIDS disease. To resolve more genetic factors related to AIDS, a canonical correlation analysis was used to determine the correlation between AIDS restriction and metabolic pathway gene expression. The results show that HIV-1 postentry cellular viral cofactors from AIDS restriction genes are coexpressed in human transcriptome microarray datasets. Further, the purine metabolism pathway comprises novel host factors that are coexpressed with AIDS restriction genes. Using a canonical correlation analysis for expression is a reliable approach to exploring the mechanism underlying AIDS. PMID:27462363

  18. Purine biosynthetic enzyme ATase2 is involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis.

    PubMed

    Yang, Zhipan; Shang, Zengzhen; Wang, Lei; Lu, Qingtao; Wen, Xiaogang; Chi, Wei; Zhang, Lixin; Lu, Congming

    2015-12-01

    To investigate the molecular mechanism of chloroplast biogenesis and development, we characterized an Arabidopsis mutant (dg169, delayed greening 169) which showed growth retardation and delayed greening phenotype in leaves. Newly emerged chlorotic leaves recovered gradually with leaf development in the mutant, and the mature leaves showed similar phenotype to those of wild-typewild-type plants. Compared with wild-type, the chloroplasts were oval-shaped and smaller and the thylakoid membranes were less abundant in yellow section of young leaves of dg169. In addition, the functions of photosystem II (PSII) and photosystem I (PSI) were also impaired. Furthermore, the amount of core subunits of PSII and PSI, as well as PSII and PSI complexes reduced in yellow section of young leaves of dg169. Map-based positional cloning identified that phenotype of dg169 was attributed to a point mutation of ATase2 which converts the conserved Ile-155 residue to Asn. ATase2 catalyzes the first step of de novo purine biosynthesis. This mutation resulted in impaired purine synthesis and a significant decrease in ATP, ADP, GTP and GDP contents. The analysis of ATase2-GFP protein fusion showed that ATase2 was localized to nucleoid of chloroplasts. Our results further demonstrated that the levels of PEP-dependent transcripts in yellow section of young leaves of dg169 were decreased while NEP-dependent and both PEP- and NEP-dependent transcripts and chloroplast DNA replications were increased. The results in this study suggest that ATase2 plays an essential role in early chloroplast development through maintaining PEP function.

  19. Purine biosynthesis in L1210 leukemia cells is inhibited by 7-hydroxymethotrexate (7-OH-MTX) polyglutamates (PGS)

    SciTech Connect

    Seither, R.L.; Matherly, L.H.; Goldman, I.D.

    1986-05-01

    The biochemical basis for 7-OH-MTX cytotoxicity was examined in L1210 tumor cells. Cells were exposed to 100 ..mu..M 7-OH-MTX (approx. 50% growth inhibition) or 10 ..mu..M methotrexate (MTX) (approx. 95% growth inhibition) for 6 hrs to allow high levels of PGS to accumulate. Dihydrofolate reductase (DHFR) activity was assessed by dihydrofolate (FH/sub 2/) pools labeled with 5-formyl-(/sup 3/H)-tetrahydrofolate (5..mu..M) or /sup 3/H-folic acid (1 ..mu..M). FH/sub 2/ was not elevated above control levels in 7-OH-MTX treated cells, in contrast to MTX treated cells in which FH/sub 2/ increased 4- to 7-fold. /sup 3/H-Deoxyuridine incorporation into DNA was not inhibited in cells containing high levels (11.5 nmol/g dry wt.) of 7-OH-MTX tetraglutamate (7-OH-4-NH/sub 2/-10-CH/sub 3/-PteGlu/sub 4/), well in excess of the DHFR-binding capacity (7.3 +/- 0.9 nmol/g), indicating a normal rate of thymidylate synthesis. Although small amounts of 7-OH-MTX and its PGS were bound to DHFR in L1210 cells, as assessed by gel filtration, there was evidence for the preferential binding of 7-OH-MTX tetraglutamate. In all cases this was well below the DHFR binding capacity, consistent with normal rates of deoxyuridine metabolism and FH/sub 2/ levels in the cell. Incorporation of /sup 14/C-formate (60 min) into thymidylate and amino acids was unaffected by 7-OH-MTX, yet incorporation into purines was inhibited over 50%, supporting a block(s) in de novo purine biosynthesis.

  20. Highly conserved repetitive DNA sequences are present at human centromeres.

    PubMed Central

    Grady, D L; Ratliff, R L; Robinson, D L; McCanlies, E C; Meyne, J; Moyzis, R K

    1992-01-01

    Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5). The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere. Images PMID:1542662

  1. How Pronounced Is Income Inequality around the World--and How Can Education Help Reduce It? Education Indicators in Focus. No. 4

    ERIC Educational Resources Information Center

    OECD Publishing (NJ1), 2012

    2012-01-01

    How pronounced is income inequality around the world--and how can education help reduce it? This paper reports the following: (1) Across OECD (Organisation for Economic Cooperation and Development) countries, the average income of the richest 10% of the population was about nine times that of the poorest 10% before the onset of the global economic…

  2. A robust approach to correct for pronounced errors in temperature measurements by controlling radiation damping feedback fields in solution NMR.

    PubMed

    Wolahan, Stephanie M; Li, Zhao; Hsu, Chao-Hsiung; Huang, Shing-Jong; Clubb, Robert; Hwang, Lian-Pin; Lin, Yung-Ya

    2014-11-01

    Accurate temperature measurement is a requisite for obtaining reliable thermodynamic and kinetic information in all NMR experiments. A widely used method to calibrate sample temperature depends on a secondary standard with temperature-dependent chemical shifts to report the true sample temperature, such as the hydroxyl proton in neat methanol or neat ethylene glycol. The temperature-dependent chemical shift of the hydroxyl protons arises from the sensitivity of the hydrogen-bond network to small changes in temperature. The frequency separation between the alkyl and the hydroxyl protons are then converted to sample temperature. Temperature measurements by this method, however, have been reported to be inconsistent and incorrect in modern NMR, particularly for spectrometers equipped with cryogenically-cooled probes. Such errors make it difficult or even impossible to study chemical exchange and molecular dynamics or to compare data acquired on different instruments, as is frequently done in biomolecular NMR. In this work, we identify the physical origins for such errors to be unequal amount of dynamical frequency shifts on the alkyl and the hydroxyl protons induced by strong radiation damping (RD) feedback fields. Common methods used to circumvent RD may not suppress such errors. A simple, easy-to-implement solution was demonstrated that neutralizes the RD effect on the frequency separation by a "selective crushing recovery" pulse sequence to equalize the transverse magnetization of both spin species. Experiments using cryoprobes at 500 MHz and 800 MHz demonstrated that this approach can effectively reduce the errors in temperature measurements from about ±4.0 K to within ±0.4 K in general.

  3. Murine norovirus (MNV-1) exposure in vitro to the purine nucleoside analog Ribavirin increases quasispecies diversity.

    PubMed

    Julian, Timothy R; Baugher, Joseph D; Rippinger, Christine M; Pinekenstein, Rebecca; Kolawole, Abimbola O; Mehoke, Thomas S; Wobus, Christiane E; Feldman, Andrew B; Pineda, Fernando J; Schwab, Kellogg J

    2016-01-04

    Ribavirin is a pharmaceutical antiviral used for the treatment of RNA virus infections including norovirus, hepatitis C virus, hepatitis E virus, Lassa virus, respiratory syncytial virus, and rhinovirus. Despite the drug's history and documented efficacy, the antiviral mechanism of Ribavirin remains unclear. Mechanisms proposed include depletion of the intracellular GTP pool, immunomodulatory effects, induction of error catastrophe, inhibition of viral polymerase activity, and/or inhibition of viral capping. In the present study, we leveraged deep sequencing data to demonstrate that Ribavirin increases murine norovirus (MNV-1) viral diversity. By serial passaging MNV-1 in RAW 264.7 cells for twenty generations in the presence of Ribavirin, we demonstrated statistically significant increases in both the number of unique haplotypes and the average pairwise difference (APD). Based on statistically significant differences in the probability of nucleotide mutations based on Roche 454 sequencing, we also demonstrated that single nucleotide substitutions are increased in the presence of Ribavirin. Finally, we demonstrated Ribavirin's impact on statistically significantly reducing the relative proportion of the dominant sequence within the quasispecies.

  4. Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

    PubMed Central

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-01-01

    The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. PMID:9410897

  5. Synthesis and structure-activity relationships of 2-substituted-6-(dimethylamino)-9-(4-methylbenzyl)-9H-purines with antirhinovirus activity.

    PubMed

    Kelley, J L; Linn, J A; Selway, J W

    1989-01-01

    A series of 2-substituted-6-(dimethylamino)-9-(4-methylbenzyl)-9H-purines where the 2-substituent was H, F, Cl, CF3, CH3, CH2CH3, NH2, NHCH3, N(CH3)2, SCH3, or SO2CH3 was synthesized and tested for antirhinovirus activity to evaluate the effect of 2-substituents on antiviral activity. Intuitive and quantitative structure-activity relationship (QSAR) analysis showed that optimum antirhinovirus serotype 1B activity was associated with 9-benzylpurines that contained a C-2 lipophilic, electron-withdrawing substituent. The most active compound, 6-(dimethylamino)-9-(4-methylbenzyl)-2-(trifluoromethyl)-9H-purine (14), had an IC50 = 0.03 microM against serotype 1B, but its activity against 18 other serotypes was not uniform; the IC50s ranged over 260-fold.

  6. The immunosuppressives FK 506 and cyclosporin A inhibit the generation of protein factors binding to the two purine boxes of the interleukin 2 enhancer.

    PubMed Central

    Brabletz, T; Pietrowski, I; Serfling, E

    1991-01-01

    Like Cyclosporin A (CsA), the macrolide FK 506 is a potent immunosuppressive that inhibits early steps of T cell activation, including the synthesis of Interleukin 2 (II-2) and numerous other lymphokines. The block of II-2 synthesis occurs at the transcriptional level. At concentrations that block T cell activation, FK 506 and CsA inhibit the proto-enhancer activity of Purine boxes of the II-2 promoter and the generation of lymphocyte-specific factors binding to the Purine boxes. Under the same conditions, the DNA binding of other II-2 enhancer factors remains unaffected by both compounds. These results support the view that FK 506 and CsA, which both inhibit the activity of peptidylprolyl cis/trans isomerases, suppress T cell activation by a similar, if not identical mechanism. Images PMID:1707162

  7. X-ray structure, NMR and stability-in-solution study of 6-(furfurylamino)-9-(tetrahydropyran-2-yl)purine - A new active compound for cosmetology

    NASA Astrophysics Data System (ADS)

    Walla, Jan; Szüčová, Lucie; Císařová, Ivana; Gucký, Tomáš; Zatloukal, Marek; Doležal, Karel; Greplová, Jarmila; Massino, Frank J.; Strnad, Miroslav

    2010-06-01

    The crystal and molecular structure of 6-(furfurylamino)-9-(tetrahydropyran-2-yl)purine ( 1) was determined at 150(2) K. The compound crystallizes in monoclinic P2 1/ c space group with a = 10.5642(2), b = 13.6174(3), c = 10.3742(2) Å, V = 1460.78(5) Å 3, Z = 4, R( F) = for 3344 unique reflections. The purine moiety and furfuryl ring are planar and the tetrahydropyran-2-yl is disordered in the ratio 1:3, probably due to the chiral carbon atom C(17). The individual 1H and 13C NMR signals were assigned by 2D correlation experiments such as 1H- 1H COSY and ge-2D HSQC. Stability-in-solution was determined in methanol/water in acidic pH (3-7).

  8. Crystal structure of disodium 2-amino-6-oxo-6,7-di-hydro-1H-purine-1,7-diide hepta-hydrate.

    PubMed

    Gur, Dvir; Shimon, Linda J W

    2015-03-01

    In the title compound, disodium 2-amino-6-oxo-6,7-di-hydro-1H-purine-1,7-diide hepta-hydrate, 2Na(+)·C5H3N5O(2-)·7H2O, the structure is composed of alternating (100) layers of guanine mol-ecules and hydrated Na(+) ions. Within the guanine layer, the mol-ecules are arranged in centrosymmetric pairs, with a partial overlap between the guanine rings. In this compound, guanine exists as the amino-keto tautomer from which deprotonation from N1 and N7 has occurred (purine numbering). There are no direct inter-actions between the Na(+) cations and the guanine anions. Guanine mol-ecules are linked to neighboring water mol-ecules by O-H⋯N and O-H⋯O hydrogen bonds into a network structure.

  9. Purine and folate metabolism as a potential target of sex-specific nutrient allocation in Drosophila and its implication for lifespan-reproduction tradeoff.

    PubMed

    Bauer, Matthias; Katzenberger, Jörg D; Hamm, Anne C; Bonaus, Melanie; Zinke, Ingo; Jaekel, Jens; Pankratz, Michael J

    2006-05-16

    The reallocation of metabolic resources is important for survival during periods of limited nutrient intake. This has an influence on diverse physiological processes, including reproduction, repair, and aging. One important aspect of resource allocation is the difference between males and females in response to nutrient stress. We identified several groups of genes that are regulated in a sex-biased manner under complete or protein starvation. These range from expected differences in genes involved in reproductive physiology to those involved in amino acid utilization, sensory perception, immune response, and growth control. A striking difference was observed in purine and the tightly interconnected folate metabolism upon protein starvation. From these results, we conclude that the purine and folate metabolic pathway is a major point of transcriptional regulation during resource allocation and may have relevance for understanding the physiological basis for the observed tradeoff between reproduction and longevity.

  10. In Vitro Ligation of Oligodeoxynucleotides Containing C8-Oxidized Purine Lesions using Bacteriophage T4 DNA Ligase†

    PubMed Central

    Zhao, Xiaobei; Muller, James G.; Halasyam, Mohan; David, Sheila S.; Burrows, Cynthia J.

    2008-01-01

    Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3′ end of the nick compared to the 5′ end and to lesions located in the intact template strand. In general, substrates containing the OG·A mismatch were more readily ligated than OG·C. Ligations of duplexes containing the OA·T base pair (OA=8-oxo-7,8- dihydroadenosine) that could adopt an anti-anti conformation proceeded in high efficiencies. An OI·Acontaining duplex (OI = 8-oxo-7,8-dihydroinosine) behaved similarly to OG·A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G although the overall ligation efficiencies were much lower than most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90–100-fold for OG·C, OI·C, OI·A and OA·T containing duplexes compared to G·C. Substrates containing Gh·A, Gh·G, Sp·A and Sp·G base pairs showed Km values 20–70-fold higher than G·C while the Km value for OG·A was 5 times lower than G·C. PMID:17323928

  11. Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine: involvement of adenosine and dopamine receptors.

    PubMed

    Feduccia, Allison A; Wang, Yuanyuan; Simms, Jeffrey A; Yi, Henry Y; Li, Rui; Bjeldanes, Leonard; Ye, Chuangxing; Bartlett, Selena E

    2012-08-01

    Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor

  12. Sequence selectivity of azinomycin B in DNA alkylation and cross-linking: a QM/MM study.

    PubMed

    Senthilnathan, Dhurairajan; Kalaiselvan, Anbarasan; Venuvanalingam, Ponnambalam

    2013-01-01

    Azinomycin B--a well-known antitumor drug--forms cross-links with DNA through alkylation of purine bases and blocks tumor cell growth. This reaction has been modeled using the ONIOM (B3LYP/6-31+g(d):UFF) method to understand the mechanism and sequence selectivity. ONIOM results have been checked for reliability by comparing them with full quantum mechanics calculations for selected paths. Calculations reveal that, among the purine bases, guanine is more reactive and is alkylated by aziridine ring through the C10 position, followed by alkylation of the epoxide ring through the C21 position of Azinomycin B. While the mono alkylation is controlled kinetically, bis-alkylation is controlled thermodynamically. Solvent effects were included using polarized-continuum-model calculations and no significant change from gas phase results was observed.

  13. An Interpretation of the Ancestral Codon from Miller’s Amino Acids and Nucleotide Correlations in Modern Coding Sequences

    PubMed Central

    Carels, Nicolas; de Leon, Miguel Ponce

    2015-01-01

    Purine bias, which is usually referred to as an “ancestral codon”, is known to result in short-range correlations between nucleotides in coding sequences, and it is common in all species. We demonstrate that RWY is a more appropriate pattern than the classical RNY, and purine bias (Rrr) is the product of a network of nucleotide compensations induced by functional constraints on the physicochemical properties of proteins. Through deductions from universal correlation properties, we also demonstrate that amino acids from Miller’s spark discharge experiment are compatible with functional primeval proteins at the dawn of living cell radiation on earth. These amino acids match the hydropathy and secondary structures of modern proteins. PMID:25922573

  14. Synthesis and antirhinovirus activity of 8-substituted analogues of 6-(dimethylamino)-9-(4-methylbenzyl)-2-(trifluoromethyl)-9H-purine.

    PubMed

    Kelley, J L; Linn, J A; Selway, J W

    1991-01-01

    Several 8-substituted analogues of 6-(dimethylamino) -9-(4-methylbenzyl)-2-(trifluoromethyl)-9H-purine (1) were synthesized and tested for activity against rhinovirus type 1B. Among 16 8-substituted analogues, the 8-amino (3) and 8-bromo (2) analogues were most active with IC50s of 0.36 and 1.4 microM, respectively, under conditions where 1 had an IC50 of 0.03 microM.

  15. Theoretical and experimental studies on the corrosion inhibition potentials of some purines for aluminum in 0.1 M HCl

    PubMed Central

    Eddy, Nnabuk O.; Momoh-Yahaya, H.; Oguzie, Emeka E.

    2014-01-01

    Experimental aspect of the corrosion inhibition potential of adenine (AD), guanine (GU) and, hypoxanthine (HYP) was carried out using weight loss, potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) methods while the theoretical aspect of the work was carried out by calculations of semi-empirical parameters (for AM1, MNDO, CNDO, PM3 and RM1 Hamiltonians), Fukui functions and inhibitor–metal interaction energies. Results obtained from the experimental studies were in good agreement and indicated that adenine (AD), guanine (GU) and hypoxanthine (HYP) are good adsorption inhibitors for the corrosion of aluminum in solutions of HCl. Data obtained from electrochemical experiment revealed that the studied purines functioned by adsorption on the aluminum/HCl interface and inhibited the cathodic half reaction to a greater extent and anodic half reaction to a lesser extent. The adsorption of the purines on the metal surface was found to be exothermic and spontaneous. Deviation of the adsorption characteristics of the studied purines from the Langmuir adsorption model was compensated by the fitness of Flory Huggins and El Awardy et al. adsorption models. Quantum chemical studies revealed that the experimental inhibition efficiencies of the studied purines are functions of some quantum chemical parameters including total energy of the molecules (TE), energy gap (EL–H), electronic energy of the molecule (EE), dipole moment and core–core repulsion energy (CCR). Fukui functions analysis through DFT and MP2 theories indicated slight complications and unphysical results. However, results obtained from calculated Huckel charges, molecular orbital and interaction energies, the adsorption of the inhibitors proceeded through the imine nitrogen (N5) in GU, emanine nitrogen (N7) in AD and the pyridine nitrogen (N5) in HPY. PMID:25750754

  16. Theoretical and experimental studies on the corrosion inhibition potentials of some purines for aluminum in 0.1 M HCl.

    PubMed

    Eddy, Nnabuk O; Momoh-Yahaya, H; Oguzie, Emeka E

    2015-03-01

    Experimental aspect of the corrosion inhibition potential of adenine (AD), guanine (GU) and, hypoxanthine (HYP) was carried out using weight loss, potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) methods while the theoretical aspect of the work was carried out by calculations of semi-empirical parameters (for AM1, MNDO, CNDO, PM3 and RM1 Hamiltonians), Fukui functions and inhibitor-metal interaction energies. Results obtained from the experimental studies were in good agreement and indicated that adenine (AD), guanine (GU) and hypoxanthine (HYP) are good adsorption inhibitors for the corrosion of aluminum in solutions of HCl. Data obtained from electrochemical experiment revealed that the studied purines functioned by adsorption on the aluminum/HCl interface and inhibited the cathodic half reaction to a greater extent and anodic half reaction to a lesser extent. The adsorption of the purines on the metal surface was found to be exothermic and spontaneous. Deviation of the adsorption characteristics of the studied purines from the Langmuir adsorption model was compensated by the fitness of Flory Huggins and El Awardy et al. adsorption models. Quantum chemical studies revealed that the experimental inhibition efficiencies of the studied purines are functions of some quantum chemical parameters including total energy of the molecules (TE), energy gap (E L-H), electronic energy of the molecule (EE), dipole moment and core-core repulsion energy (CCR). Fukui functions analysis through DFT and MP2 theories indicated slight complications and unphysical results. However, results obtained from calculated Huckel charges, molecular orbital and interaction energies, the adsorption of the inhibitors proceeded through the imine nitrogen (N5) in GU, emanine nitrogen (N7) in AD and the pyridine nitrogen (N5) in HPY.

  17. Synergistic effects of the purine analog sulfinosine and curcumin on the multidrug resistant human non-small cell lung carcinoma cell line (NCI-H460/R).

    PubMed

    Andjelkovic, Tijana; Pesic, Milica; Bankovic, Jasna; Tanic, Nikola; Markovic, Ivanka D; Ruzdijic, Sabera

    2008-07-01

    Multidrug resistance (MDR) is the main obstacle to a successful chemotherapy of lung cancer. We tested the potential of sulfinosine and curcumin, alone and in combination, for modulating MDR in the human resistant, non-small cell lung carcinoma cell line (NCI-H460/R). First, we determined the mutational status of the p53 gene in NCI-H460/R cells by PCR-SSCP and DNA sequencing and identified mutations which could at least partially contribute to the development of the MDR phenotype. The effects of sulfinosine and curcumin were studied, both separately and in combination, at the level of cytotoxicity, cell cycle distribution and gene expression. Sulfinosine displayed dose-dependent growth inhibition in both resistant and control sensitive cell lines, whereas curcumin considerably inhibited their growth only at relatively high doses. When sulfinosine was combined with a low dose of curcumin the drugs exerted a synergistic cytotoxic effect in NCI-H460/R cells. The expression of MDR-related genes mdr1, gst-pi and topo IIalpha, was altered by sulfinosine and curcumin. The most pronounced effect was observed when the agents were applied together. Sulfinosine and curcumin caused perturbations in cell cycle distribution in the NCI-H460/R cell line. The combination of the two drugs induced a more pronounced cell cycle arrest in S and G(2)/M in NCI-H460/R cells. Our results show that sulfinosine and curcumin overcome MDR in non-small cell lung carcinoma cell line (NSCLC), especially in combination despite the presence of a mutated p53 gene.

  18. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

    PubMed

    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-02

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.

  19. Evaluation of effects of bivalent cations on the formation of purine-rich triple-helix DNA by ESI-FT-MS.

    PubMed

    Wan, Cuihong; Cui, Meng; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

    2009-07-01

    The GGA triplet repeats are widely dispersed throughout eukaryotic genomes. (GGA)n or (GGT)n oligonucleotides can interact with double-stranded DNA containing (GGA:CCT)n to form triple-stranded DNA. The effects of 8 divalent metal ions (3 alkaline-earth metals and 5 transition metals) on formation of these purine-rich triple-helix DNA were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-MS). In the absence of metal ions, no triplex but single-strand, duplex, and purine homodimer ions were observed in mass spectra. The triple-helix DNA complexes were observed only in the presence of certain divalent ions. The effects of different divalent cations on the formation of purine-rich triplexes were compared. Transition-metal ions, especially Co(2+) and Ni(2+), significantly boost the formation of triple-helix DNA, whereas alkaline-earth metal ions have no positive effects on triplex formation. In addition, Ba(2+) is notably beneficial to the formation of homodimer instead of triplex.

  20. Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution

    SciTech Connect

    Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi; Fujii, Kentaro; Yokoya, Akinari; Fukuda, Yoshihiro; Saitoh, Yuji

    2014-08-07

    The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5{sup ′}-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5{sup ′}-monophosphate, and adenosine 5{sup ′}-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

  1. Influence of experimental canine ehrlichiosis on the E-ADA activity and purine levels in serum and possible functional correlations with pathogenesis.

    PubMed

    Da Silva, Aleksandro S; Munhoz, Thiago D; Faria, Joice L M; Vargas-Hérnandez, Giovanni; Machado, Rosangela Z; Luz, Nathalia C; Moritz, Cesar E J; Casali, Emerson A; Bottari, Nathieli B; Stefani, Lenita M; Tinucci-Costa, Mirela

    2013-10-25

    The aim of this study was to evaluate adenosine deaminase activity and purines levels in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups with five animals each: healthy animals and animals infected by E. canis. The concentration of purines (adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, inosine, hypoxanthine, xanthine and uric acid), and adenosine deaminase (E-ADA) activity in sera were evaluated. Samples were collected on days 12 and 30 post-infection (PI). The E-ADA activity showed a significant reduction on day 12 PI, and increased on day 30 PI in dogs infected with E. canis. On day 12, an increase in seric concentration of ATP, ADP and adenosine was verified, and different levels of hypoxanthine, xanthine and uric acid had a drastic reduction in infected compared healthy dogs (P<0.05). However, on day 30 PI, the levels of seric ADP and AMP decreased, unlike the concentration of xanthine and uric acid that increased significantly in infected dogs (P<0.05). Therefore, the activity of E-ADA and purine levels are altered in experimental canine ehrlichiosis, probably with the purpose of modulating the pathogenesis of the disease related to immune response, oxidative stress and coagulation disorders in acute phase.

  2. Purine analogs sensitize the multidrug resistant cell line (NCI-H460/R) to doxorubicin and stimulate the cell growth inhibitory effect of verapamil.

    PubMed

    Pesić, Milica; Podolski, Ana; Rakić, Ljubisa; Ruzdijić, Sabera

    2010-08-01

    The resistant cell line NCI-H460/R and its counterpart NCI-H460 were used to investigate the ability of purine analogs to overcome multidrug resistance (MDR) that seriously limit the efficacy of lung cancer regimens with chemotherapeutic agents. Two purine analogs, sulfinosine (SF) and 8-Cl-cAMP, exerted dose-dependent effects on cell growth in both parental and resistant cell lines. They significantly decreased mdr1 expression in NCI-H460/R cells. Low concentrations (1 microM) of SF and 8-Cl-cAMP in combination with doxorubicin (DOX) exerted synergistic growth inhibition in both cell lines. Pretreatment with SF and 8-Cl-cAMP improved the sensitivity to DOX more than verapamil (VER), the standard modulator of MDR. The increased accumulation of DOX observed after the treatment with SF and 8-Cl-cAMP was consistent with the results obtained with VER. VER stimulated the effect of 8-Cl-cAMP on DOX cytotoxicity and mdr1 expression. Combinations of either SF or 8-Cl-cAMP with VER at clinically acceptable concentrations exhibited synergistic effects on cell growth inhibition in the resistant cell line. SF and 8-Cl-cAMP modulated MDR in NCI-H460/R cells, especially when applied before DOX administration. This feature, together with their ability to reverse MDR, renders the purine analogs (in combination with VER) as potential candidates for improving the clinical activity of existing lung cancer therapeutics.

  3. A thermodynamic and mutational analysis of an RNA purine loop as a protein binding site.

    PubMed

    White, S A; Li, H; Rauch, M E

    1995-10-01

    The thermal stability and protein binding of a 36 nucleotide RNA hairpin containing an internal loop were studied under various solution conditions. Yeast ribosomal protein L32 binds to its transcript and small RNAs which reproduce the L32 transcript's secondary structure have been examined. Replacement of the internal loop with canonical base pairs did not affect the salt dependence of the melting temperature suggesting that both molecules adopt a linear shape. Several electrostatic contacts are formed on binding to a ribosomal fusion protein, but Mg+2 is not required for binding. The RNA protein complex is stable up to 50 degrees C. Two internal loop deletion mutants have similar thermodynamic stabilities and chemical and enzymatic reactivities, but fail to bind the fusion protein. However, several of the internal loop bases of the deletion mutants are moderately reactive to chemical agents whereas the wild type loop sequence displayed a mixed pattern of protection and hyperreactivity.

  4. Hydrogen-bond-assisted controlled C-H functionalization via adaptive recognition of a purine directing group.

    PubMed

    Kim, Hyun Jin; Ajitha, Manjaly J; Lee, Yongjae; Ryu, Jaeyune; Kim, Jin; Lee, Yunho; Jung, Yousung; Chang, Sukbok

    2014-01-22

    We have developed the Rh-catalyzed selective C-H functionalization of 6-arylpurines, in which the purine moiety directs the C-H bond activation of the aryl pendant. While the first C-H amination proceeds via the N1-chelation assistance, the subsequent second C-H bond activation takes advantage of an intramolecular hydrogen-bonding interaction between the initially formed amino group and one nitrogen atom, either N1 or N7, of the purinyl part. Isolation of a rhodacycle intermediate and the substrate variation studies suggest that N1 is the main active site for the C-H functionalization of both the first and second amination in 6-arylpurines, while N7 plays an essential role in controlling the degree of functionalization serving as an intramolecular hydrogen-bonding site in the second amination process. This pseudo-Curtin-Hammett situation was supported by density functional calculations, which suggest that the intramolecular hydrogen-bonding capability helps second amination by reducing the steric repulsion between the first installed ArNH and the directing group.

  5. A Purine Analog Synergizes with Chloroquine (CQ) by Targeting Plasmodium falciparum Hsp90 (PfHsp90)

    PubMed Central

    Shahinas, Dea; Folefoc, Asongna; Taldone, Tony; Chiosis, Gabriela; Crandall, Ian; Pillai, Dylan R.

    2013-01-01

    Background Drug resistance, absence of an effective vaccine, and inadequate public health measures are major impediments to controlling Plasmodium falciparum malaria worldwide. The development of antimalarials to which resistance is less likely is paramount. To this end, we have exploited the chaperone function of P. falciparum Hsp90 (PfHsp90) that serves to facilitate the expression of resistance determinants. Methods The affinity and activity of a purine analogue Hsp90 inhibitor (PU-H71) on PfHsp90 was determined using surface plasmon resonance (SPR) studies and an ATPase activity assay, respectively. In vitro, antimalarial activity was quantified using flow cytometry. Interactors of PfHsp90 were determined by LC-MS/MS. In vivo studies were conducted using the Plasmodium berghei infection mouse model. Results PU-H71 exhibited antimalarial activity in the nanomolar range, displayed synergistic activity with chloroquine in vitro. Affinity studies reveal that the PfHsp90 interacts either directly or indirectly with the P. falciparum chloroquine resistance transporter (PfCRT) responsible for chloroquine resistance. PU-H71 synergized with chloroquine in the P.berghei mouse model of malaria to reduce parasitemia and improve survival. Conclusions We propose that the interaction of PfHsp90 with PfCRT may account for the observed antimalarial synergy and that PU-H71 is an effective adjunct for combination therapy. PMID:24098696

  6. Transcriptional Signature of an Altered Purine Metabolism in the Skeletal Muscle of a Huntington's Disease Mouse Model

    PubMed Central

    Mielcarek, Michal; Smolenski, Ryszard T.; Isalan, Mark

    2017-01-01

    Huntington's disease (HD) is a fatal neurodegenerative disorder, caused by a polyglutamine expansion in the huntingtin protein (HTT). HD has a peripheral component to its pathology: skeletal muscles are severely affected, leading to atrophy, and malfunction in both pre-clinical and clinical settings. We previously used two symptomatic HD mouse models to demonstrate the impairment of the contractile characteristics of the hind limb muscles, which was accompanied by a significant loss of function of motor units. The mice displayed a significant reduction in muscle force, likely because of deteriorations in energy metabolism, decreased oxidation, and altered purine metabolism. There is growing evidence suggesting that HD-related skeletal muscle malfunction might be partially or completely independent of CNS degeneration. The pathology might arise from mutant HTT within muscle (loss or gain of function). Hence, it is vital to identify novel peripheral biomarkers that will reflect HD skeletal muscle atrophy. These will be important for upcoming clinical trials that may target HD peripherally. In order to identify potential biomarkers that might reflect muscle metabolic changes, we used qPCR to validate key gene transcripts in different skeletal muscle types. Consequently, we report a number of transcript alterations that are linked to HD muscle pathology. PMID:28303108

  7. Ambiguous base pairing of the purine analogue 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide during PCR.

    PubMed

    Sala, M; Pezo, V; Pochet, S; Wain-Hobson, S

    1996-09-01

    In principle the hydrogen bonding capacities of 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (dY), and its N-propyl derivative (dYPr), allow them to pair to all four deoxynucleosides. Their triphosphate derivatives (dYTP and dYPrTP) are preferentially incorporated as dATP analogues in a PCR reaction. However, once incorporated into a DNA template their ambiguous hydrogen bonding potential gave rise to misincorporation at frequencies of approximately 3 x 10(-2) per base per amplification. Most of the substitutions were transitions resulting from rotation about the carboxamide bond when part of the template. Between 11-15% of transversions were noted implying rotation of purine or imidazole moieties about the glycosidic bond. As part of a DNA template, dYPr behaved in the same way as dY, despite its propyl moiety. These deoxyimidazole derivatives are among the most radical departures from the canonical bases used so far as substrates in PCR and could be used to generate mutant gene libraries.

  8. Ambiguous base pairing of the purine analogue 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide during PCR.

    PubMed Central

    Sala, M; Pezo, V; Pochet, S; Wain-Hobson, S

    1996-01-01

    In principle the hydrogen bonding capacities of 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (dY), and its N-propyl derivative (dYPr), allow them to pair to all four deoxynucleosides. Their triphosphate derivatives (dYTP and dYPrTP) are preferentially incorporated as dATP analogues in a PCR reaction. However, once incorporated into a DNA template their ambiguous hydrogen bonding potential gave rise to misincorporation at frequencies of approximately 3 x 10(-2) per base per amplification. Most of the substitutions were transitions resulting from rotation about the carboxamide bond when part of the template. Between 11-15% of transversions were noted implying rotation of purine or imidazole moieties about the glycosidic bond. As part of a DNA template, dYPr behaved in the same way as dY, despite its propyl moiety. These deoxyimidazole derivatives are among the most radical departures from the canonical bases used so far as substrates in PCR and could be used to generate mutant gene libraries. PMID:8811081

  9. 9-(Arenethenyl)purines as dual Src/Abl kinase inhibitors targeting the inactive conformation: design, synthesis, and biological evaluation.

    PubMed

    Huang, Wei-Sheng; Zhu, Xiaotian; Wang, Yihan; Azam, Mohammad; Wen, David; Sundaramoorthi, Raji; Thomas, R Mathew; Liu, Shuangying; Banda, Geetha; Lentini, Scott P; Das, Sasmita; Xu, Qihong; Keats, Jeff; Wang, Frank; Wardwell, Scott; Ning, Yaoyu; Snodgrass, Joseph T; Broudy, Marc I; Russian, Karin; Daley, George Q; Iuliucci, John; Dalgarno, David C; Clackson, Tim; Sawyer, Tomi K; Shakespeare, William C

    2009-08-13

    A novel series of potent dual Src/Abl kinase inhibitors based on a 9-(arenethenyl)purine core has been identified. Unlike traditional dual Src/Abl inhibitors targeting the active enzyme conformation, these inhibitors bind to the inactive, DFG-out conformation of both kinases. Extensive SAR studies led to the discovery of potent and orally bioavailable inhibitors, some of which demonstrated in vivo efficacy. Once-daily oral administration of inhibitor 9i (AP24226) significantly prolonged the survival of mice injected intravenously with wild type Bcr-Abl expressing Ba/F3 cells at a dose of 10 mg/kg. In a separate model, oral administration of 9i to mice bearing subcutaneous xenografts of Src Y527F expressing NIH 3T3 cells elicited dose-dependent tumor shrinkage with complete tumor regression observed at the highest dose. Notably, several inhibitors (e.g., 14a, AP24163) exhibited modest cellular potency (IC50 = 300-400 nM) against the Bcr-Abl mutant T315I, a variant resistant to all currently marketed therapies for chronic myeloid leukemia.

  10. Structure of eukaryotic purine/H+ symporter UapA suggests a role for homodimerization in transport activity

    NASA Astrophysics Data System (ADS)

    Alguel, Yilmaz; Amillis, Sotiris; Leung, James; Lambrinidis, George; Capaldi, Stefano; Scull, Nicola J.; Craven, Gregory; Iwata, So; Armstrong, Alan; Mikros, Emmanuel; Diallinas, George; Cameron, Alexander D.; Byrne, Bernadette

    2016-04-01

    The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1-11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

  11. Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates.

    PubMed

    Heinrich, Timo; Buchstaller, Hans-Peter; Cezanne, Bertram; Rohdich, Felix; Bomke, Jörg; Friese-Hamim, Manja; Krier, Mireille; Knöchel, Thorsten; Musil, Djordje; Leuthner, Birgitta; Zenke, Frank

    2017-02-01

    The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC50: 1μM) led to the most potent compound 27 (IC50: 0.038μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.

  12. Molecular mechanism of diaminomaleonitrile to diaminofumaronitrile photoisomerization: an intermediate step in the prebiotic formation of purine nucleobases.

    PubMed

    Szabla, Rafał; Góra, Robert W; Sponer, Jiří; Sponer, Judit E

    2014-02-24

    The photoinduced isomerization of diaminomaleonitrile (DAMN) to diaminofumaronitrile (DAFN) was suggested to play a key role in the prebiotically plausible formation of purine nucleobases and nucleotides. In this work we analyze two competitive photoisomerization mechanisms on the basis of state-of-the-art quantum-chemical calculations. Even though it was suggested that this process might occur on the triplet potential-energy surface, our results indicate that the singlet reaction channel should not be disregarded either. In fact, the peaked topography of the S1 /S0 conical intersection suggests that the deexcitation should most likely occur on a sub-picosecond timescale and the singlet photoisomerization mechanism might effectively compete even with a very efficient intersystem crossing. Such a scenario is further supported by the relatively small spin-orbit coupling of the S1 and T2 states in the Franck-Condon region, which does not indicate a very effective triplet bypass for this photoreaction. Therefore, we conclude that the triplet reaction channel in DAMN might not be as prominent as was previously thought.

  13. Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase.

    PubMed

    Suarez, Javier; Schramm, Vern L

    2015-09-08

    Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

  14. Purine nucleoside phosphorylase and the enzymatic antioxidant defense system in breast milk from women with different levels of arsenic exposure.

    PubMed

    Gaxiola-Robles, Ramón; Labrada-Martagón, Vanessa; Bitzer-Quintero, Oscar Kurt; Zenteno-Savín, Tania; Méndez-Rodríguez, Lía Celina

    2015-05-01

    Purine nucleoside phosphorylase (PNP) is an ubiquitous enzyme which plays an important role in arsenic (As) detoxification. As is a toxic metalloid present in air, soil and water; is abundant in the environment and is readily transferred along the trophic chain, being found even in human breast milk. Milk is the main nutrient source for the growth and development of neonates. Information on breast milk synthesis and its potential defense mechanism against As toxicity is scarce. In this study, PNP and antioxidant enzymes activities, as well as glutathione (GSH) and total arsenic (TAs) concentrations, were quantified in breast milk samples. PNP, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) activities and GSH concentration were determined spectrophotometrically; TAs concentration ([TAs]) was measured by atomic absorption spectrometry. Data suggest an increase in PNP activity (median = 0.034 U mg protein-1) in the presence of TAs (median = 1.16 g L(-1)). To explain the possible association of PNP activity in breast milk with the activity of the antioxidant enzymes as well as with GSH and TAs concentrations, generalized linear models were built. In the adjusted model, GPx and GR activities showed a statistically significant (p<0.01) association with PNP activity. These results may suggest that PNP activity increases in the presence of TAs as part of the detoxification mechanism in breast milk.

  15. Structure of eukaryotic purine/H(+) symporter UapA suggests a role for homodimerization in transport activity.

    PubMed

    Alguel, Yilmaz; Amillis, Sotiris; Leung, James; Lambrinidis, George; Capaldi, Stefano; Scull, Nicola J; Craven, Gregory; Iwata, So; Armstrong, Alan; Mikros, Emmanuel; Diallinas, George; Cameron, Alexander D; Byrne, Bernadette

    2016-04-18

    The uric acid/xanthine H(+) symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1-11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.

  16. Structure of eukaryotic purine/H+ symporter UapA suggests a role for homodimerization in transport activity

    PubMed Central

    Alguel, Yilmaz; Amillis, Sotiris; Leung, James; Lambrinidis, George; Capaldi, Stefano; Scull, Nicola J.; Craven, Gregory; Iwata, So; Armstrong, Alan; Mikros, Emmanuel; Diallinas, George; Cameron, Alexander D.; Byrne, Bernadette

    2016-01-01

    The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1–11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin. PMID:27088252

  17. Purine (N)-Methanocarba Nucleoside Derivatives Lacking an Exocyclic Amine as Selective A3 Adenosine Receptor Agonists

    PubMed Central

    2016-01-01

    Purine (N)-methanocarba-5′-N-alkyluronamidoriboside A3 adenosine receptor (A3AR) agonists lacking an exocyclic amine resulted from an unexpected reaction during a Sonogashira coupling and subsequent aminolysis. Because the initial C6-Me and C6-styryl derivatives had unexpectedly high A3AR affinity, other rigid nucleoside analogues lacking an exocyclic amine were prepared. Of these, the C6-Me-(2-phenylethynyl) and C2-(5-chlorothienylethynyl) analogues were particularly potent, with human A3AR Ki values of 6 and 42 nM, respectively. Additionally, the C2-(5-chlorothienyl)-6-H analogue was potent and selective at A3AR (MRS7220, Ki 60 nM) and also completely reversed mouse sciatic nerve mechanoallodynia (in vivo, 3 μmol/kg, po). The lack of a C6 H-bond donor while maintaining A3AR affinity and efficacy could be rationalized by homology modeling and docking of these hypermodified nucleosides. The modeling suggests that a suitable combination of stabilizing features can partially compensate for the lack of an exocyclic amine, an otherwise important contributor to recognition in the A3AR binding site. PMID:26890707

  18. Radioimmunoassays for the modified nucleosides N[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine and 2-methylthioadenosine.

    PubMed Central

    Vold, B S

    1979-01-01

    Radioimmunoassays were established for the modified nucleosides N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, t6A, and 2-methylthioadenosine, ms2A. The assays depended on the production of antisera specific for t6A and ms2A that have not been previously reported. The nitrocellulose membrane filtration and saturated ammonium sulfate RIA techniques were compared for efficiency. Various radioactive antigens were employed to establish which type of antigen would give the best binding. The tritium post-labeling procedure of Randerath and Randerath was used to obtain labeled nucleosides of high enough specific activity to be useful for RIAs when the labeled nucleoside was not available commerically. The specificity of the antibodies toward nucleosides and purified tRNAs is reported. Although the titer of the t6A antiserum was low, the specificity was very sharp. An interesting finding was that threonine, a major structural component of the side-chain modification of t6A, was completely infective as an inhibitor. PMID:493139

  19. Integrating Genome-Wide Association Study and Brain Expression Data Highlights Cell Adhesion Molecules and Purine Metabolism in Alzheimer's Disease.

    PubMed

    Xiang, Zimin; Xu, Meiling; Liao, Mingzhi; Jiang, Yongshuai; Jiang, Qinghua; Feng, Rennan; Zhang, Liangcai; Ma, Guoda; Wang, Guangyu; Chen, Zugen; Zhao, Bin; Sun, Tiansheng; Li, Keshen; Liu, Guiyou

    2015-08-01

    Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly. Recently, genome-wide association studies (GWAS) have been used to investigate AD pathogenesis. However, a large proportion of AD heritability has yet to be explained. We previously identified the cell adhesion molecule (CAM) pathway as a consistent signal in two AD GWAS. However, it is unclear whether CAM is present in the Genetic and Environmental Risk for Alzheimer's Disease Consortium (GERAD) GWAS and brain expression GWAS. Meanwhile, we think integrating AD GWAS and AD brain expression datasets may provide complementary information to identify important pathways involved in AD. Here, we conducted a systems analysis using (1) KEGG pathways, (2) large-scale AD GWAS from GERAD (n = 11,789), (3) two brain expression GWAS datasets (n = 399) from the AD cerebellum and temporal cortex, and (4) previous results from pathway analysis of AD GWAS. Our results indicate that (1) CAM is a consistent signal in five AD GWAS; (2) CAM is the most significant signal in AD; (3) we confirmed previous AD risk pathways related to immune system and diseases, and cardiovascular disease, etc.; and (4) we highlighted the purine metabolism pathway in AD for the first time. We believe that our results may advance our understanding of AD mechanisms and will be very informative for future genetic studies in AD.

  20. Humanized ADEPT Comprised of an Engineered Human Purine Nucleoside Phosphorylase and a Tumor Targeting Peptide for Treatment of Cancer

    PubMed Central

    Afshar, Sepideh; Asai, Tsuneaki; Morrison, Sherie L.

    2009-01-01

    Immunogenicity caused by the use of non-human enzymes in Antibody Directed Enzyme Prodrug Therapy (ADEPT) has limited its clinical application. To overcome this problem, we have developed a mutant human purine nucleoside phosphorylase (PNP), which unlike the wild-type enzyme, accepts (deoxy)adenosine-based prodrugs as substrates. Amongst the different mutants of human PNP tested, a double mutant with amino acid substitutions E201Q:N243D (hDM) is most efficient in cleaving (deoxy)adenosine-based prodrugs. While hDM is capable of utilizing multiple prodrugs as substrates, it is most effective at cleaving 2-fluoro-2′-deoxyadenosine to a cytotoxic drug. To target hDM to the tumor site, the enzyme was fused to an Anti-HER2/neu Peptide mimetic (AHNP). Treatment of HER2/neu expressing tumor cells with hDM-AHNP results in cellular localization of enzyme activity. As a consequence, harmless prodrug is converted to a cytotoxic drug in the vicinity of the tumor cells, resulting in tumor cell apoptosis. Unlike the non-human enzymes, the hDM should have minimal immunogenicity when used in ADEPT thus providing a novel promising therapeutic agent for the treatment of tumors. PMID:19139128

  1. Sequencing technologies and genome sequencing.

    PubMed

    Pareek, Chandra Shekhar; Smoczynski, Rafal; Tretyn, Andrzej

    2011-11-01

    The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of $1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research.

  2. Energies of interactions between purine and pyrimidine bases in B- and Z-DNA

    NASA Astrophysics Data System (ADS)

    Förner, W.; Otto, P.; Ladik, J.

    The results of ab initio SCF LCAO calculations on the dimers of the nucleotide bases for all possible sixteen stacked configurations in B-DNA geometry, on eight stacked conformations in Z-DNA geometry and on the Watson-Crick-type pairs in both DNA configurations are reported. In addition to the SCF interaction energy in the supermolecule approach, the contributions of the dispersion energy have been computed using London's formula with empirical parameters. In the case of the Watson-Crick-type pairs corrections for the basis-set superposition error are reported, which seem to be negligible for the stacked configuration as a calculation on the adenine dimer shows. The results for the Watson-Crick-type pairs are in good agreement with experimental values and with theoretical ones, obtained by other methods. The stacking energies are mostly attractive but show no significant stabilization of special sequences for B- or Z-DNA. Four dimers are recalculated with the Clementi minimal basis which leads to no considerable qualitative changes in the results. As a better approach to the situation in real DNA we took at least in a qualitative approximation the charge transfer from the sugar phosphate group to the bases into account. Qualitatively this charge transfer destabilizes the pairs to some extent, namely it leads in our approximation to a small repulsion between the stacked bases and lowers the attraction in the Watson-Crick base pairs to some extent.

  3. [Foetal akinesia-hypokinesia deformation sequence].

    PubMed

    Bayat, Allan; Petersen, Astrid; Møller, Margrethe; Andersen, Graziella; Ebbesen, Finn

    2010-05-10

    Foetal akinesia-hypokinesia deformation sequence (FADS) involves arthrogryposis, facial deformations, pulmonary hypoplasia, intrauterine growth retardation, polyhydramnios and short umbilical cord. FADS is caused by lack of foetal movements, most often due to neuromuscular diseases. FADS is associated with a high mortality rate, and the infants usually die due to pulmonary hypoplasia. Antenatal diagnosis by ultrasound is possible when the condition is pronounced, or by genetic investigation, on suspicion of a specific underlying disease with known genetics.

  4. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  5. PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium †

    PubMed Central

    London, Fredda S.; Marcinkiewicz, Mariola; Walsh, Peter N.

    2008-01-01

    We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 seconds and positive factor IXa binding by 2 minutes, with calcium transients sustained for 45 minutes. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365 or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5 to 20 minutes before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (Kd 160 nM) had minimal effects, 100 μM 5, 5′-dimethylBAPTA/AM (Kd 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients. PMID:16752917

  6. Sequence specificity of streptozotocin-induced mutations.

    PubMed Central

    Mack, S L; Fram, R J; Marinus, M G

    1988-01-01

    The isolation and characterization of streptozotocin (STZ)-induced mutations in the phage P22 mnt repressor gene is described. Cells carrying the plasmid-borne mnt gene were exposed to STZ to give 10-20 percent survival and at least an eleven-fold increase in mutation frequency. DNA sequence analysis showed that 50 of 51 STZ-induced mutations were GC to AT transitions, and one was an AT to GC transition. We have also compared the STZ mutational spectrum to that for N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). There are sites in the mnt gene which are mutated only by STZ; only by MNNG, or by both agents. Sites at which only STZ induced GC to AT transition mutations occur were in sequences that are pyrimidine rich 5' to the mutated site and purine rich 3' to the mutated site. Induction of mutations by both STZ and MNNG should be considered to maximize the number of mutable sites. PMID:2972994

  7. DNA Sequencing by Hexagonal Boron Nitride Nanopore: A Computational Study

    PubMed Central

    Zhang, Liuyang; Wang, Xianqiao

    2016-01-01

    The single molecule detection associated with DNA sequencing has motivated intensive efforts to identify single DNA bases. However, little research has been reported utilizing single-layer hexagonal boron nitride (hBN) for DNA sequencing. Here we employ molecular dynamics simulations to explore pathways for single-strand DNA (ssDNA) sequencing by nanopore on the hBN sheet. We first investigate the adhesive strength between nucleobases and the hBN sheet, which provides the foundation for the hBN-base interaction and nanopore sequencing mechanism. Simulation results show that the purine base has a more remarkable energy profile and affinity than the pyrimidine base on the hBN sheet. The threading of ssDNA through the hBN nanopore can be clearly identified due to their different energy profiles and conformations with circular nanopores on the hBN sheet. The sequencing process is orientation dependent when the shape of the hBN nanopore deviates from the circle. Our results open up a promising avenue to explore the capability of DNA sequencing by hBN nanopore.

  8. Technical note: fatty acids and purine profile of cecum and colon bacteria as indicators of equine microbial metabolism.

    PubMed

    Santos, A S; Jerónimo, E; Ferreira, L M; Rodrigues, M A M; Bessa, R J B

    2013-04-01

    The potential use of odd- and branched-chain fatty acids (OBCFA) and purine bases (PB) as microbial markers in the equine hindgut was studied. For this purpose, feed particles adherent bacteria [solid associated bacteria (SAB)] and planktonic bacteria [liquid associated bacteria (PAB)] were isolated from total cecum and colon contents of 8 healthy, crossbred horses (9 ± 3 yr). Horses were fasted for 12 to 15 h before slaughter, and the cecum and colon were identified and clamped in their extremities to avoid mixing of digesta contents. The total cecum or colon contents was collected into thermal containers previously filled with CO2, immediately transported to the laboratory, and subjected to separation of solid and liquid phases to obtain bacterial PAB and SAB pellets from each horse. Overall differences observed were mainly between site of bacterial collection (cecum vs. colon) rather than between type of bacterial population (PAB vs. SAB). Cecal bacteria fraction had greater (P < 0.05) OM, PB, and branched-chain fatty acids (BCFA):odd-chain fatty acids (OFA) ratio but less (P < 0.05) BCFA, OFA, BCFA:PB ratio, and adenine:guanine ratio than colon bacterial biomass. Results indicated that the composition of cecal and colon bacteria is very different from that of similar ecosystems (e.g., rumen). These differences can be a reflection of different growth stages or nutrition of particular populations as well as different bacterial metabolic activities. Results presented herein provide evidence that PB and fatty acids can be used as microbial markers in equine studies.

  9. Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

    PubMed Central

    de Giuseppe, Priscila O.; Martins, Nadia H.; Meza, Andreia N.; dos Santos, Camila R.; Pereira, Humberto D’Muniz; Murakami, Mario T.

    2012-01-01

    The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg43* side chain contributes for the ribosyl radical to adopt an unusual C3’-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl6 and Br8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr91) is responsible for the lack of negative cooperativity of phosphate binding in this

  10. Efficient electrogene therapy for pancreatic adenocarcinoma treatment using the bacterial purine nucleoside phosphorylase suicide gene with fludarabine.

    PubMed

    Deharvengt, Sophie; Rejiba, Soukaina; Wack, Séverine; Aprahamian, Marc; Hajri, Amor

    2007-06-01

    The aim of this study was to demonstrate the potential of electrogene therapy with the bacterial purine nucleoside phosphorylase gene (ePNP), on pancreatic carcinoma (PC) large tumors. The in vivo electroporation (EP) conditions and efficacy were investigated on both subcutaneous xenografts of human PC cells in immunocompromised mice and orthotopic intrapancreatic grafts of rat PC cells in syngenic rats. After intratumoral injection of naked plasmid DNA, EP was performed using a two-needle array with 25-msec pulses and either a 300 V/cm field strength for subcutaneous or a 500 V/cm field strength for orthotopic PC, parameters providing the best electrotransfer as reflected by the measurements of both luciferase activity and ePNP mRNA. As expected, tumors developed sensitivity to prodrug treatment (6-methylpurine deoxyribose or fludarabine phosphate). We observed both significant inhibition of tumor growth and extended survival of treated mice. In fact, after prodrug treatment, PC growth in the subcutaneous model was delayed by 50-70% for ePNP-expressing tumors. In an orthotopic pancreatic tumor model, the animal survival was significantly prolonged after ePNP electrogene transfer followed by fludarabine treatment, with one animal out of 10 being tumor-free 6 months thereafter. The current study demonstrates for the first time on PC the in vivo feasibility of electrogene transfer and its therapeutic efficiency using the suicide gene/prodrug system, ePNP/fludarabine. These findings suggest that electrogene therapy strategy must be considered for pancreatic cancer treatment, particularly at advanced stages of the disease.

  11. LC-MS-Based Metabolomics Discovers Purine Endogenous Associations with Low-Dose Salbutamol in Urine Collected for Antidoping Tests.

    PubMed

    Wang, Yaoyao; Caldwell, Richard; Cowan, David A; Legido-Quigley, Cristina

    2016-02-16

    Current antidoping analytical methods are tailored mainly to the targeting of known drugs and endogenous molecules. This causes difficulties in rapidly reacting to emerging threats, such as designer drugs, biological therapeutic agents, and technologies. Biomarkers are considered as a promising approach for the fight against these threats to sport. The main purpose of this study was to find surrogate biomarkers induced by the intake of small amounts of the model compound salbutamol and explore a sensitive approach to help screen for possible drug misuse. Urine samples (91) from athletes with detectable salbutamol (30) and negative samples (61) were analyzed using a UHPLC-MS. A third group (30) was created by spiking salbutamol into negative samples to eliminate confounding effects. Data were then analyzed in XCMS to extract metabolic features. Orthogonal partial least squares-discriminant analysis was performed to select features correlated with detectable salbutamol (p(corr) > 0.5) and ROC analysis was performed to measure the predictive potential of the markers. Univariate analysis including Mann-Whitney U test and Spearman's correlation was conducted on selected markers. A total of 7000 metabolic features were parsed, one feature identified as hypoxanthine increased with salbutamol (p < 0.001). The ROC curve of hypoxanthine returned an AUC of 0.79 (p < 0.001). Correlation with salbutamol (r = 0.415, p < 0.01, Spearman's correlation) showed hypoxanthine and purine metabolism have association with salbutamol administration. This surrogate discovery approach needs further PK studies but in the meantime can be used as an intelligence-based complementary approach for targeting of athletes to be further tested.

  12. From formamide to purine: a self-catalyzed reaction pathway provides a feasible mechanism for the entire process.

    PubMed

    Wang, Jing; Gu, Jiande; Nguyen, Minh Tho; Springsteen, Greg; Leszczynski, Jerzy

    2013-08-15

    A formamide self-catalyzed mechanistic pathway that transforms formamide to purine through a five-membered ring intermediate has been explored by density functional theory calculations. The highlight of the mechanistic route detailed here is that the proposed pathway represents the simplest and lowest energy reaction pathway. All necessary reactants, including catalysts, are generated from a single initial compound, formamide. The most catalytically effective form of formamide is found to be the imidic acid isomer. The catalytic effect of formamide has been found to be much more significant than that of water. The self-catalytic mechanism revealed here provides a pathway with the lowest energy barriers among all reaction routes previously published. Several important reaction steps are involved in this mechanistic route: formylation-dehydration, Leuckart reduction, five- and six-member ring-closing, and deamination. Overall, a five-membered ring-closing is the rate-determining step in the present catalytic route, which is consistent with our previous mechanistic investigations. The activation energy of this rate-controlling step (ca. 27 kcal/mol) is significantly lower than the rate-determining step (ca. 34 kcal/mol) in the pathway from 4-aminoimidazole-5-carboxamidine described by Schleyer's group (Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17272-17277) and in the pyrimidine pathway (ca. 44 kcal/mol) reported by Sponer et al. (J. Phys. Chem. A 2012, 116, 720-726). The self-catalyzed mechanistic pathway reported herein is less energetically demanding than previously proposed routes.

  13. Solving the Tautomeric Equilibrium of Purine Through the Analysis of the Complex Hyperfine Structure of the Four 14N Nuclei

    NASA Astrophysics Data System (ADS)

    Cocinero, Emilio J.; Uriarte, Iciar; Ecija, Patricia; Favero, Laura B.; Spada, Lorenzo; Calabrese, Camilla; Caminati, Walther

    2016-06-01

    Microwave spectroscopy has been restricted to the investigation of small molecules in the last years. However, with the advent of FTMW and CP-FTMW spectroscopies coupled with laser vaporization techniques it has turned into a very competitive methodology in the studies of moderate-size biomolecules. Here, we present the study of purine, characterized by two aromatic rings, one six- and one five-membered, fused together to give a planar aromatic bicycle. Biologically, it is the mainframe of two of the five nucleobases of DNA and RNA. Two tautomers were observed by FTMW spectroscopy coupled to UV ultrafast laser vaporization system. The population ratio of the two main tautomers [N(7)H]/[N(9)H] is about 1/40 in the gas phase. It contrasts with the solid state where only the N(7)H species is present, or in solution where a mixture of both tautomers is observed. For both species, a full quadrupolar hyperfine analysis has been performed. This has led to the determination of the full sets of diagonal quadrupole coupling constants of the four 14N atoms, which have provided crucial information for the unambiguous identification of both species. T. J. Balle and W. H. Flygare Rev. Sci. Instrum. 52, 33-45, 1981 J.-U. Grabow, W. Stahl and H. Dreizler Rev. Sci. Instrum. 67, 4072-4084, 1996 G. G. Brown, B. D. Dian, K. O. Douglass, S. M. Geyer, S. T. Shipman and B. H. Pate Rev. Sci. Instrum. 79, 0531031/1-053103/13, 2008 E. J. Cocinero, A. Lesarri, P. écija, F. J. Basterretxea, J. U. Grabow, J. A. Fernández and F. Castaño Angew. Chem. Int. Ed. 51, 3119-3124, 2012

  14. Theacrine, a purine alkaloid obtained from Camellia assamica var. kucha, attenuates restraint stress-provoked liver damage in mice.

    PubMed

    Li, Wei-Xi; Li, Yi-Fang; Zhai, Yu-Jia; Chen, Wei-Min; Kurihara, Hiroshi; He, Rong-Rong

    2013-07-03

    Theacrine (1,3,7,9-tetramethyluric acid), a purine alkaloid, has proven to be beneficial in maintaining several brain functions and is being studied for potential medicinal uses in recent years. In this study, we isolated theacrine from Camellia assamica var. kucha and investigated its protective effects on liver damage induced by restraint stress in mice. Results showed that 18 h of restraint stress could induce liver damage, with an obvious increase in levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST). This finding was further confirmed by hepatic pathological examination, which showed inflammatory cell infiltration and focal necrosis of hepatocytes. However, oral administration of theacrine (10, 20, 30 mg/kg for 7 consecutive days) was found to decrease plasma ALT and AST levels, reduce hepatic mRNA levels of inflammatory mediators (IL-1β, TNF-α, IL-6, and IFN-γ), and reverse the histologic damages in stressed mice. Simultaneously, theacrine also significantly decreased the content of malondialdehyde and increased oxygen radical absorbance capacity (ORAC) level in the plasma and liver of stressed mice. These results suggested that the protective effects of theacrine on stress-induced liver damage might be correlated with its antioxidative activity. The antioxidative capacity of theacrine was further evaluated by in vitro ORAC and cellular antioxidant activity assay. The results suggested that the antioxidative capacity of theacrine was not due to the direct action on free radical clearance. Moreover, the elevated activities and gene expressions of superoxide dismutase, catalase, and glutathione peroxidase, as well as the reduced activity of xanthine oxidase by theacrine treatment in stressed mice suggested that the antioxidative activity might be due to the strengthening of the antioxidant system in vivo. On the basis of the above results, theacrine is possibly a good candidate for protecting against or treating lifestyle

  15. Purine receptor antagonist modulates serology and affective behaviors in lupus-prone mice: evidence of autoimmune-induced pain?

    PubMed Central

    Ballok, David A.; Sakic, Boris

    2008-01-01

    Neurologic and psychiatric (NP) manifestations are severe complications of systemic lupus erythematosus (SLE). As commonly seen in patients, spontaneous disease onset in the MRL/MpJ-Faslpr/ J (MRL-lpr) mouse model of NP-SLE is accompanied by increased autoantibodies, proinflammatorycytokines and behavioral dysfunction which precede neuroinflammation and structural brain lesions. The role of purinergic receptors in the regulation of immunity and behavior remains largely unexplored in the field of neuropsychiatry. To examine the possibility that purinoception is involved in the development of affective behaviors, the P2X purinoceptor antagonist, suramin, was administered to lupus-prone mice from 5 to 14 weeks of age. In addition to food and water measures, novel object and sucrose preference tests were performed to assess neophobic anxiety- and anhedonic-like behaviors. Enzyme-linked immunosorbant assays for anti-nuclear antibodies (ANA) and pro-inflammatory cytokines were employed in immunopathological analyses. Changes in dendritic morphology in the hippocampal CA1 region were examined by a Golgi impregnation method. Suramin significantly lowered serum ANA and prevented behavioral deficits, but did not prevent neuronal atrophy in MRL-lpr animals. In a new batch of asymptomatic mice, systemic administration of corticosterone was found to induce aberrations in CA1 dendrites, comparable to the “stress” of chronic disease. The precise mechanism(s) through which purine receptor inhibition exerted beneficial effects is not known. The present data supports the hypothesis that activation of the peripheral immune system induces nociceptive-related behavioral symptomatology which is attenuated by the analgesic effects of suramin. Hypercortisolemia may also initiate neuronal damage, and metabolic perturbations may underlie neuro-immuno-endocrine imbalances in MRL-lpr mice. PMID:18601998

  16. Distortional binding of transition state analogs to human purine nucleoside phosphorylase probed by magic angle spinning solid-state NMR

    PubMed Central

    Vetticatt, Mathew J.; Itin, Boris; Evans, Gary B.; Schramm, Vern L.

    2013-01-01

    Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4′-deaza-1′-aza-2′-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to “achieve” the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known transition state. PMID:24043827

  17. Pronounced enhancement of glucocorticoid-induced gene expression following severe heat shock of heat-conditioned cells hints to intricate cell survival tactics.

    PubMed

    Mitsiou, Dimitra J; Florentin, Ida; Baki, Lia; Georgakopoulos, Anastasios; Alexis, Michael N

    2005-02-01

    We have previously reported that severe heat shock of HeLa cells stably transfected with a chloramphenicol acetyltransferase (CAT) gene, transcription of which is controlled by two glucocorticoid-responsive elements and a minimal promoter, pronouncedly enhanced glucocorticoid-induced CAT expression compared to that of non-heated cells, in spite of the glucocorticoid-receptor-mediated transcription of the gene being temporarily compromised by the shock. We now report that prolonged severe heat shock of properly heat-conditioned cells resulted in far more pronounced enhancement of glucocorticoid-induced CAT mRNA and protein expressions, in spite of a similar heat-induced loss of receptor-mediated CAT gene transcription. During recovery from the shock the hormonal activation of transcription exceeded that of non-heated cells. While CAT mRNA translation was restored appreciably later than CAT gene transcription, mRNA and protein expressions were thermally enhanced to a comparable extent, consistent with the integrity of CAT mRNA being preserved during recovery. CAT mRNA turnover was fully impaired during early recovery, suggesting that stabilisation of CAT mRNA as well as stimulation of the hormonal activation of CAT gene transcription account for the thermal enhancement of glucocorticoid-induced CAT expression. This data hint to cell survival tactics designed to safeguard high expression of genes of stress-enduring function.

  18. Pronounced weight gain in insulin-treated patients with type 2 diabetes mellitus is associated with an unfavourable cardiometabolic risk profile.

    PubMed

    Jansen, H J; Vervoort, G; van der Graaf, M; Tack, C J

    2010-11-01

    Pronounced weight gain after start of insulin therapy in patients with type 2 diabetes mellitus (T2DM) may offset beneficial effects conferred by the improvement of glycaemic control. This hypothesis was tested by comparing the cardiometabolic risk profile of a group of type 2 diabetes patients with a marked increase in body weight ('gainers) after the start of insulin treatment and a similar group without any or only minimal weight gain ('non-gainers'). In a cross-sectional study, we compared two predefined groups of patients with T2DM who had been on insulin therapy for a mean of 4.0 years: 'gainers' vs 'non-gainers'. Cardiometabolic risk was assessed by measuring fat content and distribution (physical examination, bioelectrical impedance analysis, dual energy X-ray absorption, and magnetic resonance imaging), liver fat content (magnetic resonance spectroscopy), physical activity levels (Sensewear® armband) and plasma markers. Each subgroup consisted of 14 patients. Gainers had significantly more total body and trunk fat (especially subcutaneous fat) compared with no-gainers. Gainers had similar liver fat content, and slightly higher levels of fat hormones. Furthermore, gainers performed significantly less physical activity. Lastly, gainers had higher total cholesterol, low-density lipoprotein cholesterol, and alanine aminotransferase levels with similar cholesterol-lowering treatment. Patients with T2DM who show pronounced weight gain during insulin therapy have a less favourable cardiometabolic risk profile compared with patients who show no or minimal weight gain.

  19. Structural Analysis of DFG-in and DFG-out Dual Src-Abl Inhibitors Sharing a Common Vinyl Purine Template

    SciTech Connect

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Sawyer, Tomi K.; Shakespeare, William C.; Clackson, Tim; Zhu, Xiaotian; Dalgarno, David C.

    2010-09-30

    Bcr-Abl is the oncogenic protein tyrosine kinase responsible for chronic myeloid leukemia (CML). Treatment of the disease with imatinib (Gleevec) often results in drug resistance via kinase mutations at the advanced phases of the disease, which has necessitated the development of new mutation-resistant inhibitors, notably against the T315I gatekeeper mutation. As part of our efforts to discover such mutation resistant Abl inhibitors, we have focused on optimizing purine template kinase inhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are also potent Src inhibitors. Here we present crystal structures of Abl bound by two such inhibitors, based on a common N9-arenyl purine, and that represent both DFG-in and -out binding modes. In each structure the purine template is bound deeply in the adenine pocket and the novel vinyl linker forms a non-classical hydrogen bond to the gatekeeper residue, Thr315. Specific template substitutions promote either a DFG-in or -out binding mode, with the kinase binding site adjusting to optimize molecular recognition. Bcr-Abl T315I mutant kinase is resistant to all currently marketed Abl inhibitors, and is the focus of intense drug discovery efforts. Notably, our DFG-out inhibitor, AP24163, exhibits modest activity against this mutant, illustrating that this kinase mutant can be inhibited by DFG-out class inhibitors. Furthermore our DFG-out inhibitor exhibits dual Src-Abl activity, absent from the prototypical DFG-out inhibitor, imatinib as well as its analog, nilotinib. The data presented here provides structural guidance for the further design of novel potent DFG-out class inhibitors against Src, Abl and Abl T315I mutant kinases.

  20. Structure of a mutant human purine nucleoside phosphorylase with the prodrug, 2-fluoro-2-deoxyadenosine and the cytotoxic drug, 2-fluoroadenine

    SciTech Connect

    Afshar, Sepideh; Sawaya, Michael R.; Morrison, Sherie L.

    2009-06-30

    A double mutant of human purine nucleoside phosphorylase (hDM) with the amino acid mutations Glu201Gln:Asn243Asp cleaves adenosine-based prodrugs to their corresponding cytotoxic drugs. When fused to an anti-tumor targeting component, hDM is targeted to tumor cells, where it effectively catalyzes phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine (F-dAdo) to the cytotoxic drug, 2-fluoroadenine (F-Ade). This cytotoxicity should be restricted only to the tumor microenvironment, because the endogenously expressed wild type enzyme cannot use adenosine-based prodrugs as substrates. To gain insight into the interaction of hDM with F-dAdo, we have determined the crystal structures of hDM with F-dAdo and F-Ade. The structures reveal that despite the two mutations, the overall fold of hDM is nearly identical to the wild type enzyme. Importantly, the residues Gln201 and Asp243 introduced by the mutation form hydrogen bond contacts with F-dAdo that result in its binding and catalysis. Comparison of substrate and product complexes suggest that the side chains of Gln201 and Asp243 as well as the purine base rotate during catalysis possibly facilitating cleavage of the glycosidic bond. The two structures suggest why hDM, unlike the wild-type enzyme, can utilize F-dAdo as substrate. More importantly, they provide a critical foundation for further optimization of cleavage of adenosine-based prodrugs, such as F-dAdo by mutants of human purine nucleoside phosphorylase.

  1. Ultrafast time resolved spectroscopic studies on the generation of the ketyl-sugar biradical by intramolecular hydrogen abstraction among ketoprofen and purine nucleoside dyads.

    PubMed

    Li, Ming-De; Dang, Li; Liu, Mingyue; Du, Lili; Zheng, Xuming; Phillips, David Lee

    2015-04-03

    Intramolecular hydrogen abstraction reactions among ketoprofen (KP) and purine nucleoside dyads have been proposed to form ketyl-sugar biradical intermediates in acetonitrile. Femtosecond transient absorption studies on KP and purine nucleoside dyads reveal that the triplet state of the KP moiety of the dyads with cisoid structure decay faster (due to an intramolecular hydrogen abstraction reaction to produce a ketyl-sugar biradical intermediate) than the triplet state of the KP moiety of the dyads with transoid structure detected in acetonitrile solvent. For the cisoid 5-KP-dG dyad, the triplet state of the KP moiety decays too fast to be observed by ns-TR(3); only the ketyl-sugar biradical intermediates are detected by ns-TR(3) in acetonitrile. For the cisoid 5-KP-dA dyad, the triplet states of the KP moiety could be observed at early nanosecond delay times, and then it quickly undergoes intramolecular hydrogen abstraction to produce a ketyl-sugar biradical intermediate. For the cisoid 5-KPGly-dA and transoid 3-KP-dA dyads, the triplet state of the KP moiety had a longer lifetime due to the long distance chain between the KP moiety and the purine nucleoside (5-KPGly-dA) and the transoid structure (3-KP-dA). The experimental and computational results suggest that the ketyl-sugar biradical intermediate is generated with a higher efficiency for the cisoid dyad. However, the transoid dyad exhibits similar photochemistry behavior as the KP molecule, and no ketyl-sugar biradical intermediate was observed in the ns-TR(3) experiments for the transoid 3-KP-dA dyad.

  2. Allantoin, a stress-related purine metabolite, can activate jasmonate signaling in a MYC2-regulated and abscisic acid-dependent manner

    PubMed Central

    Takagi, Hiroshi; Ishiga, Yasuhiro; Watanabe, Shunsuke; Konishi, Tomokazu; Egusa, Mayumi; Akiyoshi, Nobuhiro; Matsuura, Takakazu; Mori, Izumi C.; Hirayama, Takashi; Kaminaka, Hironori; Shimada, Hiroshi; Sakamoto, Atsushi

    2016-01-01

    Allantoin is a metabolic intermediate of purine catabolism that often accumulates in stressed plants. Recently, we used Arabidopsis knockout mutants (aln) of ALLANTOINASE to show that this purine metabolite activates abscisic acid (ABA) production, thereby stimulating stress-related gene expression and enhancing seedling tolerance to abiotic stress. A detailed re-examination of the microarray data of an aln mutant (aln-1) confirmed the increased expression of ABA-related genes and also revealed altered expression of genes involved in jasmonic acid (JA) responses, probably under the control of MYC2, a master switch in the JA signaling pathway. Consistent with the transcriptome profiles, the aln-1 mutant displayed increased JA levels and enhanced responses to mechanical wounding and exogenous JA. Moreover, aln mutants demonstrated modestly increased susceptibility to Pseudomonas syringae and Pectobacterium carotovorum, probably reflecting the antagonistic action of MYC2 on the defense against these bacterial phytopathogens. Exogenously administered allantoin elicited the expression of JA-responsive genes, including MYC2, in wild-type plants, supporting the idea that allantoin might be responsible for the observed JA-related phenotypes of aln mutants. However, mutants deficient in bioactive JA (jar1-1), insensitive to JA (myc2-3), or deficient in ABA (aba2-1 and bglu18) suppressed the effect of exogenous allantoin. The suppression was further confirmed in aln-1 jar1-1 and aln-1 bglu18 double mutants. These results indicate that allantoin can activate the MYC2-regulated JA signaling pathway through ABA production. Overall, this study suggests a possible connection of purine catabolism with stress hormone homeostasis and signaling, and highlights the potential importance of allantoin in these interactions. PMID:26931169

  3. Effects of chicory inulin on serum metabolites of uric acid, lipids, glucose, and abdominal fat deposition in quails induced by purine-rich diets.

    PubMed

    Lin, Zhijian; Zhang, Bing; Liu, Xiaoqing; Jin, Rui; Zhu, Wenjing

    2014-11-01

    Inulin, a group of dietary fibers, is reported to improve the metabolic disorders. In the present study, we investigated the effects of chicory inulin on serum metabolites of uric acid (UA), lipids, glucose, and abdominal fat deposition in quail model induced by a purine-rich diet. In this study, 60 male French quails were randomly allocated to five groups: CON (control group), MOD (model group), BEN (benzbromarone-treated group), CHI-H (high-dosage chicory inulin-treated group), and CHI-L (low-dosage chicory inulin-treated group). The serum UA level was significantly increased in the model group from days 7 to 28, as well as triglyceride (TG) and free fatty acid (FFA) increased later in the experimental period. The abdominal fat ratio was increased on day 28. Benzbromarone can decrease UA levels on days 14 and 28. The high and low dosage of chicory inulin also decreased serum UA levels on days 7, 14, and 28. The abdominal fat ratio, activity, and protein of acetyl-CoA carboxylase (ACC) were decreased in chicory inulin-treated groups. The activities of xanthine oxidase (XOD) and fatty acid synthase (FAS) were increased in the model group and decreased in the benzbromarone and chicory inulin groups. This study evaluated a quail model of induced hyperuricemia with other metabolic disorders caused by a high-purine diet. The results indicated that a purine-rich diet might contribute to the development of hyperuricemia, hypertriglyceridemia, and abdominal obesity. Chicory inulin decreased serum UA, TG, and abdominal fat deposition in a quail model of hyperuricemia by altering the ACC protein expression and FAS and XOD activities.

  4. Concurrent profiling of indole-3-acetic acid, abscisic acid, and cytokinins and structurally related purines by high-performance-liquid-chromatography tandem electrospray mass spectrometry

    PubMed Central

    2012-01-01

    Background Cytokinins (CKs) are a group of plant growth regulators that are involved in several plant developmental processes. Despite the breadth of knowledge surrounding CKs and their diverse functions, much remains to be discovered about the full potential of CKs, including their relationship with the purine salvage pathway, and other phytohormones. The most widely used approach to query unknown facets of CK biology utilized functional genomics coupled with CK metabolite assays and screening of CK associated phenotypes. There are numerous different types of assays for determining CK quantity, however, none of these methods screen for the compendium of metabolites that are necessary for elucidating all roles, including purine salvage pathway enzymes in CK metabolism, and CK cross-talk with other phytohormones. Furthermore, all published analytical methods have drawbacks ranging from the required use of radiolabelled compounds, or hazardous derivatization reagents, poor sensitivity, lack of resolution between CK isomers and lengthy run times. Results In this paper, a method is described for the concurrent extraction, purification and analysis of several CKs (freebases, ribosides, glucosides, nucleotides), purines (adenosine monophosphate, inosine, adenosine, and adenine), indole-3-acetic acid, and abscisic acid from hundred-milligram (mg) quantities of Arabidopsis thaliana leaf tissue. This method utilizes conventional Bieleski solvents extraction, solid phase purification, and is unique because of its diverse range of detectable analytes, and implementation of a conventional HPLC system with a fused core column that enables good sensitivity without the requirement of a UHPLC system. Using this method we were able to resolve CKs about twice as fast as our previous method. Similarly, analysis of adenosine, indole-3-acetic acid, and abscisic acid, was comparatively rapid. A further enhancement of the method was the utilization of a QTRAP 5500 mass analyzer, which

  5. Computational Methods for De novo Protein Design and its Applications to the Human Immunodeficiency Virus 1, Purine Nucleoside Phosphorylase, Ubiquitin Specific Protease 7, and Histone Demethylases

    PubMed Central

    Bellows, M.L.; Floudas, C.A.

    2010-01-01

    This paper provides an overview of computational de novo protein design methods, highlighting recent advances and successes. Four protein systems are described that are important targets for drug design: human immunodeficiency virus 1, purine nucleoside phosphorylase, ubiquitin specific protease 7, and histone demethylases. Target areas for drug design for each protein are described, along with known inhibitors, focusing on peptidic inhibitors, but also describing some small-molecule inhibitors. Computational design methods that have been employed in elucidating these inhibitors for each protein are outlined, along with steps that can be taken in order to apply computational protein design to a system that has mainly used experimental methods to date. PMID:20210752

  6. Anticancer activity and cDNA microarray studies of a (RS)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9H-purine, and an acyclic (RS)-O,N-acetalic 6-chloro-7H-purine.

    PubMed

    Caba, Octavio; Díaz-Gavilán, Mónica; Rodríguez-Serrano, Fernando; Boulaiz, Houria; Aránega, Antonia; Gallo, Miguel A; Marchal, Juan A; Campos, Joaquín M

    2011-09-01

    Completing a SAR study, a series of (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H or 9H-purines was previously prepared. The most potent antiproliferative agent against the MCF-7 adenocarcinoma cell line that belongs to the benzoxazepine O,N-acetalic family is (RS)-9-[1-(9H-fluorenyl-9-methoxycarbonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9H-purine (16, IC(50) = 0.67 ± 0.18 μM), whilst (RS)-7-{2-(N-hydroxymethylphenyl)-2-nitrobenzenesulfonamido]-1-methoxyethyl}-6-chloro-7H-purine (37) shows the lowest IC(50) value between the family of acyclic O,N-acetals (IC(50) = 3.25 ± 0.23 μM). Moreover, 16 showed the better in vitro Therapeutic Index in breast cell lines (3.19), whilst 37 was found to be 3.69-fold more active against HT-29 human colon cancer cell line than versus IEC-6 normal rat intestinal epithelial cell line. The global apoptotic cells caused by 16 and 37 against MCF-7 were 80.08% and 54.85% of cell population after 48 h, respectively. cDNA microarray technology reveals potential drug targets, which are mainly centred on positive apoptosis regulatory pathway genes, and the repression of genes involved in carcinogenesis, proliferation and tumour invasion.

  7. Transformation of humus substances in the long-drained surface-gleyed soddy-podzolic soils under conditions of pronounced microrelief and different agrogenic loads

    NASA Astrophysics Data System (ADS)

    Ovchinnikova, M. F.

    2016-08-01

    The transformation of humus substances resulting from artificial drainage of the surface-gleyed soddy-podzolic soils under conditions of pronounced microtopography and different agrogenic loads was studied. The studied soil characteristics included acid-base conditions, the content and group composition of humus, the ratios between the fractions of humus acids, and optical density of humic acids. The features attesting to humus degradation were found in the soils of microdepressions periodically subjected to excessive surface moistening, in the soils of different landforms upon the construction of drainage trenches, and in the plowed non-fertilized soils. The response of humus characteristics to the changes in the ecological situation in the period of active application of agrochemicals for reclamation of the agrotechnogenically disturbed soils was traced. It was shown that the long-term dynamics of the particular parameters of the biological productivity of the soil depend on the hydrological and agrogenic factors, as well as on the weather conditions.

  8. Abrupt transition to heightened poliomyelitis epidemicity in England and Wales, 1947-1957, associated with a pronounced increase in the geographical rate of disease propagation.

    PubMed

    Smallman-Raynor, M R; Cliff, A D

    2014-03-01

    The abrupt transition to heightened poliomyelitis epidemicity in England and Wales, 1947-1957, was associated with a profound change in the spatial dynamics of the disease. Drawing on the complete record of poliomyelitis notifications in England and Wales, we use a robust method of spatial epidemiological analysis (swash-backwash model) to evaluate the geographical rate of disease propagation in successive poliomyelitis seasons, 1940-1964. Comparisons with earlier and later time periods show that the period of heightened poliomyelitis epidemicity corresponded with a sudden and pronounced increase in the spatial rate of disease propagation. This change was observed for both urban and rural areas and points to an abrupt enhancement in the propensity for the geographical spread of polioviruses. Competing theories of the epidemic emergence of poliomyelitis in England and Wales should be assessed in the light of this evidence.

  9. Learning to Pronounce First Words in Three Languages: An Investigation of Caregiver and Infant Behavior Using a Computational Model of an Infant

    PubMed Central

    Howard, Ian S.; Messum, Piers

    2014-01-01

    Words are made up of speech sounds. Almost all accounts of child speech development assume that children learn the pronunciation of first language (L1) speech sounds by imitation, most claiming that the child performs some kind of auditory matching to the elements of ambient speech. However, there is evidence to support an alternative account and we investigate the non-imitative child behavior and well-attested caregiver behavior that this account posits using Elija, a computational model of an infant. Through unsupervised active learning, Elija began by discovering motor patterns, which produced sounds. In separate interaction experiments, native speakers of English, French and German then played the role of his caregiver. In their first interactions with Elija, they were allowed to respond to his sounds if they felt this was natural. We analyzed the interactions through phonemic transcriptions of the caregivers' utterances and found that they interpreted his output within the framework of their native languages. Their form of response was almost always a reformulation of Elija's utterance into well-formed sounds of L1. Elija retained those motor patterns to which a caregiver responded and formed associations between his motor pattern and the response it provoked. Thus in a second phase of interaction, he was able to parse input utterances in terms of the caregiver responses he had heard previously, and respond using his associated motor patterns. This capacity enabled the caregivers to teach Elija to pronounce some simple words in their native languages, by his serial imitation of the words' component speech sounds. Overall, our results demonstrate that the natural responses and behaviors of human subjects to infant-like vocalizations can take a computational model from a biologically plausible initial state through to word pronunciation. This provides support for an alternative to current auditory matching hypotheses for how children learn to pronounce. PMID

  10. Learning to pronounce first words in three languages: an investigation of caregiver and infant behavior using a computational model of an infant.

    PubMed

    Howard, Ian S; Messum, Piers

    2014-01-01

    Words are made up of speech sounds. Almost all accounts of child speech development assume that children learn the pronunciation of first language (L1) speech sounds by imitation, most claiming that the child performs some kind of auditory matching to the elements of ambient speech. However, there is evidence to support an alternative account and we investigate the non-imitative child behavior and well-attested caregiver behavior that this account posits using Elija, a computational model of an infant. Through unsupervised active learning, Elija began by discovering motor patterns, which produced sounds. In separate interaction experiments, native speakers of English, French and German then played the role of his caregiver. In their first interactions with Elija, they were allowed to respond to his sounds if they felt this was natural. We analyzed the interactions through phonemic transcriptions of the caregivers' utterances and found that they interpreted his output within the framework of their native languages. Their form of response was almost always a reformulation of Elija's utterance into well-formed sounds of L1. Elija retained those motor patterns to which a caregiver responded and formed associations between his motor pattern and the response it provoked. Thus in a second phase of interaction, he was able to parse input utterances in terms of the caregiver responses he had heard previously, and respond using his associated motor patterns. This capacity enabled the caregivers to teach Elija to pronounce some simple words in their native languages, by his serial imitation of the words' component speech sounds. Overall, our results demonstrate that the natural responses and behaviors of human subjects to infant-like vocalizations can take a computational model from a biologically plausible initial state through to word pronunciation. This provides support for an alternative to current auditory matching hypotheses for how children learn to pronounce.

  11. DNA sequence-dependent ionic currents in ultra-small solid-state nanopores

    NASA Astrophysics Data System (ADS)

    Comer, Jeffrey; Aksimentiev, Aleksei

    2016-05-01

    Measurements of ionic currents through nanopores partially blocked by DNA have emerged as a powerful method for characterization of the DNA nucleotide sequence. Although the effect of the nucleotide sequence on the nanopore blockade current has been experimentally demonstrated, prediction and interpretation of such measurements remain a formidable challenge. Using atomic resolution computational approaches, here we show how the sequence, molecular conformation, and pore geometry affect the blockade ionic current in model solid-state nanopores. We demonstrate that the blockade current from a DNA molecule is determined by the chemical identities and conformations of at least three consecutive nucleotides. We find the blockade currents produced by the nucleotide triplets to vary considerably with their nucleotide sequences despite having nearly identical molecular conformations. Encouragingly, we find blockade current differences as large as 25% for single-base substitutions in ultra small (1.6 nm × 1.1 nm cross section; 2 nm length) solid-state nanopores. Despite the complex dependence of the blockade current on the sequence and conformation of the DNA triplets, we find that, under many conditions, the number of thymine bases is positively correlated with the current, whereas the number of purine bases and the presence of both purines and pyrimidines in the triplet are negatively correlated with the current. Based on these observations, we construct a simple theoretical model that relates the ion current to the base content of a solid-state nanopore. Furthermore, we show that compact conformations of DNA in narrow pores provide the greatest signal-to-noise ratio for single base detection, whereas reduction of the nanopore length increases the ionic current noise. Thus, the sequence dependence of the nanopore blockade current can be theoretically rationalized, although the predictions will likely need to be customized for each nanopore type.Measurements of ionic

  12. Flanking sequences modulate diepoxide and mustard cross-linking efficiencies at the 5'-GNC site.

    PubMed

    Sawyer, Gregory A; Frederick, Elizabeth D; Millard, Julie T

    2004-08-01

    Diepoxybutane, diepoxyoctane, and mechlorethamine are cytotoxic agents that induce interstrand cross-links between the N7 positions of deoxyguanosine residues on opposite strands of the DNA duplex preferentially at 5'-GNC sequences. We have systematically varied the identity of either the base 5' to the cross-linked deoxyguanosine residues or the intervening base pair to determine flanking sequence effects on cross-linking efficiency. We used synthetic DNA oligomers containing four 5'-N(1)GN(2)C sites that varied either N(1) or N(2). Interstrand cross-links were purified through denaturing polyacrylamide gel electrophoresis and then subjected to piperidine cleavage. The amount of cleavage at each deoxyguanosine residue, representative of cross-linking efficiency at that site, was determined by sequencing gel analysis. Our data suggest that cross-linking efficiency varies with the identity of N(1) similarly (purines > pyrimidines) for diepoxybutane, diepoxyoctane, and mechlorethamine but that the effects of N(2) differ for the three compounds.

  13. MSLICE Sequencing

    NASA Technical Reports Server (NTRS)

    Crockett, Thomas M.; Joswig, Joseph C.; Shams, Khawaja S.; Norris, Jeffrey S.; Morris, John R.

    2011-01-01

    MSLICE Sequencing is a graphical tool for writing sequences and integrating them into RML files, as well as for producing SCMF files for uplink. When operated in a testbed environment, it also supports uplinking these SCMF files to the testbed via Chill. This software features a free-form textural sequence editor featuring syntax coloring, automatic content assistance (including command and argument completion proposals), complete with types, value ranges, unites, and descriptions from the command dictionary that appear as they are typed. The sequence editor also has a "field mode" that allows tabbing between arguments and displays type/range/units/description for each argument as it is edited. Color-coded error and warning annotations on problematic tokens are included, as well as indications of problems that are not visible in the current scroll range. "Quick Fix" suggestions are made for resolving problems, and all the features afforded by modern source editors are also included such as copy/cut/paste, undo/redo, and a sophisticated find-and-replace system optionally using regular expressions. The software offers a full XML editor for RML files, which features syntax coloring, content assistance and problem annotations as above. There is a form-based, "detail view" that allows structured editing of command arguments and sequence parameters when preferred. The "project view" shows the user s "workspace" as a tree of "resources" (projects, folders, and files) that can subsequently be opened in editors by double-clicking. Files can be added, deleted, dragged-dropped/copied-pasted between folders or projects, and these operations are undoable and redoable. A "problems view" contains a tabular list of all problems in the current workspace. Double-clicking on any row in the table opens an editor for the appropriate sequence, scrolling to the specific line with the problem, and highlighting the problematic characters. From there, one can invoke "quick fix" as described

  14. Influence of infection by Toxoplasma gondii on purine levels and E-ADA activity in the brain of mice experimentally infected mice.

    PubMed

    Tonin, Alexandre A; Da Silva, Aleksandro S; Casali, Emerson A; Silveira, Stephanie S; Moritz, Cesar E J; Camillo, Giovana; Flores, Mariana M; Fighera, Rafael; Thomé, Gustavo R; Morsch, Vera M; Schetinger, Maria Rosa C; Rue, Mario De La; Vogel, Fernanda S F; Lopes, Sonia T A

    2014-07-01

    The aim of this study was to assess the purine levels and E-ADA activity in the brain of mice (BALB/c) experimentally infected with Toxoplasma gondii. In experiment I (n=24) the mice were infected with RH strain of T. gondii, while in experiment II (n=36) they were infected with strain ME-49 of T. gondii. Our results showed that, for RH strain (acute phase), an increase in both periods in the levels of ATP, ADP, AMP, adenosine, hypoxanthine, xanthine (only on day 6 PI) and uric acid (only on day 6 PI). By the other hand, the RH strain led, on days 4 and 6 PI, to a reduction in the concentration of inosine. ME-49, a cystogenic strain, showed some differences in acute and chronic phase, since on day 6 PI the levels of ATP and ADP were increased, while on day 30 these same nucleotides were reduced. On day 60 PI, ME-49 induced a reduction in the levels of ATP, ADP, AMP, adenosine, inosine and xanthine, while uric acid was increased. A decrease of E-ADA activity was observed in brain on days 4 and 6 PI (RH), and 30 PI (ME-49); however on day 60 PI E-ADA activity was increased for infection by ME-49 strain. Therefore, it was possible to conclude that infection with T. gondii changes the purine levels and the activity of E-ADA in brain, which may be associated with neurological signs commonly observed in this disease.

  15. Protection of midbrain dopaminergic neurons by the end-product of purine metabolism uric acid: potentiation by low-level depolarization.

    PubMed

    Guerreiro, Serge; Ponceau, Aurélie; Toulorge, Damien; Martin, Elodie; Alvarez-Fischer, Daniel; Hirsch, Etienne C; Michel, Patrick P

    2009-05-01

    High plasma levels of the end product of purine metabolism uric acid (UA) predict a reduced risk of developing Parkinson's disease suggesting that UA may operate as a protective factor for midbrain dopaminergic neurons. Consistent with this view, UA exerted partial but long-term protection in a culture model in which these neurons die spontaneously. The rescued neurons were functional as they accumulated dopamine, efficiently. The use of the fluorescent probe dihydrorhodamine-123 revealed that UA operated by an antioxidant mechanism. The iron chelating agent desferrioxamine, the H(2)O(2) scavenger enzyme catalase and the inhibitor of lipid peroxidation Trolox mimicked the effects of UA, suggesting that UA neutralized reactive oxygen species produced via a Fenton-type chemical reaction. UA was, however, not significantly accumulated into neurons, which indicates that the antioxidant effect occurred probably extracellularly. Structure - activity relationships among purine derivatives revealed that the antioxidant properties of UA resulted from the presence of a 8-one substituent in its chemical structure. Of interest, the stimulation of L-type Ca(2+) channels by high K(+)-induced depolarization and the ensuing activation of extracellular signal-regulated kinases 1/2 strongly improved the neuroprotective effect of UA whereas the depolarizing signal alone had no effect. In summary, our data indicate that UA may interfere directly with the disease's pathomechanism.

  16. DNA sequence-dependent ionic currents in ultra-small solid-state nanopores.

    PubMed

    Comer, Jeffrey; Aksimentiev, Aleksei

    2016-05-05

    Measurements of ionic currents through nanopores partially blocked by DNA have emerged as a powerful method for characterization of the DNA nucleotide sequence. Although the effect of the nucleotide sequence on the nanopore blockade current has been experimentally demonstrated, prediction and interpretation of such measurements remain a formidable challenge. Using atomic resolution computational approaches, here we show how the sequence, molecular conformation, and pore geometry affect the blockade ionic current in model solid-state nanopores. We demonstrate that the blockade current from a DNA molecule is determined by the chemical identities and conformations of at least three consecutive nucleotides. We find the blockade currents produced by the nucleotide triplets to vary considerably with their nucleotide sequences despite having nearly identical molecular conformations. Encouragingly, we find blockade current differences as large as 25% for single-base substitutions in ultra small (1.6 nm × 1.1 nm cross section; 2 nm length) solid-state nanopores. Despite the complex dependence of the blockade current on the sequence and conformation of the DNA triplets, we find that, under many conditions, the number of thymine bases is positively correlated with the current, whereas the number of purine bases and the presence of both purines and pyrimidines in the triplet are negatively correlated with the current. Based on these observations, we construct a simple theoretical model that relates the ion current to the base content of a solid-state nanopore. Furthermore, we show that compact conformations of DNA in narrow pores provide the greatest signal-to-noise ratio for single base detection, whereas reduction of the nanopore length increases the ionic current noise. Thus, the sequence dependence of the nanopore blockade current can be theoretically rationalized, although the predictions will likely need to be customized for each nanopore type.

  17. Insertion Sequences

    PubMed Central

    Mahillon, Jacques; Chandler, Michael

    1998-01-01

    Insertion sequences (ISs) constitute an important component of most bacterial genomes. Over 500 individual ISs have been described in the literature to date, and many more are being discovered in the ongoing prokaryotic and eukaryotic genome-sequencing projects. The last 10 years have also seen some striking advances in our understanding of the transposition process itself. Not least of these has been the development of various in vitro transposition systems for both prokaryotic and eukaryotic elements and, for several of these, a detailed understanding of the transposition process at the chemical level. This review presents a general overview of the organization and function of insertion sequences of eubacterial, archaebacterial, and eukaryotic origins with particular emphasis on bacterial elements and on different aspects of the transposition mechanism. It also attempts to provide a framework for classification of these elements by assigning them to various families or groups. A total of 443 members of the collection have been grouped in 17 families based on combinations of the following criteria: (i) similarities in genetic organization (arrangement of open reading frames); (ii) marked identities or similarities in the enzymes which mediate the transposition reactions, the recombinases/transposases (Tpases); (iii) similar features of their ends (terminal IRs); and (iv) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length). A brief description of the mechanism(s) involved in the mobility of individual ISs in each family and of the structure-function relationships of the individual Tpases is included where available. PMID:9729608

  18. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    PubMed

    Paugh, Steven W; Coss, David R; Bao, Ju; Laudermilk, Lucas T; Grace, Christy R; Ferreira, Antonio M; Waddell, M Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F; Panetta, John C; Wilkinson, Mark R; Pui, Ching-Hon; Naeve, Clayton W; Uberbacher, Edward C; Bonten, Erik J; Evans, William E

    2016-02-01

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16)) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

  19. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

    PubMed Central

    Grace, Christy R.; Ferreira, Antonio M.; Waddell, M. Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F.; Panetta, John C.; Wilkinson, Mark R.; Pui, Ching-Hon; Naeve, Clayton W.; Uberbacher, Edward C.; Bonten, Erik J.; Evans, William E.

    2016-01-01

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10−16) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

  20. Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA.

    PubMed

    Kretschy, Nicole; Sack, Matej; Somoza, Mark M

    2016-03-16

    The fluorescent intensity of Cy3 and Cy5 dyes is strongly dependent on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent fluorescence may significantly influence the data obtained from many common experimental methods based on fluorescence detection of nucleic acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence dependent fluorescence, we have measured the fluorescence intensity of Cy3 and Cy5 bound to the 5' end of all 1024 possible double-stranded DNA 5mers. The fluorescence intensity was also determined for these dyes bound to the 5' end of fixed-sequence double-stranded DNA with a variable sequence 3' overhang adjacent to the dye. The labeled DNA oligonucleotides were made using light-directed, in situ microarray synthesis. The results indicate that the fluorescence intensity of both dyes is sensitive to all five bases or base pairs, that the sequence dependence is stronger for double- (vs single-) stranded DNA, and that the dyes are sensitive to both the adjacent dsDNA sequence and the 3'-ssDNA overhang. Purine-rich sequences result in higher fluorescence. The results can be used to estimate measurement error in experiments with fluorescent-labeled DNA, as well as to optimize the fluorescent signal by considering the nucleobase environment of the labeling cyanine dye.

  1. The immediate upstream region of the 5′-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana

    PubMed Central

    Kim, Younghyun; Lee, Goeun; Jeon, Eunhyun; Sohn, Eun ju; Lee, Yongjik; Kang, Hyangju; Lee, Dong wook; Kim, Dae Heon; Hwang, Inhwan

    2014-01-01

    The nucleotide sequence around the translational initiation site is an important cis-acting element for post-transcriptional regulation. However, it has not been fully understood how the sequence context at the 5′-untranslated region (5′-UTR) affects the translational efficiency of individual mRNAs. In this study, we provide evidence that the 5′-UTRs of Arabidopsis genes showing a great difference in the nucleotide sequence vary greatly in translational efficiency with more than a 200-fold difference. Of the four types of nucleotides, the A residue was the most favourable nucleotide from positions −1 to −21 of the 5′-UTRs in Arabidopsis genes. In particular, the A residue in the 5′-UTR from positions −1 to −5 was required for a high-level translational efficiency. In contrast, the T residue in the 5′-UTR from positions −1 to −5 was the least favourable nucleotide in translational efficiency. Furthermore, the effect of the sequence context in the −1 to −21 region of the 5′-UTR was conserved in different plant species. Based on these observations, we propose that the sequence context immediately upstream of the AUG initiation codon plays a crucial role in determining the translational efficiency of plant genes. PMID:24084084

  2. Emotional scenes elicit more pronounced self-reported emotional experience and greater EPN and LPP modulation when compared to emotional faces.

    PubMed

    Thom, Nathaniel; Knight, Justin; Dishman, Rod; Sabatinelli, Dean; Johnson, Douglas C; Clementz, Brett

    2014-06-01

    Emotional faces and scenes carry a wealth of overlapping and distinct perceptual information. Despite widespread use in the investigation of emotional perception, expressive face and evocative scene stimuli are rarely assessed in the same experiment. Here, we evaluated self-reports of arousal and pleasantness, as well as early and late event-related potentials (e.g., N170, early posterior negativity [EPN], late positive potential [LPP]) as subjects viewed neutral and emotional faces and scenes, including contents representing anger, fear, and joy. Results demonstrate that emotional scenes were rated as more evocative than emotional faces, as only scenes produced elevated self-reports of arousal. In addition, viewing scenes resulted in more extreme ratings of pleasantness (and unpleasantness) than did faces. EEG results indicate that both expressive faces and emotional scenes evoke enhanced negativity in the N170 component, while the EPN and LPP components show significantly enhanced modulation only by scene, relative to face stimuli. These data suggest that viewing emotional scenes results in a more pronounced emotional experience that is associated with reliable modulation of visual event-related potentials that are implicated in emotional circuits in the brain.

  3. Photosynthetic temperature responses of tree species in Rwanda: evidence of pronounced negative effects of high temperature in montane rainforest climax species

    NASA Astrophysics Data System (ADS)

    Vårhammar, Angelica; Wallin, Göran; McLean, Christopher M.; Dusenge, Mirindi Eric; Medlyn, Belinda E.; Hasper, Thomas B.; Nsabimana, Donat; Uddling, Johan

    2015-04-01

    The sensitivity of photosynthetic metabolism to temperature has been identified as a key uncertainty for projecting the magnitude of the terrestrial feedback on future climate change. While temperature responses of photosynthetic capacities have been comparatively well investigated in temperate species, the responses of tropical tree species remain unexplored. We compared the responses of seedlings of native cold-adapted tropical montane rainforest tree species to exotic warm-adapted plantation species, all growing in an intermediate temperature common garden in Rwanda. Leaf gas exchange responses to CO2 at different temperatures (20 - 40 C) were used to assess the temperature responses of biochemical photosynthetic capacities. Analyses revealed a lower optimum temperature for photosynthetic electron transport rates than for Rubisco carboxylation rates, along with lower electron transport optima in the native cold-adapted than in the exotic warm-adapted species. The photosynthetic optimum temperatures were generally exceeded by daytime peak leaf temperatures, in particular in the native montane rainforest climax species. This study thus provides evidence of pronounced negative effects of high temperature in tropical trees and indicates high susceptibility of montane rainforest climax species to future global warming. (Reference: New Phytologist, in press)

  4. Photosynthetic temperature responses of tree species in Rwanda: evidence of pronounced negative effects of high temperature in montane rainforest climax species.

    PubMed

    Vårhammar, Angelica; Wallin, Göran; McLean, Christopher M; Dusenge, Mirindi Eric; Medlyn, Belinda E; Hasper, Thomas B; Nsabimana, Donat; Uddling, Johan

    2015-05-01

    The sensitivity of photosynthetic metabolism to temperature has been identified as a key uncertainty for projecting the magnitude of the terrestrial feedback on future climate change. While temperature responses of photosynthetic capacities have been comparatively well investigated in temperate species, the responses of tropical tree species remain unexplored. We compared the responses of seedlings of native cold-adapted tropical montane rainforest tree species with those of exotic warm-adapted plantation species, all growing in an intermediate temperature common garden in Rwanda. Leaf gas exchange responses to carbon dioxide (CO2 ) at different temperatures (20-40°C) were used to assess the temperature responses of biochemical photosynthetic capacities. Analyses revealed a lower optimum temperature for photosynthetic electron transport rates than for Rubisco carboxylation rates, along with lower electron transport optima in the native cold-adapted than in the exotic warm-adapted species. The photosynthetic optimum temperatures were generally exceeded by daytime peak leaf temperatures, in particular in the native montane rainforest climax species. This study thus provides evidence of pronounced negative effects of high temperature in tropical trees and indicates high susceptibility of montane rainforest climax species to future global warming.

  5. Early sensory–perceptual processing deficits for affectively valenced inputs are more pronounced in schizophrenia patients with a history of violence than in their non-violent peers

    PubMed Central

    Foxe, John J.; Czobor, Pal; Wylie, Glenn R.; Kamiel, Stephanie M.; Huening, Jessica; Nair-Collins, Mike; Krakowski, Menahem I.

    2013-01-01

    Individuals with schizophrenia are more prone to violent behaviors than the general population. It is increasingly recognized that processing of emotionally valenced stimuli is impaired in schizophrenia, a deficit that may play a role in aggressive behavior. Our goal was to establish whether patients with a history of violence would show more severe deficits in processing emotionally valenced inputs than non-violent patients. Using event-related potentials, we measured how early during processing of emotional valence, evidence of aberrant function was observed. A total of 42 schizophrenia patients (21 with history of violence; 21 without) and 28 healthy controls were tested. Participants performed an inhibitory control task, making speeded responses to pictorial stimuli. Pictures occasionally repeated twice and participants withheld responses to these repeats. Valenced pictures from the International Affective Picture System were presented. Results in controls showed modulations during the earliest phases of sensory processing (<100 ms) for negatively valenced pictures. A cascade of modulations ensued, involving sensory and perceptual processing stages. In contrast, neither schizophrenia group showed early differentiation. Non-violent patients showed earliest modulations beginning ∼150 ms. For violent patients, however, earliest modulations were further delayed and highly attenuated. The current study reveals sensory–perceptual processing dysfunction for negatively valenced inputs, which is particularly pronounced in aggressive patients. PMID:22563006

  6. Delay of postnatal maturation sensitizes the mouse prostate to testosterone-induced pronounced hyperplasia: protective role of estrogen receptor-beta.

    PubMed

    Savolainen, Saija; Pakarainen, Tomi; Huhtaniemi, Ilpo; Poutanen, Matti; Mäkelä, Sari

    2007-09-01

    The role of estrogens in the etiology of prostate cancer is controversial. To demonstrate the specific effects of estrogens and androgens on the development of the prostatic epithelial hyperplasia, we used luteinizing hormone receptor knockout mice (LuRKO), which are resistant to pituitary regulation mediated by luteinizing hormone, lack postnatal androgen production, and have rudimentary accessory sex glands, the growth of which can be induced with exogenous androgen replacement. This model is thus ideal for the investigation of direct hormonal effects on the prostate. Testosterone, but not 5alpha-dihydrotestosterone, replacement from 21 days of life for 8 weeks induced pronounced hyperplasia and inflammation in the prostates of LuRKO mice. Interestingly, 5alpha-dihydrotestosterone combined with 17beta-estradiol did not induce hyperplasia or inflammation, and treatments with inhibitors of estrogen action, aromatase inhibitor, and ICI 182780 further exacerbated testosterone-induced hyperplastic growth. However, the activation of estrogen receptor (ER)-beta with a specific agonist, DPN [2,3-bis(4-hydroxyphenol)-propionitrile], prevented the development of prostatic hyperplasia and inflammation in testosterone-treated LuRKO mice. Thus, it seems that in the presence of sufficient androgenic stimulation, it is the balance between ER-alpha- and ER-beta-mediated signaling that determines whether estrogens promote hyperplasia or protect the prostate against hyperplastic changes.

  7. The big ban on bituminous coal sales revisited: Serious epidemics and pronounced trends feign excess mortality previously attributed to heavy black-smoke exposure

    SciTech Connect

    Wittmaack, K.

    2007-07-01

    The effect of banning bituminous coal sales on the black-smoke concentration and the mortality rates in Dublin, Ireland, has been analyzed recently. Based on the application of standard epidemiological procedures, the authors concluded that, as a result of the ban, the total nontrauma death rate was reduced strongly (-8.0% unadjusted, -5.7% adjusted). The purpose of this study was to reanalyze the original data with the aim of clarifying the three most important aspects of the study, (a) the effect of epidemics, (b) the trends in mortality rates due to advances in public health care, and (c) the correlation between mortality rates and black-smoke concentrations. Particular attention has been devoted to a detailed evaluation of the time dependence of mortality rates, stratified by season. Death rates were found to be strongly enhanced during three severe pre-ban winter-spring epidemics. The cardiovascular mortality rates exhibited a continuous decrease over the whole study period, in general accordance with trends in the rest of Ireland. These two effects can fully account for the previously identified apparent correlation between reduced mortality and the very pronounced ban-related lowering of the black-smoke concentration. The third important finding was that in nonepidemic pre-ban seasons even large changes in the concentration of black smoke had no detectable effect on mortality rates. The reanalysis suggests that epidemiological studies exploring the effect of ambient particulate matter on mortality require improved tools allowing proper adjustment for epidemics and trends.

  8. Chronic pro-oxidative state and mitochondrial dysfunctions are more pronounced in fibroblasts from Down syndrome foeti with congenital heart defects.

    PubMed

    Piccoli, Claudia; Izzo, Antonella; Scrima, Rosella; Bonfiglio, Ferdinando; Manco, Rosanna; Negri, Rosa; Quarato, Giovanni; Cela, Olga; Ripoli, Maria; Prisco, Marina; Gentile, Flaviana; Calì, Gaetano; Pinton, Paolo; Conti, Anna; Nitsch, Lucio; Capitanio, Nazzareno

    2013-03-15

    Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.

  9. De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo

    PubMed Central

    Yan, Donghong; Katakam, Anand K.; Reichelt, Mike; Lin, Baiwei; Kim, Janice; Park, Summer; Date, Shailesh V.; Monk, Ian R.; Xu, Min; Austin, Cary D.; Maurer, Till

    2016-01-01

    ABSTRACT De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5′-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo. In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCE De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus. We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from

  10. Functional Analysis of 14 Genes That Constitute the Purine Catabolic Pathway in Bacillus subtilis and Evidence for a Novel Regulon Controlled by the PucR Transcription Activator

    PubMed Central

    Schultz, Anna C.; Nygaard, Per; Saxild, Hans H.

    2001-01-01

    The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285° on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B

  11. Quantum spin Hall insulators in functionalized arsenene (AsX, X = F, OH and CH3) monolayers with pronounced light absorption

    NASA Astrophysics Data System (ADS)

    Zhao, Jun; Li, Yanle; Ma, Jing

    2016-05-01

    The search for new two-dimensional topological insulators (2D-TIs) with large band gaps is of great interest and importance. Our first-principles calculations predicted three candidates for 2D-TIs, arsenene functionalized with F, OH and CH3 groups (AsX, X = F, OH and CH3), which preserved large bulk band gaps from 100 to 160 meV (up to 260 meV) derived from the spin-orbit coupling (SOC) within the px,y orbitals. This picture is similar to what was reported for an AsH monolayer with a band gap of 193 meV. Ab initio molecular dynamic (AIMD) simulations demonstrated the thermal stabilities of the AsX monolayers even at 500 K. The nontrivial topological phase was confirmed by the topological invariant Z2 and topological edge state. The topological electronic bandgap of the AsF monolayer can be effectively modulated by biaxial tensile strain and vertical external electric field. In addition, pronounced light absorption in the near-infrared and visible range of the solar spectrum was expected for the AsX (X = H, F) monolayers from the adsorption peaks at 0.45-1.6 eV, which is attractive for light harvesting. The nontrivial quantum spin Hall (QSH) insulators AsX could be promising candidates for practical room-temperature applications in dissipationless transport devices and photovoltaics.The search for new two-dimensional topological insulators (2D-TIs) with large band gaps is of great interest and importance. Our first-principles calculations predicted three candidates for 2D-TIs, arsenene functionalized with F, OH and CH3 groups (AsX, X = F, OH and CH3), which preserved large bulk band gaps from 100 to 160 meV (up to 260 meV) derived from the spin-orbit coupling (SOC) within the px,y orbitals. This picture is similar to what was reported for an AsH monolayer with a band gap of 193 meV. Ab initio molecular dynamic (AIMD) simulations demonstrated the thermal stabilities of the AsX monolayers even at 500 K. The nontrivial topological phase was confirmed by the topological

  12. Pronounced reduction of fluoride exposure in free-ranging deer in North Bohemia (Czech Republic) as indicated by the biomarkers skeletal fluoride content and dental fluorosis.

    PubMed

    Kierdorf, Uwe; Bahelková, Petra; Sedláček, František; Kierdorf, Horst

    2012-01-01

    Wild deer have been recommended as bioindicators of fluoride pollution. We compared bone fluoride concentrations and prevalence and severity of dental fluorosis in free-ranging European roe deer (Capreolus capreolus) and red deer (Cervus elaphus) from five counties in the northwestern part of the Czech Republic that had been collected by hunters and whose mandibles were presented at trophy exhibitions in the years 1996/1997 ("early period") and 2009 ("late period"). Data on atmospheric fluoride deposition suggested that the deer from the early period had been exposed to markedly higher fluoride levels than those from the late period. We therefore predicted a decline in skeletal fluoride levels and prevalence of dental fluorosis for both species from the early to the late period. Fluoride concentrations were determined in the coronoid process of the mandible, and assessment of dental fluorosis was performed on the permanent cheek teeth. A pronounced drop in fluoride concentrations from the early period (roe deer (n = 157), median: 3147 mg F(-)/kg of dry bone; red deer (n = 127), median: 1263 mg F(-)/kg of dry bone) to the late period (roe deer (n = 117), median: 350 mg F(-)/kg of dry bone; red deer (n = 72), median: 288 mg F(-)/kg of dry bone) was recorded. Prevalence of dental fluorosis also markedly declined from the early to the late period (roe deer: from 93% to 12%, red deer: from 87% to 28%). The reduction of fluoride deposition in the study area and, in consequence, fluoride exposure of the resident deer populations, is attributed largely to the implementation of emission control devices in the brown coal-fired power plants located in North Bohemia from the mid 1990s onwards. The findings of the present study demonstrate that wild deer are well suited for monitoring temporal changes in fluoride pollution of their habitats.

  13. A Tree-Ring Temperature Reconstruction from the Wrangell Mountains, Alaska (1593-1992): Evidence for Pronounced Regional Cooling During the Maunder Minimum

    NASA Astrophysics Data System (ADS)

    DArrigo, R.; Davi, N.; Jacoby, G.; Wiles, G.

    2002-05-01

    The Maunder Minimum interval (from the mid-1600s-early 1700s) is believed to have been one of the coldest periods of the past thousand years in the Northern Hemisphere. A maximum latewood density temperature reconstruction for the Wrangell Mountains, southern Alaska (1593-1992) provides information on regional temperature change during the Maunder Minimum and other periods of severe cold over the past four centuries. The Wrangell density record, which reflects warm season (July-September) temperatures, shows an overall cooling over the Maunder Minimum period with annual values reaching as low as -1.8oC below the long-term mean. Ring widths, which can integrate annual as well as summer conditions, also show pronounced cooling at the Wrangell site during this time, as do Arctic and hemispheric-scale temperature reconstructions based on tree rings and other proxy data. Maximum ages of glacial advance based on kill dates from overrun logs (which reflect cooler temperatures) coincide temporally with the cooling seen in the density and ring width records. In contrast, a recent modeling study indicates that during this period there was cold season (November-April) warming over much of Alaska, but cooling over other northern continental regions, as a result of decreased solar irradiance initiating low Arctic Oscillation index conditions. The influence of other forcings on Alaskan climate, the absence of ocean dynamical feedbacks in the model, and the different seasonality represented by the model and the trees may be some of the possible explanations for the different model and proxy results.

  14. Comparing antioxidant capacity of purine alkaloids: a new, efficient trio for screening and discovering potential antioxidants in vitro and in vivo.

    PubMed

    Tsoi, Bun; Yi, Ruo-Nan; Cao, Ling-Fang; Li, Shan-Bing; Tan, Rui-Rong; Chen, Min; Li, Xiao-Xiao; Wang, Chen; Li, Yi-Fang; Kurihara, Hiroshi; He, Rong-Rong

    2015-06-01

    The most commonly applied strategies for the evaluation of antioxidant capacity are the chemical- or cell-based approaches. However, the results obtained from these methods might not reflect the antioxidant ability of test samples within organisms. In this study, we propose a combination of experiments, including oxygen radical absorbance capacity (ORAC), cellular antioxidant activity assay (CAA), and the chick embryo model, as an efficient trio to evaluate antioxidant capacity of food components. Taking purine alkaloids as example, results demonstrate that chemical and cellular method might misinterpret their true ability on antioxidation. In chick embryo model, caffeine and theacrine can significantly improve vessel density on chorioallantoic membrane and myocardial apoptosis. The mechanism can be involving multiple targets within the organism. We believe that the trio proposed can be widely utilized in screening massive number of antioxidant in a cost-effective way. It will also help discovering new antioxidants that are easily being omitted due to their relatively poor in vitro activities.

  15. Adenine versus guanine DNA adducts of aristolochic acids: role of the carcinogen-purine linkage in the differential global genomic repair propensity.

    PubMed

    Kathuria, Preetleen; Sharma, Purshotam; Wetmore, Stacey D

    2015-09-03

    Computational modeling is employed to provide a plausible structural explanation for the experimentally-observed differential global genome repair (GGR) propensity of the ALII-N(2)-dG and ALII-N(6)-dA DNA adducts of aristolochic acid II. Our modeling studies suggest that an intrinsic twist at the carcinogen-purine linkage of ALII-N(2)-dG induces lesion site structural perturbations and conformational heterogeneity of damaged DNA. These structural characteristics correlate with the relative repair propensities of AA-adducts, where GGR recognition occurs for ALII-N(2)-dG, but is evaded for intrinsically planar ALII-N(6)-dA that minimally distorts DNA and restricts the conformational flexibility of the damaged duplex. The present analysis on the ALII adduct model systems will inspire future experimental studies on these adducts, and thereby may extend the list of structural factors that directly correlate with the propensity for GGR recognition.

  16. Design and SAR of new substituted purines bearing aryl groups at N9 position as HIV-1 Tat-TAR interaction inhibitors.

    PubMed

    Pang, Ruifang; Zhang, Chunlei; Yuan, Dekai; Yang, Ming

    2008-09-01

    Twenty-four purine derivatives bearing aryl groups at N9 position were designed and synthesized as HIV-1 Tat-TAR interaction inhibitors. All the compounds showed high antiviral activities in inhibiting the formation of SIV-induced syncytium in CEM174 cells. Ten of them with low cytotoxicities were evaluated by Tat dependent HIV-1 LTR-driven CAT gene expression colorimetric enzyme assay in human 293T cells at a concentration of 30 microM, indicating effective inhibitory activities of blocking the Tat-TAR interaction. The aryl groups at N9 position affected the binding affinities between compounds and TAR RNA, showing some specificities of aryl groups to TAR RNA.

  17. Exposure, drowning, and missed beats'': Shelf-edge sequence stratigraphy, Middle Ordovician non-cycle carbonates, east Tennessee

    SciTech Connect

    Steinhauff, D.M.; Walker, K.R. . Dept. of Geological Sciences)

    1993-03-01

    Stratigraphic sequences in a Middle Ordovician platform interior carbonate succession can be correlated to the shelf-edge. Surfaces of subaerial exposure are correlated among seven stratigraphic sections, 4 at on-shelf positions and 3 at or near the shelf-edge. This analysis of carbonate facies geometry is based on outcrop observation, analysis of thin-sections, and consideration of water-depth curves. In platform successions, most sequences are bounded by exposure surfaces, but in more continuously subtidal areas, period of shallowing and drowning are discerned in bathymetric curves based on multiple lines of evidence. Cephalopod-rich limestone beds that overlie subtidal limestones and contain black, iron-manganese oxide rich sediment and pavements provide additional evidence for drowning followed by condensed sedimentation. These non-cyclic carbonates have 4th order sequences that are grouped into 3rd order sequences based on the occurrence of pronounced and less pronounced exposure surfaces. For example, 7 pronounced surfaces of subaerial exposure define six 3rd order sequences at on-shelf positions at Thorn Hill, TN. Within the lower four 3rd order sequences, 19 less pronounced surfaces occur with a total of 23 surfaces recognized in about 400 m. These less pronounced surfaces define 4th order sequences that are correlative across the shelf. Within these 3rd and 4th order sequences, 5th order packages occur. Fifth order sequences are not correlative across the shelf or from the shelf to the shelf-edge and many are probably the result of lateral environmental migration. Stratigraphic sequences define shallowing-upward packages and are used to construct Fischer plots, substituting sequences for cycles. The modified Fischer plots show that some sequences are missing. Sequences are missed in 3 ways: Sea-level stays below the shelf edge even though sea level is oscillating; Tectonic uplift exposes the shelf until subsidence allows submergence; and During drowning.

  18. New 7-arylpiperazinylalkyl-8-morpholin-4-yl-purine-2,6-dione derivatives with anxiolytic activity - Synthesis, crystal structure and structure-activity study

    NASA Astrophysics Data System (ADS)

    Chłoń-Rzepa, Grażyna; Żmudzki, Paweł; Pawłowski, Maciej; Wesołowska, Anna; Satała, Grzegorz; Bojarski, Andrzej J.; Jabłoński, Mateusz; Kalinowska-Tłuścik, Justyna

    2014-06-01

    On the basis of our earlier studies with serotonin (5-HT) receptor ligands in the group of long-chain arylpiperazines (LCAPs), a new series of 7-arylpiperazinylalkyl-8-morpholin-4-yl-purine-2,6-dione derivatives (5-12) has been designed, synthesised and studied in vitro for their affinity for 5-HT1A, 5-HT2A, 5-HT6 and 5-HT7 receptors. The introduction of o-OCH3 and m-Cl into the phenylpiperazinyl moiety as well as the elongation of the linker between purine-2,6-dione core and arylpiperazine fragment modified the affinity for the tested 5-HT receptors. The structures of compounds 9-11 (hydrochloride salts) were confirmed by an X-ray diffraction method. All molecules adopted a different conformation in the crystal. The strongest observed type of interaction is a charge assisted hydrogen bond N+-H⋯Cl-. Additionally, the π-π interactions between 1,3-dimethyl-3,7-dihydropurine-2,6-dione cores of the neighbouring molecules were also observed. As it is observed in the presented crystal structures, the morpholine ring (a potential donor and acceptor of the hydrogen bonds) seems to be an attractive substituent, that may support binding to the non-specific sites of 5-HT receptors. Another interesting feature is the mutual orientation of rings in the arylpiperazine fragment, with plausible influence on ligand-receptor recognition. For compound 10, with strong 5-HT1A binding affinity, the mutual orientation of rings is determined by the intramolecular weak C-H⋯O hydrogen bond. This observation may contribute to a better understanding of the more selective binding of o-OCH3 arylpiperazine derivatives to the 5-HT1A receptor.

  19. Investigation of free amino acid, total phenolics, antioxidant activity and purine alkaloids to assess the health properties of non-Camellia tea

    PubMed Central

    Bi, Wu; He, Chunnian; Ma, Yunyun; Shen, Jie; Zhang, Linghua Harris; Peng, Yong; Xiao, Peigen

    2015-01-01

    To find novel functional beverages from folk teas, 33 species of frequently used non-Camellia tea (plants other than Camellia) were collected and compared with Camellia tea (green tea, pu-erh tea and black tea) for the first time. Data are reported here on the quantities of 20 free amino acids (FAAs) and three purine alkaloids (measured by UHPLC), total polyphenols (measured by Folin-Ciocalteu assay), and antioxidant activity (DPPH). The total amounts of FAAs in non-Camellia tea (0.62–18.99 mg/g) are generally less than that of Camellia tea (16.55–24.99 mg/g). However, for certain FAAs, the quantities were much higher in some non-Camellia teas, such as γ-aminobutyric acid in teas from Ampelopsis grossedentata, Isodon serra and Hibiscus sabdariffa. Interestingly, theanine was detected in tea from Potentilla fruticosa (1.16±0.81 mg/g). Furthermore, the content of polyphenols in teas from A. grossedentata, Acer tataricum subsp. ginnala are significantly higher than those from Camellia tea; teas from I. serra, Pistacia chinensis and A. tataricum subsp. ginnala have remarkable antioxidant activities similar to the activities from green tea (44.23 μg/mL). Purine alkaloids (caffeine, theobromine and theophylline) were not detected in non-Camellia teas. The investigation suggest some non-Camellia teas may be great functional natural products with potential for prevention of chronic diseases and aging, by providing with abundant polyphenols, antioxidants and specific FAAs. PMID:27006902

  20. Structure of a beta-alanine-linked polyamide bound to a full helical turn of purine tract DNA in the 1:1 motif.

    PubMed

    Urbach, Adam R; Love, John J; Ross, Scott A; Dervan, Peter B

    2002-06-28

    Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methylhydroxypyrrole (Hp) amino acids linked by beta-alanine (beta) bind the minor groove of DNA in 1:1 and 2:1 ligand to DNA stoichiometries. Although the energetics and structure of the 2:1 complex has been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. We present here the 1:1 solution structure of ImPy-beta-Im-beta-ImPy-beta-Dp bound in a single orientation to its match site within the DNA duplex 5'-CCAAAGAGAAGCG-3'.5'-CGCTTCTCTTTGG-3' (match site in bold), as determined by 2D (1)H NMR methods. The representative ensemble of 12 conformers has no distance constraint violations greater than 0.13 A and a pairwise RMSD over the binding site of 0.80 A. Intermolecular NOEs place the polyamide deep inside the minor groove, and oriented N-C with the 3'-5' direction of the purine-rich strand. Analysis of the high-resolution structure reveals the ligand bound 1:1 completely within the minor groove for a full turn of the DNA helix. The DNA is B-form (average rise=3.3 A, twist=38 degrees ) with a narrow minor groove closing down to 3.0-4.5 A in the binding site. The ligand and DNA are aligned in register, with each polyamide NH group forming bifurcated hydrogen bonds of similar length to purine N3 and pyrimidine O2 atoms on the floor of the minor groove. Each imidazole group is hydrogen bonded via its N3 atom to its proximal guanine's exocyclic amino group. The important roles of beta-alanine and imidazole for 1:1 binding are discussed.

  1. Effect of feeding tannin degrading bacterial culture (Streptococcus gallolyticus strain TDGB 406) on nutrient utilization, urinary purine derivatives and growth performance of goats fed on Quercus semicarpifolia leaves.

    PubMed

    Kumar, K; Chaudhary, L C; Agarwal, N; Kamra, D N

    2014-10-01

    To study the effect of supplementation of tannin degrading bacterial culture (Streptococcus gallolyticus strain TDGB 406) on growth performance, nutrient utilization and urinary purine derivatives of goats fed on oak (Quercus semicarpifolia) leaves. For growth study, eighteen billy goats (4 month old, average body weight 9.50 ± 1.50 kg) were distributed into three groups of six animals each. The animals of group 1 served as control while animals of groups 2 (T1) and 3 (T2) were given (@ 5 ml/kg live weight) autoclaved and live culture of isolate TDGB 406 (10(6) cells/ml) respectively. The animals were fed measured quantity of dry oak leaves as the main roughage source and ad libitum maize hay along with fixed quantity of concentrate mixture. The feeding of live culture of isolate TDGB 406 (probiotic) did not affect dry matter intake and digestibility of nutrients except that of dry matter and crude protein, which was higher in T2 group as compared to control. All the animals were in positive nitrogen balance. There was no significant effect of feeding isolate TDGB 406 on urinary purine derivatives (microbial protein production) in goats. The body weight gain and average live weight gain was significantly higher (p = 0.071) in T2 group as compared to control. Feed conversion efficiency was also better in the goats fed on live culture of TDGB 406 (T2). The feeding of tannin degrading bacterial isolate TDGB 406 as probiotic resulted in improved growth performance and feed conversion ratio in goats fed on oak leaves as one of the main roughage source.

  2. Effect of high-dose nano-selenium and selenium-yeast on feed digestibility, rumen fermentation, and purine derivatives in sheep.

    PubMed

    Xun, Wenjuan; Shi, Liguang; Yue, Wenbin; Zhang, Chunxiang; Ren, Youshe; Liu, Qiang

    2012-12-01

    The aim of this study was to evaluate the effect of nano-selenium (NS) and yeast-selenium (YS) supplementation on feed digestibility, rumen fermentation, and urinary purine derivatives in sheep. Six male ruminally cannulated sheep, average 43.32 ± 4.8 kg of BW, were used in a replicated 3 × 3 Latin square experiment. The treatments were control (without NS and YS), NS with 4 g nano-Se (provide 4 mg Se), and YS with 4 g Se-yeast (provide 4 mg Se) per kilogram of diet dry matter (DM), respectively. Experimental periods were 25 days with 15 days of adaptation and 10 days of sampling. Ruminal pH, ammonia N concentration, molar proportion of propionate, and ratio of acetate to propionate were decreased (P < 0.01), and total ruminal VFA concentration was increased with NS and YS supplementation (P < 0.01). In situ ruminal neutral detergent fiber (aNDF) degradation of Leymus chinensis (P < 0.01) and crude protein (CP) of soybean meal (P < 0.01) were significantly improved by Se supplementation. Digestibilities of DM, organic matter, crude protein, ether extract, aNDF, and ADF in the total tract and urinary excretion of purine derivatives were also affected by feeding Se supplementation diets (P < 0.01). Ruminal fermentation was improved by feeding NS, and feed conversion efficiency was also increased compared with YS (P < 0.01). We concluded that nano-Se can be used as a preferentially available selenium source in ruminant nutrition.

  3. Trapping of a cross-link formed by a major purine adduct of a metabolite of the carcinogen N-nitrosomorpholine by inorganic and biological reductants.

    PubMed

    Koissi, Niangoran; Fishbein, James C

    2013-05-20

    3-Hydroperoxy-N-nitrosomorpholine in buffered aqueous media in the presence of calf thymus DNA was treated with a phosphine reductant to generate the transient α-hydroxynitrosamine and subsequent diazonium ion that alkylated the DNA, as previously reported. Subsequent addition of hydride donors, for 30 min, followed by acid hydrolysis of the mixture allowed detection and quantification of 6-(2-{2-[(9H-purin-6-yl)amino]ethoxy}ethoxy)-9H-purin-2-amine, the reduced cross-link formed from deposition, via the diazonium ion, of a 3-oxapentanal fragment on O(6)-Gua, and condensation with N(6)-Ade, presumably in the vicinity. Decreasing the temperature of the reaction mixtures and decreasing the pH modestly increased the yields of the trapped cross-link. Among three borohydride reductants, NaNCBH3 is superior, being ∼4 times more effective on a molar basis, as opposed to a hydride equivalent basis, than NaBH4 or Na(AcO)3BH. For trapping with NaNCBH3, it is deduced that the reaction likely occurs with the iminium ion that is in protonic equilibrium with its conjugate base imine. In an experiment in which the hydroperoxide was decomposed and NaNCBH3 was introduced after various periods of time, the amount of cross-link was observed to increase, nearly linearly, by ∼4-fold over 1 week. These data indicate that there are a minimum of two populations of cross-links, one that forms rapidly, in minutes, and another that grows in with time, over days. Reduced nicotinamide cofactors and ascorbate are observed to effect reduction (over 3 days) of the cross-links, confirming the possibility that otherwise reversible cross-links might be immortalized under biological conditions.

  4. Purification and Characterization of a White Laccase with Pronounced Dye Decolorizing Ability and HIV-1 Reverse Transcriptase Inhibitory Activity from Lepista nuda.

    PubMed

    Zhu, Mengjuan; Zhang, Guoqing; Meng, Li; Wang, Hexiang; Gao, Kexiang; Ng, Tb

    2016-03-26

    A strain LN07 with high laccase yield was identified as basidiomycete fungus Lepista nuda from which a white laccase without type I copper was purified and characterized. The laccase was a monomeric protein with a molecular mass of 56 kDa. Its N-terminal amino acid sequence was AIGPAADLHIVNKDISPDGF. Besides, eight inner peptide sequences were determined and lac4, lac5 and lac6 sequences were in the Cu(2+) combination and conservation zones of laccases. HIV-1 reverse transcriptase was inhibited by the laccase with a half-inhibitory concentration of 0.65 μM. Cu(2+) ions (1.5 mM) enhanced the laccase production and the optimal pH and temperature of the laccase were pH 3.0 and 50 °C, respectively. The Km and Vmax of the laccase using ABTS as substrate were respectively 0.19 mM and 195 μM. Several dyes including laboratory dyes and textile dyes used in this study, such as Methyl red, Coomassie brilliant blue, Reactive brilliant blue and so on, were decolorized in different degrees by the purified laccase. By LC-MS analysis, Methyl red was structurally degraded by the laccase. Moreover, the laccase affected the absorbance at the maximum wavelength of many pesticides. Thus, the white laccase had potential commercial value for textile finishing and wastewater treatment.

  5. Comparison of the global gene expression of choroid plexus and meninges and associated vasculature under control conditions and after pronounced hyperthermia or amphetamine toxicity

    PubMed Central

    2013-01-01

    may not be as pronounced as they are in the MAV, particularly for AMPH. Expression profiles in the MAV and choroid plexus differed to some extent and differences were not restricted to vascular related genes. PMID:23497014

  6. The Risk of Coronary Heart Disease Associated With Glycosylated Hemoglobin ≥ 6.5% is Pronounced in the Haptoglobin 2-2 Genotype

    PubMed Central

    Cahill, Leah E; Jensen, Majken K; Chiuve, Stephanie E; Shalom, Hadar; Pai, Jennifer K; Flint, Alan J; Mukamal, Kenneth J; Rexrode, Kathryn M; Levy, Andrew P; Rimm, Eric B

    2015-01-01

    BACKGROUND Research targeting glycosylated hemoglobin (HbA1c) to < 6.5% to prevent coronary heart disease (CHD) events has conflicting results. We previously observed the haptoglobin (Hp) Hp2-2 genotype is associated with a ~10-fold increased CHD risk among individuals with HbA1c ≥ 6.5%, and thus might be useful in identifying those at high risk of CHD who would benefit from maintaining HbA1c < 6.5%. OBJECTIVES We modeled whether HbA1c ≥ 6.5% in the Hp2-2 genotype is associated with CHD in a prospective case-control study nested within the Health Professionals Follow-Up Study (HPFS). METHODS HbA1c concentration and Hp genotype were determined for 695 incident cases of CHD from 1994–2010 and matched controls. Logistic regression models calculated relative risk (RR) and 95% confidence intervals (CI), for the first and second halves of follow-up, adjusting for confounding variables. A dataset from the Nurses’ Health Study (NHS) served as a replication cohort. RESULTS Prevalence of Hp2-2 genotype in HPFS was 39%. Compared to HbA1c < 6.5%, RR of CHD for HbA1c ≥ 6.5% for the Hp2-2 genotype over full follow-up was 3.07 (1.37–6.86)-- 3.88 (1.31–11.52) during the first half of follow-up and 2.16 (0.61–7.61) in the second half. The corresponding RRs (95% CI) for the Hp1-1 + Hp2-1 genotypes were 2.19 (1.14–4.24) (full follow-up), 1.60 (0.73–3.53) (first half), and 4.72 (1.26–17.65) (second half). CONCLUSIONS In 2 independent cohorts, risk of CHD associated with HbA1c ≥ 6.5% is pronounced in the Hp2-2 genotype, particularly in early cases. The Hp2-2 genotype may identify individuals at greatest CHD risk from hyperglycemia. PMID:26483103

  7. Plastid sequence evolution: a new pattern of nucleotide substitutions in the Cucurbitaceae.

    PubMed

    Decker-Walters, Deena S; Chung, Sang-Min; Staub, Jack E

    2004-05-01

    Nucleotide substitutions (i.e., point mutations) are the primary driving force in generating DNA variation upon which selection can act. Substitutions called transitions, which entail exchanges between purines (A = adenine, G = guanine) or pyrimidines (C = cytosine, T = thymine), typically outnumber transversions (e.g., exchanges between a purine and a pyrimidine) in a DNA strand. With an increasing number of plant studies revealing a transversion rather than transition bias, we chose to perform a detailed substitution analysis for the plant family Cucurbitaceae using data from several short plastid DNA sequences. We generated a phylogenetic tree for 19 taxa of the tribe Benincaseae and related genera and then scored conservative substitution changes (e.g., those not exhibiting homoplasy or reversals) from the unambiguous branches of the tree. Neither the transition nor (A+T)/(G+C) biases found in previous studies were supported by our overall data. More importantly, we found a novel and symmetrical substitution bias in which Gs had been preferentially replaced by A, As by C, Cs by T, and Ts by G, resulting in the G-->A-->C-->T-->G substitution series. Understanding this pattern will lead to new hypotheses concerning plastid evolution, which in turn will affect the choices of substitution models and other tree-building algorithms for phylogenetic analyses based on nucleotide data.

  8. The sequence of sequencers: The history of sequencing DNA.

    PubMed

    Heather, James M; Chain, Benjamin

    2016-01-01

    Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way.

  9. Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken.

    PubMed

    van het Schip, F; Strijker, R; Samallo, J; Gruber, M; Geert, A B

    1986-11-11

    The vitellogenin and apoVLDLII yolk protein genes of chicken are transcribed in the liver upon estrogenization. To get information on putative regulatory elements, we compared more than 2 kb of their 5' flanking DNA sequences. Common sequence motifs were found in regions exhibiting estrogen-induced changes in chromatin structure. Stretches of alternating pyrimidines and purines of about 30-nucleotides long are present at roughly similar positions. A distinct box of sequence homology in the chicken genes also appears to be present at a similar position in front of the vitellogenin genes of Xenopus laevis, but is absent from the estrogen-responsive egg-white protein genes expressed in the oviduct. In front of the vitellogenin (position -595) and the VLDLII gene (position -548), a DNA element of about 300 base-pairs was found, which possesses structural characteristics of a mobile genetic element and bears homology to the transposon-like Vi element of Xenopus laevis.

  10. Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

    PubMed Central

    Prakash, Ashwin; Bechtel, Jason; Fedorov, Alexei

    2011-01-01

    Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova & Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009). Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition. PMID:21610667

  11. Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor.

    PubMed

    Andersen, S O; Rafn, K; Roepstorff, P

    1997-02-01

    Complete amino acid sequences have been determined for six larval-pupal cuticular proteins from Tenebrio molitor. The sequenced proteins are major components in both larval and pupal cuticle, and both basic and slightly acidic proteins are represented. The proteins show pronounced similarities to some of the proteins sequenced from other insect cuticles. Three slightly acidic larval-pupal Tenebrio cuticular proteins contain a 66-residue central, hydrophilic region, resembling regions in cuticular proteins from insect species of four different orders (Coleoptera, Diptera, Lepidoptera and Orthoptera), and three basic proteins from larval-pupal Tenebrio cuticle have a 51-residue hydrophilic region in common with two proteins from cuticle of pharate adult locusts (Locusta migratoria). The Tenebrio larval-pupal cuticular proteins are also similar to locust adult cuticular proteins, by frequent occurrence of the short sequence motif Ala-Ala-Pro-Ala/Val. The pronounced sequence similarities between cuticular proteins from different insect orders indicate that the conserved regions are functionally important.

  12. Effects of perinatal exposure to lead (Pb) on purine receptor expression in the brain and gliosis in rats tolerant to morphine analgesia.

    PubMed

    Baranowska-Bosiacka, Irena; Listos, Joanna; Gutowska, Izabela; Machoy-Mokrzyńska, Anna; Kolasa-Wołosiuk, Agnieszka; Tarnowski, Maciej; Puchałowicz, Kamila; Prokopowicz, Adam; Talarek, Sylwia; Listos, Piotr; Wąsik, Agnieszka; Chlubek, Dariusz

    2016-01-02

    The aim of the present study was to investigate the molecular effects of perinatal exposure to lead (Pb) on protein and mRNA expression of purine receptors: P2X4, P2X7, adenosine receptor A1; and astrocytes (GFAP mRNA expression) and on microglia activation (Iba1 mRNA expression) in several structures of the mesolimbic system (striatum, hippocampus, prefrontal cortex) in rats expressing tolerance to the antinociceptive effect of morphine. Rat mothers were orally treated with 0.1% lead acetate from conception, through gestation, and postnatally, as well as to offspring up to day (PND) 28; subsequently molecular studies were conducted on adult (PND 60) male rats. Morphine tolerance developed more strongly in rats perinatally exposed to Pb. The analysis revealed a significant up-regulation of protein and mRNA P2X4 receptor expression in the striatum and prefrontal cortex but not in the hippocampus; P2X7 protein and mRNA receptor expression in the striatum and hippocampus, but not in the prefrontal cortex; A1 protein receptor expression in all investigated structures and A1 mRNA expression in the striatum and hippocampus; Iba1 mRNA expression in the striatum and hippocampus; and GFAP mRNA expression in the striatum and prefrontal cortex. Immunohistochemical analysis has also revealed significant alterations. Strong expressions of P2X4, P2X7, A1 receptors, astrocytes and microglia activation were observed in the hippocampus in Pb and/or morphine treated rats. The higher expression of purine receptors and glial cell activation are important markers of neuroinflammatory processes. Therefore, we conclude that Pb-induced neuroinflammation may be responsible for the intensification of morphine tolerance in the Pb-treated rats. Additionally, the dysregulation of A1 adenosine receptors, mainly in the hippocampus, may also be involved in the intensification of morphine tolerance in Pb-treated rats. Our study demonstrates the significant participation of environmental factors in

  13. Inosine, an Endogenous Purine Nucleoside, Suppresses Immune Responses and Protects Mice from Experimental Autoimmune Encephalomyelitis: a Role for A2A Adenosine Receptor.

    PubMed

    Junqueira, Stella Célio; Dos Santos Coelho, Igor; Lieberknecht, Vicente; Cunha, Mauricio Peña; Calixto, João B; Rodrigues, Ana Lúcia S; Santos, Adair Roberto Soares; Dutra, Rafael Cypriano

    2016-04-30

    Multiple sclerosis (MS) is a T cell autoimmune, inflammatory, and demyelinating disease of the central nervous system (CNS). Currently available therapies have partially effective actions and numerous side reactions. Inosine, an endogenous purine nucleoside, has immunomodulatory, neuroprotective, and analgesic properties. Herein, we evaluated the effect of inosine on the development and progression of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Inosine (1 or 10 mg/kg, i.p.) was administrated twice a day for 40 days. Immunological and inflammatory responses were evaluated by behavioral, histological, immunohistochemical, ELISA, RT-PCR, and Western blotting analysis. The administration of inosine exerted neuroprotective effects against EAE by diminishing clinical signs, including thermal and mechanical hyperalgesia, as well as weight loss typical of the disease. These beneficial effects of inosine seem to be associated with the blockade of inflammatory cell entry into the CNS, especially lymphocytes, thus delaying the demyelinating process and astrocytes activation. In particular, up-regulation of IL-17 levels in the secondary lymphoid tissues, a result of EAE, was prevented by inosine treatment in EAE mice. Additionally, inosine consistently prevented A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. Altogether, these results allow us to propose that this endogenous purine might be a putative novel and helpful tool for the prevention of autoimmune and neurodegenerative diseases, such as MS. Thus, inosine could have considerable implications for future therapies of MS, and this study may represent the starting point for further investigation into the role of inosine and adenosinergic receptors in neuroinflammation processes. Graphical Abstract Preventive treatment with inosine inhibits the development and progression of EAE in C57Bl/6 mice. Furthermore, neuroinflammation and demyelinating processes

  14. Simultaneous quantification of purine and pyrimidine bases, nucleosides and their degradation products in bovine blood plasma by high performance liquid chromatography tandem mass spectrometry.

    PubMed

    Stentoft, Charlotte; Vestergaard, Mogens; Løvendahl, Peter; Kristensen, Niels Bastian; Moorby, Jon M; Jensen, Søren Krogh

    2014-08-22

    Improved nitrogen utilization in cattle is important in order to secure a sustainable cattle production. As purines and pyrimidines (PP) constitute an appreciable part of rumen nitrogen, an improved understanding of the absorption and intermediary metabolism of PP is essential. The present work describes the development and validation of a sensitive and specific method for simultaneous determination of 20 purines (adenine, guanine, guanosine, inosine, 2'-deoxyguanosine, 2'-deoxyinosine, xanthine, hypoxanthine), pyrimidines (cytosine, thymine, uracil, cytidine, uridine, thymidine, 2'-deoxyuridine), and their degradation products (uric acid, allantoin, β-alanine, β-ureidopropionic acid, β-aminoisobutyric acid) in blood plasma of dairy cows. The high performance liquid chromatography-based technique coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) was combined with individual matrix-matched calibration standards and stable isotopically labelled reference compounds. The quantitative analysis was preceded by a novel pre-treatment procedure consisting of ethanol precipitation, filtration, evaporation and reconstitution. Parameters for separation and detection during the LC-MS/MS analysis were investigated. It was confirmed that using a log-calibration model rather than a linear calibration model resulted in lower CV% and a lack of fit test demonstrated a satisfying linear regression. The method covers concentration ranges for each metabolite according to that in actual samples, e.g. guanine: 0.10-5.0 μmol/L, and allantoin: 120-500 μmol/L. The CV% for the chosen quantification ranges were below 25%. The method has good repeatability (CV%≤25%) and intermediate precision (CV%≤25%) and excellent recoveries (91-107%). All metabolites demonstrated good long-term stability and good stability within-runs (CV%≤10%). Different degrees of absolute matrix effects were observed in plasma, urine and milk. The determination of relative matrix effects

  15. Contrasting Sequence Groups by Emerging Sequences

    NASA Astrophysics Data System (ADS)

    Deng, Kang; Zaïane, Osmar R.

    Group comparison per se is a fundamental task in many scientific endeavours but is also the basis of any classifier. Contrast sets and emerging patterns contrast between groups of categorical data. Comparing groups of sequence data is a relevant task in many applications. We define Emerging Sequences (ESs) as subsequences that are frequent in sequences of one group and less frequent in the sequences of another, and thus distinguishing or contrasting sequences of different classes. There are two challenges to distinguish sequence classes: the extraction of ESs is not trivially efficient and only exact matches of sequences are considered. In our work we address those problems by a suffix tree-based framework and a similar matching mechanism. We propose a classifier based on Emerging Sequences. Evaluating against two learning algorithms based on frequent subsequences and exact matching subsequences, the experiments on two datasets show that our model outperforms the baseline approaches by up to 20% in prediction accuracy.

  16. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  17. Synthesis and biological evaluation of 2-fluoro and 3-trifluoromethyl-phenyl-piperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione as potential antidepressant agents.

    PubMed

    Zagórska, Agnieszka; Bucki, Adam; Kołaczkowski, Marcin; Siwek, Agata; Głuch-Lutwin, Monika; Starowicz, Gabriela; Kazek, Grzegorz; Partyka, Anna; Wesołowska, Anna; Słoczyńska, Karolina; Pękala, Elżbieta; Pawłowski, Maciej

    2016-01-01

    A series of 2-fluoro and 3-trifluoromethylphenylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (4-21) were synthesized and evaluated for their serotonin (5-HT1A/5-HT7) receptor affinity and phosphodiesterase (PDE4B and PDE10A) inhibitor activity. The study enabled the identification of potent 5-HT1A, 5-HT7 and mixed 5-HT1A/5-HT7 receptor ligands with weak inhibitory potencies for PDE4B and PDE10A. The tests have been completed with the determination of lipophilicity and metabolic stability using micellar electrokinetic chromatography (MEKC) system and human liver microsomes (HLM) model. In preliminary pharmacological in vivo studies, selected compound 8-(5-(4-(2-fluorophenyl)piperazin-1-yl)pentyl)-1,3,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (9) behaved as a potential antidepressant in forced swim test (FST) in mice. Moreover, potency of antianxiety effects evoked by 9 (2.5 mg/kg) is greater than that of the reference anxiolytic drug, diazepam. Molecular modeling revealed that fluorinated arylpiperazinylalkyl derivatives of 1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione have major significance for the provision of lead compounds for antidepressant and/or anxiolytic application.

  18. The sequence of sequencers: The history of sequencing DNA

    PubMed Central

    Heather, James M.; Chain, Benjamin

    2016-01-01

    Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way. PMID:26554401

  19. Multimodal sequence learning.

    PubMed

    Kemény, Ferenc; Meier, Beat

    2016-02-01

    While sequence learning research models complex phenomena, previous studies have mostly focused on unimodal sequences. The goal of the current experiment is to put implicit sequence learning into a multimodal context: to test whether it can operate across different modalities. We used the Task Sequence Learning paradigm to test whether sequence learning varies across modalities, and whether participants are able to learn multimodal sequences. Our results show that implicit sequence learning is very similar regardless of the source modality. However, the presence of correlated task and response sequences was required for learning to take place. The experiment provides new evidence for implicit sequence learning of abstract conceptual representations. In general, the results suggest that correlated sequences are necessary for implicit sequence learning to occur. Moreover, they show that elements from different modalities can be automatically integrated into one unitary multimodal sequence.

  20. Discovery of mono- and disubstituted 1H-pyrazolo[3,4]pyrimidines and 9H-purines as catalytic inhibitors of human DNA topoisomerase IIα.

    PubMed

    Pogorelčnik, Barbara; Brvar, Matjaž; Žegura, Bojana; Filipič, Metka; Solmajer, Tom; Perdih, Andrej

    2015-02-01

    Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Based on structural data regarding the binding mode of AMP-PNP (5'-adenylyl-β,γ-imidodiphosphate) to htIIα, we designed a two-stage virtual screening campaign that combines structure-based pharmacophores and molecular docking. In the first stage, we identified several monosubstituted 9H-purine compounds and a novel class of 1H-pyrazolo[3,4]pyrimidines as inhibitors of htIIα. In the second stage, disubstituted analogues with improved cellular activities were discovered. Compounds from both classes were shown to inhibit htIIα-mediated DNA decatenation, and surface plasmon resonance (SPR) experiments confirmed binding of these two compounds on the htIIα ATPase domain. Proposed complexes and interaction patterns between both compounds and htIIα were further analyzed in molecular dynamics simulations. Two compounds identified in the second stage showed promising anticancer activities in hepatocellular carcinoma (HepG2) and breast cancer (MCF-7) cell lines. The discovered compounds are suitable starting points for further hit-to-lead development in anticancer drug discovery.

  1. SURVEY AND SUMMARY: Roles of 5-substituents of tRNA wobble uridines in the recognition of purine-ending codons

    PubMed Central

    Takai, Kazuyuki; Yokoyama, Shigeyuki

    2003-01-01

    Many tRNA molecules that recognize the purine-ending codons but not the pyrimidine-ending codons have a modified uridine at the wobble position, in which a methylene carbon is attached directly to position 5 of the uracil ring. Although several models have been proposed concerning the mechanism by which the 5-substituents regulate codon-reading properties of the tRNAs, none could explain recent results of the experiments utilizing well-characterized modification-deficient strains of Escherichia coli. Here, we first summarize previous studies on the codon-reading properties of tRNA molecules with a U derivative at the wobble position. Then, we propose a hypothetical mechanism of the reading of the G-ending codons by such tRNA molecules that could explain the experimental results. The hypothesis supposes unconventional base pairs between a protonated form of the modified uridines and the G at the third position of the codon stabilized by two direct hydrogen bonds between the bases. The hypothesis also addresses differences between the prokaryotic and eukaryotic decoding systems. PMID:14602896

  2. Structure and dynamics of near-threshold leptons driven by dipolar interactions: an accurate computational study for the DNA purinic bases

    NASA Astrophysics Data System (ADS)

    Carelli, Fabio; Gianturco, Francesco Antonio

    2016-06-01

    The interaction of low-energy scattering electrons/positrons with molecular targets characterized by a "supercritical" permanent dipole moment (≳2.0 D) presents special physical characteristics that affect their spatial distributions, around the nuclear network of the molecular partners, both above and below the energy thresholds. Such special states are described as either dipole scattering states (DSS) above thresholds or as dipole bound states (DBS) below thresholds. The details of their respective behaviour will be presented and discussed in this work in the case of the purinic DNA bases of adenine and guanine. The behavior of the additional electron, in particular, will be discussed in detail by providing new computational results that will be related to the findings from recent experiments on the same DNA bases, confirming the transient electron's behaviour surmised by them. This work is affectionately dedicated to Michael Allan on the occasion of his official retirement. We wish to this dear friend and outstanding scientist many years to come in the happy pursuit of his many scientific interests.Contribution to the Topical Issue "Advances in Positron and Electron Scattering", edited by Paulo Limao-Vieira, Gustavo Garcia, E. Krishnakumar, James Sullivan, Hajime Tanuma and Zoran Petrovic.

  3. Di-μ-but-2-enoato-bis­[diaqua­bis(but-2-enoato)neodymium(III)] 2,6-diamino­purine disolvate

    PubMed Central

    Atria, Ana María; Astete, Alan; Garland, Maria Teresa; Baggio, Ricardo

    2011-01-01

    The title Nd complex [Nd2(C4H5O2)6(H2O)4]·2C5H6N6 is isotypic with two previously reported Dy and Ho isologues. It is composed of [Nd(crot)3(H2O)2]2 dimers [crot(onate) = but-2-enoate = C4H5O2], built up around symmetry centres and completed by 2,6-diamine­purine mol­ecules acting as solvates. The neodymium cations are coordinated by three chelating crotonato units and two water mol­ecules. One of the chelating carboxyl­ates acts also in a bridging mode, sharing one oxygen with both cations, and the final result is a pair of NdO9 tricapped prismatic polyhedra linked to each other through a central (Nd—O)2 loop. A most attractive aspect of the structures resides in the existence of a complex inter­molecular hydrogen-bonding interaction scheme involving two sets of tightly inter­linked, non-inter­secting one-dimensional structures, one of them formed by the [Nd(crot)3(H2O)2]2 dimers running along [100] and the second by the solvate mol­ecules evolving along [010]. PMID:22058842

  4. Coordinate cytokine regulatory sequences

    DOEpatents

    Frazer, Kelly A.; Rubin, Edward M.; Loots, Gabriela G.

    2005-05-10

    The present invention provides CNS sequences that regulate the cytokine gene expression, expression cassettes and vectors comprising or lacking the CNS sequences, host cells and non-human transgenic animals comprising the CNS sequences or lacking the CNS sequences. The present invention also provides methods for identifying compounds that modulate the functions of CNS sequences as well as methods for diagnosing defects in the CNS sequences of patients.

  5. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  6. Determinants of Sequence-Specificity within Human AID and APOBEC3G

    PubMed Central

    Carpenter, Michael A.; Rajagurubandara, Erandi; Wijesinghe, Priyanga; Bhagwat, Ashok S.

    2010-01-01

    Human APOBEC3G (A3G) and activation-induced deaminase (AID) belong to a family of DNA-cytosine deaminases. While A3G targets the last C in a run of C’s, AID targets C in the consensus sequence WRC (W is A or T and R is a purine). Guided by the structures of the A3G carboxyl-terminal catalytic domain (A3G-CTD), we identified two potential regions (region 1 and region 2) that may interact with DNA and swapped the corresponding regions between a variant of A3G-CTD and AID. The resulting hybrids were expressed in Escherichia coli and two different genetic assays and a biochemical assay were used to determine the sequence selectivity of the hybrids in promoting C to T mutations. The results show that while the 10 amino acid region 2 of A3G was its principal sequence-specificity determinant, region 1 of A3G enhanced the target cytosine preference conferred by region 2. In contrast, neither of the two regions in AID individually or in combination were sufficient to confer the DNA sequence preference of this protein upon A3G. Instead, introduction of AID sequences in A3G relaxed the sequence-specificity of the latter protein. Our results show that the sequence-selectivity of APOBEC family of enzymes is determined by at least two separate sequence segments and there may be additional regions of the protein involved in DNA sequence recognition. PMID:20338830

  7. MRO Sequence Checking Tool

    NASA Technical Reports Server (NTRS)

    Fisher, Forest; Gladden, Roy; Khanampornpan, Teerapat

    2008-01-01

    The MRO Sequence Checking Tool program, mro_check, automates significant portions of the MRO (Mars Reconnaissance Orbiter) sequence checking procedure. Though MRO has similar checks to the ODY s (Mars Odyssey) Mega Check tool, the checks needed for MRO are unique to the MRO spacecraft. The MRO sequence checking tool automates the majority of the sequence validation procedure and check lists that are used to validate the sequences generated by MRO MPST (mission planning and sequencing team). The tool performs more than 50 different checks on the sequence. The automation varies from summarizing data about the sequence needed for visual verification of the sequence, to performing automated checks on the sequence and providing a report for each step. To allow for the addition of new checks as needed, this tool is built in a modular fashion.

  8. Different mechanisms of hydroxyl radical production susceptible to purine P2 receptor antagonists between carbon monoxide poisoning and exogenous ATP in rat striatum.

    PubMed

    Hara, S; Kobayashi, M; Kuriiwa, F; Mukai, T; Mizukami, H

    2014-11-01

    Previous studies have suggested that carbon monoxide (CO) poisoning stimulates cAMP production via purine P2Y11-like receptors in the rat striatum, activating cAMP signaling pathways, resulting in hydroxyl radical ((•)OH) production. Extracellular ATP was thought likely to trigger the cascade, but the present study has failed to demonstrate a clear increase in the extracellular ATP due to CO poisoning. The CO-induced (•)OH production was attenuated by the P2Y11 receptor antagonist NF157, in parallel with its abilities to suppress the CO-induced cAMP production. The (•)OH production was more strongly suppressed by a non-selective P2 receptor antagonist, PPADS, which had no effect on cAMP production. More selective antagonists toward the respective P2 receptors susceptible to PPADS, including NF279, had little or no effect on the CO-induced (•)OH production. The intrastriatal administration of exogenous ATP dose-dependently stimulated (•)OH production, which was dose-dependently antagonized by PPADS and NF279 but not by NF157. Exogenous GTP and CTP dose-dependently stimulated (•)OH production, though less potently. The GTP-induced (•)OH production was susceptible to both of NF279 and PPADS, but the CTP-induced (•)OH production was resistant to PPADS. The mechanism of (•)OH production may differ between CO poisoning and exogenous ATP, while multiple P2 receptors could participate in (•)OH production. The CO-induced (•)OH production was susceptible to the inhibition of NADPH oxidase, but not xanthine oxidase. Also, the NADPH oxidase inhibition suppressed (•)OH production induced by forskolin, a stimulator of intracellular cAMP formation. It is likely that (•)OH is produced by NADPH oxidase activation via cAMP signaling pathways during CO poisoning.

  9. 6-Methylpurine derived sugar modified nucleosides: Synthesis and in vivo antitumor activity in D54 tumor expressing M64V-Escherichia coli purine nucleoside phosphorylase.

    PubMed

    Hassan, Abdalla E A; Abou-Elkhair, Reham A I; Parker, William B; Allan, Paula W; Secrist, John A

    2016-01-27

    Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

  10. Oxidation of Purine Nucleotides by Triplet 3,3′,4,4′-Benzophenone Tetracarboxylic Acid in Aqueous Solution: pH-Dependence

    PubMed Central

    2014-01-01

    The photo-oxidation of purine nucleotides adenosine-5′-monophosphate (AMP) and guanosine-5′-monophosphate (GMP) by 3,3′,4,4′-benzophenone tetracarboxylic acid (TCBP) has been investigated in aqueous solutions using nanosecond laser flash photolysis (LFP) and time-resolved chemically induced dynamic nuclear polarization (CIDNP). The pH dependences of quenching rate constants and of geminate polarization are measured within a wide range of pH values. As a result, the chemical reactivity of reacting species in different protonation states is determined. In acidic solution (pH < 4.9), the quenching rate constant is close to the diffusion-controlled limit: kq = 1.3 × 109 M–1 s–1 (GMP), and kq = 1.2 × 109 M–1 s–1 (AMP), whereas in neutral and basic solutions it is significantly lower: kq = 2.6 × 108 M–1 s–1 (GMP, 4.9 < pH < 9.4), kq = 3.5 × 107 M–1 s–1 (GMP, pH > 9.4), kq = 1.0 × 108 M–1 s–1 (AMP, pH > 6.5). Surprisingly, the strong influence of the protonation state of the phosphoric group on the oxidation of adenosine-5′-monophosphate is revealed: the deprotonation of the AMP phosphoric group (6.5) decreases the quenching rate constant from 5.0 × 108 M–1 s–1 (4.9 < pH < 6.5) to 1.0 × 108 M–1 s–1 (pH > 6.5). PMID:24926567

  11. Can an Excess Electron Localise on a Purine Moiety in the Adenine-thymine Watson-Crick Base Pair? A Computational Study

    SciTech Connect

    Mazurkiewicz, Kamil; Haranczyk, Maciej; Gutowski, Maciej S.; Rak, Janusz

    2007-04-17

    The electron affinity and the propensity to electron-induced proton transfer (PT) of hydrogen-bonded complexes between the Watson–Crick adenine–thymine pair (AT) and simple organic acid (HX), attached to adenine in the Hoogsteen-type configuration, were studied at the B3LYP/6-31+G** level. Although the carboxyl group is deprotonated at physiological pH, its neutral form, COOH, resembles the peptide bond or the amide fragment in the side chain of asparagine (Asn) or glutamine (Gln). Thus, these complexes mimic the interaction between the DNA environment (e.g., proteins) and nucleobase pairs incorporated in the biopolymer. Electron attachment is thermodynamically feasible and adiabatic electron affinities range from 0.41 to 1.28 eV, while the vertical detachment energies of the resulting anions span the range of 0.39 –2.88 eV. Low-energy activation barriers separate the anionic minima: aHX(AT) from the more stable single-PT anionic geometry, aHX(AT)-SPT, and aHX(AT)-SPT from the double-PT anionic geometry, aHX(AT)-DPT. Interaction between the adenine of the Watson–Crick AT base pair with an acidic proton donor probably counterbalances the larger EA of isolated thymine, as SOMO is almost evenly delocalized over both types of nucleic bases in the aHX(AT) anions. Moreover, as a result of PT the excess electron localizes entirely on adenine. Thus, in DNA interacting with its physiological environment, damage induced by low-energy electrons could begin, contrary to the current view, with the formation of purine anions, which are not formed in isolated DNA because of the greater stability of anionic pyrimidines.

  12. Hepatic xanthine oxidase activity and purine nucleosides levels as physiological mediators to analyze a subcutaneous treatment with (PhSe)2 in mice infected by Toxoplasma gondii.

    PubMed

    Doleski, Pedro H; Leal, Daniela B R; Machado, Vanessa S; Bottari, Nathieli B; Casali, Emerson A; Moritz, Cesar E J; Camillo, Giovana; Vogel, Fernanda F; Stefani, Lenita M; da Silva, Aleksandro Schafer

    2017-03-01

    The aim of this study was to evaluate the levels of purine nucleosides and xanthine oxidase (XO) activity in the liver of mice chronically infected by Toxoplasma gondii and treated with diphenyl diselenide (PhSe)2. For this experiment, forty Swiss mice were used. Twenty animals were orally infected by approximately 50 bradizoites of a cystogenic ME-49 strain of T. gondii, and the same number of uninfected mice was used as a control group. Ten infected and ten uninfected mice were subcutaneously treated twice (days 1 and 20 post-infection (PI)) with 5 μmol kg(-1) of (PhSe)2. On day 30 PI, liver samples were collected to measure the levels of hypoxanthine (HYPO), xanthine (XAN), uric acid (UA), and XO activity. Infected animals showed increased (P < 0.05) levels of hepatic XAN and UA, as well as XO activity compared to uninfected animals. The use of (PhSe)2 in healthy mice increased the levels of all nucleosides, but decreased XO activity compared to healthy untreated animals. The group of infected and treated animals showed increased XAN and UA levels, and XO activity compared to the healthy control group, however infected and treated mice showed a decrease in the XO activity compared to the infected untreated group. We conclude that chronic infection caused by T. gondii can induce hepatic changes, such as increased UA levels and XO activity, that can increase the pro-oxidative profile. The (PhSe)2 treatment of healthy animals altered the levels of nucleosides, possibly due to low XO activity that decreased nucleoside degradation. Finally, (PhSe)2 treatment decreased XO activity in the infected group and increased nucleoside levels; however it was unable to reduce the UA levels found during the infection.

  13. Effects of dietary supplementation of rumen-protected folic acid on rumen fermentation, degradability and excretion of urinary purine derivatives in growing steers.

    PubMed

    Wang, Cong; Liu, Qiang; Guo, Gang; Huo, WenJie; Ma, Le; Zhang, YanLi; Pei, CaiXia; Zhang, ShuanLin; Wang, Hao

    2016-12-01

    The present experiment was undertaken to determine the effects of dietary addition of rumen-protected folic acid (RPFA) on ruminal fermentation, nutrient degradability, enzyme activity and the relative quantity of ruminal cellulolytic bacteria in growing beef steers. Eight rumen-cannulated Jinnan beef steers averaging 2.5 years of age and 419 ± 1.9 kg body weight were used in a replicated 4 × 4 Latin square design. The four treatments comprised supplementation levels of 0 (Control), 70, 140 and 210 mg RPFA/kg dietary dry matter (DM). On DM basis, the ration consisted of 50% corn silage, 47% concentrate and 3% soybean oil. The DM intake (averaged 8.5 kg/d) was restricted to 95% of ad libitum intake. The intake of DM, crude protein (CP) and net energy for growth was not affected by treatments. In contrast, increasing RPFA supplementation increased average daily gain and the concentration of total volatile fatty acid and reduced ruminal pH linearly. Furthermore, increasing RPFA supplementation enhanced the acetate to propionate ratio and reduced the ruminal ammonia N content linearly. The ruminal effective degradability of neutral detergent fibre from corn silage and CP from concentrate improved linearly and was highest for the highest supplementation levels. The activities of cellobiase, xylanase, pectinase and α-amylase linearly increased, but carboxymethyl-cellulase and protease were not affected by the addition of RPFA. The relative quantities of Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes increased linearly. With increasing RPFA supplementation levels, the excretion of urinary purine derivatives was also increased linearly. The present results indicated that the supplementation of RPFA improved ruminal fermentation, nutrient degradability, activities of microbial enzymes and the relative quantity of the ruminal cellulolytic bacteria in a dose-dependent manner. According to the conditions of this

  14. Genome sequence of the pea aphid Acyrthosiphon pisum.

    PubMed

    2010-02-23

    Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems.

  15. Genome Sequence of the Pea Aphid Acyrthosiphon pisum

    PubMed Central

    2010-01-01

    Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems. PMID:20186266

  16. The Connell Sum Sequence

    NASA Astrophysics Data System (ADS)

    Bullington, Grady D.

    2007-01-01

    The Connell sum sequence refers to the partial sums of the Connell sequence. In this paper, the Connell sequence, Connell sum sequence and generalizations from Iannucci and Mills-Taylor are interpreted as sums of elements of triangles, relating them to polygonal number-stuttered arithmetic progressions. The n-th element of the Connell sum sequence is established as a sharp upper bound for the value of a gamma-labeling of a graph of size n. The limiting behavior and a explicit formula for the Connell (m,r)-sum sequence are also given.

  17. Genome Sequence Databases (Overview): Sequencing and Assembly

    SciTech Connect

    Lapidus, Alla L.

    2009-01-01

    From the date its role in heredity was discovered, DNA has been generating interest among scientists from different fields of knowledge: physicists have studied the three dimensional structure of the DNA molecule, biologists tried to decode the secrets of life hidden within these long molecules, and technologists invent and improve methods of DNA analysis. The analysis of the nucleotide sequence of DNA occupies a special place among the methods developed. Thanks to the variety of sequencing technologies available, the process of decoding the sequence of genomic DNA (or whole genome sequencing) has become robust and inexpensive. Meanwhile the assembly of whole genome sequences remains a challenging task. In addition to the need to assemble millions of DNA fragments of different length (from 35 bp (Solexa) to 800 bp (Sanger)), great interest in analysis of microbial communities (metagenomes) of different complexities raises new problems and pushes some new requirements for sequence assembly tools to the forefront. The genome assembly process can be divided into two steps: draft assembly and assembly improvement (finishing). Despite the fact that automatically performed assembly (or draft assembly) is capable of covering up to 98% of the genome, in most cases, it still contains incorrectly assembled reads. The error rate of the consensus sequence produced at this stage is about 1/2000 bp. A finished genome represents the genome assembly of much higher accuracy (with no gaps or incorrectly assembled areas) and quality ({approx}1 error/10,000 bp), validated through a number of computer and laboratory experiments.

  18. Automated DNA Sequencing System

    SciTech Connect

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  19. Deficiency of the purine metabolic gene HPRT dysregulates microRNA-17 family cluster and guanine-based cellular functions: a role for EPAC in Lesch-Nyhan syndrome

    PubMed Central

    Guibinga, Ghiabe-Henri; Murray, Fiona; Barron, Nikki; Pandori, William; Hrustanovic, Gorjan

    2013-01-01

    Lesch-Nyhan syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). A series of motor, cognitive and neurobehavioral anomalies characterize this disease phenotype, which is still poorly understood. The clinical manifestations of this syndrome are believed to be the consequences of deficiencies in neurodevelopmental pathways that lead to disordered brain function. We have used microRNA array and gene ontology analysis to evaluate the gene expression of differentiating HPRT-deficient human neuron-like cell lines. We set out to identify dysregulated genes implicated in purine-based cellular functions. Our approach was based on the premise that HPRT deficiency affects preeminently the expression and the function of purine-based molecular complexes, such as guanine nucleotide exchange factors (GEFs) and small GTPases. We found that several microRNAs from the miR-17 family cluster and genes encoding GEF are dysregulated in HPRT deficiency. Most notably, our data show that the expression of the exchange protein activated by cAMP (EPAC) is blunted in HPRT-deficient human neuron-like cell lines and fibroblast cells from LNS patients, and is altered in the cortex, striatum and midbrain of HPRT knockout mouse. We also show a marked impairment in the activation of small GTPase RAP1 in the HPRT-deficient cells, as well as differences in cytoskeleton dynamics that lead to increased motility for HPRT-deficient neuron-like cell lines relative to control. We propose that the alterations in EPAC/RAP1 signaling and cell migration in HPRT deficiency are crucial for neuro-developmental events that may contribute to the neurological dysfunctions in LNS. PMID:23804752

  20. Cellulases and coding sequences

    DOEpatents

    Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

    2001-02-20

    The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

  1. Cellulases and coding sequences

    DOEpatents

    Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

    2001-01-01

    The present invention provides three fungal cellulases, their coding sequences, recombinant DNA molecules comprising the cellulase coding sequences, recombinant host cells and methods for producing same. The present cellulases are from Orpinomyces PC-2.

  2. Sequence information signal processor

    DOEpatents

    Peterson, John C.; Chow, Edward T.; Waterman, Michael S.; Hunkapillar, Timothy J.

    1999-01-01

    An electronic circuit is used to compare two sequences, such as genetic sequences, to determine which alignment of the sequences produces the greatest similarity. The circuit includes a linear array of series-connected processors, each of which stores a single element from one of the sequences and compares that element with each successive element in the other sequence. For each comparison, the processor generates a scoring parameter that indicates which segment ending at those two elements produces the greatest degree of similarity between the sequences. The processor uses the scoring parameter to generate a similar scoring parameter for a comparison between the stored element and the next successive element from the other sequence. The processor also delivers the scoring parameter to the next processor in the array for use in generating a similar scoring parameter for another pair of elements. The electronic circuit determines which processor and alignment of the sequences produce the scoring parameter with the highest value.

  3. Nonparametric Combinatorial Sequence Models

    NASA Astrophysics Data System (ADS)

    Wauthier, Fabian L.; Jordan, Michael I.; Jojic, Nebojsa

    This work considers biological sequences that exhibit combinatorial structures in their composition: groups of positions of the aligned sequences are "linked" and covary as one unit across sequences. If multiple such groups exist, complex interactions can emerge between them. Sequences of this kind arise frequently in biology but methodologies for analyzing them are still being developed. This paper presents a nonparametric prior on sequences which allows combinatorial structures to emerge and which induces a posterior distribution over factorized sequence representations. We carry out experiments on three sequence datasets which indicate that combinatorial structures are indeed present and that combinatorial sequence models can more succinctly describe them than simpler mixture models. We conclude with an application to MHC binding prediction which highlights the utility of the posterior distribution induced by the prior. By integrating out the posterior our method compares favorably to leading binding predictors.

  4. Roles of repetitive sequences

    SciTech Connect

    Bell, G.I.

    1991-12-31

    The DNA of higher eukaryotes contains many repetitive sequences. The study of repetitive sequences is important, not only because many have important biological function, but also because they provide information on genome organization, evolution and dynamics. In this paper, I will first discuss some generic effects that repetitive sequences will have upon genome dynamics and evolution. In particular, it will be shown that repetitive sequences foster recombination among, and turnover of, the elements of a genome. I will then consider some examples of repetitive sequences, notably minisatellite sequences and telomere sequences as examples of tandem repeats, without and with respectively known function, and Alu sequences as an example of interspersed repeats. Some other examples will also be considered in less detail.

  5. DNA sequencing conference, 2

    SciTech Connect

    Cook-Deegan, R.M.; Venter, J.C.; Gilbert, W.; Mulligan, J.; Mansfield, B.K.

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  6. Schur monotone decreasing sequences

    NASA Astrophysics Data System (ADS)

    Ganikhodjaev, Rasul; Saburov, Mansoor; Saburov, Khikmat

    2013-09-01

    In this paper, we introduce Schur monotone decreasing sequences in an n-dimensional space by considering a majorization pre-order. By means of down arrow mappings, we study omega limiting points of bounded Schur monotone decreasing sequences. We provide convergence criteria for such kinds of sequences. We prove that a Cesaro mean (or an arithmetic mean) of any bounded Schur monotone decreasing sequences converges to a unique limiting point.

  7. A Random Word Generator for Pronounceable Passwords

    DTIC Science & Technology

    1975-11-01

    drietoi drie-toi cywroft cy-wroft dieyedi die-ye-di drite drite dabowt da-bowt diffos dif-fos drobveje drob- veje dacoafa da-coa-fa digba dig-ba...vacquic vadfuo vadwyhey vadyjo vafjitdu ud-we-ry-ho u-dyg-ne-di uf-duj-mod uf-jea-ki uf-kod-bot uf-wa-ta u-gaz-sca ug-nea ug- veje u-je

  8. Greenland's pronounced glacier retreat not irreversible

    NASA Astrophysics Data System (ADS)

    Schultz, Colin

    2012-02-01

    In recent decades, the combined forces of climate warming and short-term variability have forced the massive glaciers that blanket Greenland into retreat, with some scientists worrying that deglaciation could become irreversible. The short history of detailed glacier observations, however, makes pinning the ice loss to either short-term dynamics or long-term change difficult. Research by Young et al. detailing the effects of two bouts of sudden and temporary cooling during an otherwise warm phase in Greenland's climate history could help answer that question by showing just how heavy a hand short-term variability can have in dictating glacier dynamics. Along the western edge of Greenland the massive Jakobshavn Isbræ glacier reaches out to the coast, its outflow dropping icebergs into Baffin Bay during the summer months. Flanking the glacier's tongue are the Tasiussaq and Marrait moraines—piles of rock marking the glacier's former extent. Researchers suspected the moraines were tied to two periods of abrupt cooling that hit Greenland 9300 and 8200 years ago, and that association was reinforced by the authors' radiocarbon and beryllium isotope analyses of the area surrounding the moraines. Beryllium-10 forms when cosmic radiation travels through the atmosphere and strikes the Earth's surface, with surface rock concentrations indicating how long it has been ice-free.

  9. [Wegener's granulomatosis with pronounced kidney involvement].

    PubMed

    Heinicke, H J; Hensel, J

    1981-06-01

    It is reported on the course of histologically ascertained Wegeners' granulomatosis in 4 patients. After initial affection of the upper respiratory tract in form of necrotizing and granulomatous inflammations in all cases a generalisation of the disease with above all early and severe participation of the kidneys developed. Maximum acceleration of sedimentation, C-reactive protein, anaemia, leucocytosis, eosinophilia, thrombocytosis, enlargement of the number of alpha 2-globulins, increase of creatinine, proteinuria, erythrocyturia and leucocyturia are the most frequent pathological laboratory findings, whereas LE-cells, ANF, rheumatoid factor and decrease of the complement never could be proved. By reason of a pathogenic immunoreaction a combined glucocorticoid therapy and immunosuppressive therapy with prednisolone and cyclophosphamide may favourably influence the course of the disease which is otherwise prognostically infaust.

  10. Career Academy Course Sequences.

    ERIC Educational Resources Information Center

    Markham, Thom; Lenz, Robert

    This career academy course sequence guide is designed to give teachers a quick overview of the course sequences of well-known career academy and career pathway programs from across the country. The guide presents a variety of sample course sequences for the following academy themes: (1) arts and communication; (2) business and finance; (3)…

  11. Low autocorrelation binary sequences

    NASA Astrophysics Data System (ADS)

    Packebusch, Tom; Mertens, Stephan

    2016-04-01

    Binary sequences with minimal autocorrelations have applications in communication engineering, mathematics and computer science. In statistical physics they appear as groundstates of the Bernasconi model. Finding these sequences is a notoriously hard problem, that so far can be solved only by exhaustive search. We review recent algorithms and present a new algorithm that finds optimal sequences of length N in time O(N {1.73}N). We computed all optimal sequences for N≤slant 66 and all optimal skewsymmetric sequences for N≤slant 119.

  12. HIV Sequence Databases

    PubMed Central

    Kuiken, Carla; Korber, Bette; Shafer, Robert W.

    2008-01-01

    Two important databases are often used in HIV genetic research, the HIV Sequence Database in Los Alamos, which collects all sequences and focuses on annotation and data analysis, and the HIV RT/Protease Sequence Database in Stanford, which collects sequences associated with the development of viral resistance against anti-retroviral drugs and focuses on analysis of those sequences. The types of data and services these two databases offer, the tools they provide, and the way they are set up and operated are described in detail. PMID:12875108

  13. Deep HST Imaging in 47 Tuc and NGC 6397: Main Sequence Turnoff Ages

    NASA Astrophysics Data System (ADS)

    Dotter, Aaron L.; Anderson, J.; Fahlman, G.; Hansen, B.; Hurley, J.; Kalirai, J.; King, I.; Reitzel, D.; Rich, R. M.; Richer, H.; Shara, M.; Stetson, P.; Woodley, K.; Zurek, D.

    2011-01-01

    The ages of Galactic globular clusters provide insight into the formation history of the Milky Way. Utilizing HST photometry of unprecendented depth and wavelength coverage, we determine the main sequence turnoff ages of the nearby globular clusters NGC 6397 and 47 Tuc. The ages are determined by comparing stellar evolution models to the main sequences with a chi-squared minimization technique. Our analysis of 47 Tuc leverages the pronounced 'kink' or 'knee' feature that appears in the lower main sequence in the near-IR. We present our age estimates as probability distributions and construct confidence intervals over input parameters such as metallicity, distance, and reddening.

  14. Self-catalyzed site-specific depurination of guanine residues within gene sequences.

    PubMed

    Amosova, Olga; Coulter, Richard; Fresco, Jacques R

    2006-03-21

    A self-catalyzed, site-specific guanine-depurination activity has been found to occur in short gene sequences with a potential to form a stem-loop structure. The critical features of that catalytic intermediate are a 5'-G-T-G-G-3' loop and an adjacent 5'-T.A-3' base pair of a short duplex stem stable enough to fix the loop structure required for depurination of its 5'-G residue. That residue is uniquely depurinated with a rate some 5 orders of magnitude faster than that of random "spontaneous" depurination. In contrast, all other purine residues in the sequence depurinate at the spontaneous background rate. The reaction requires no divalent cations or other cofactors and occurs under essentially physiological conditions. Such stem-loops can form in duplex DNA under superhelical stress, and their critical sequence features have been found at numerous sites in the human genome. Self-catalyzed stem-loop-mediated depurination leading to flexible apurinic sites may therefore serve some important biological role, e.g., in nucleosome positioning, genetic recombination, or chromosome superfolding.

  15. Cathodic Voltammetric Behavior of Pillar[5]quinone in Nonaqueous Media. Symmetry Effects on the Electron Uptake Sequence.

    PubMed

    Cheng, Beijun; Kaifer, Angel E

    2015-08-12

    The cathodic voltammetric behavior of pillar[5]quinone was investigated in dichloromethane solution. Our data show that the symmetry of the macrocycle has a pronounced effect on the electron uptake sequence. The uptake of the first five electrons follows a 2-1-2 pattern, and only a total of eight electrons could be injected into the macrocycle under our experimental conditions.

  16. Striatal Neurodevelopment Is Dysregulated in Purine Metabolism Deficiency and Impacts DARPP-32, BDNF/TrkB Expression and Signaling: New Insights on the Molecular and Cellular Basis of Lesch-Nyhan Syndrome

    PubMed Central

    Guibinga, Ghiabe-Henri; Barron, Nikki; Pandori, William

    2014-01-01

    Lesch-Nyhan Syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). This syndrome is characterized by an array of severe neurological impairments that in part originate from striatal dysfunctions. However, the molecular and cellular mechanisms underlying these dysfunctions remain largely unidentified. In this report, we demonstrate that HPRT-deficiency causes dysregulated expression of key genes essential for striatal patterning, most notably the striatally-enriched transcription factor B-cell leukemia 11b (Bcl11b). The data also reveal that the down-regulated expression of Bcl11b in HPRT-deficient immortalized mouse striatal (STHdh) neural stem cells is accompanied by aberrant expression of some of its transcriptional partners and other striatally-enriched genes, including the gene encoding dopamine- and cAMP-regulated phosphoprotein 32, (DARPP-32). Furthermore, we demonstrate that components of the BDNF/TrkB signaling, a known activator of DARPP-32 striatal expression and effector of Bcl11b transcriptional activation are markedly increased in HPRT-deficient cells and in the striatum of HPRT knockout mouse. Consequently, the HPRT-deficient cells display superior protection against reactive oxygen species (ROS)-mediated cell death upon exposure to hydrogen peroxide. These findings suggest that the purine metabolic defect caused by HPRT-deficiency, while it may provide neuroprotection to striatal neurons, affects key genes and signaling pathways that may underlie the neuropathogenesis of LNS. PMID:24804781

  17. Tautomeric purine forms of 2-amino-6-chloropurine (N9H10 and N7H10): Structures, vibrational assignments, NBO analysis, hyperpolarizability, HOMO-LUMO study using B3 based density functional calculations

    NASA Astrophysics Data System (ADS)

    Balachandran, V.; Parimala, K.

    2012-10-01

    Two purine tautomers of 2-amino-6-chloropurine (ACP), in labeled as N9H10 and N7H10, were investigated by vibrational spectroscopy and quantum chemical method. The FT-IR and FT-Raman spectra of ACP have been recorded in the regions 4000-400 cm-1 and 3500-100 cm-1, respectively. The measured spectra were interpreted by aid of a normal coordinate analysis following DFT full geometry optimization and vibrational frequency calculations at B3LYP/6-311++G(d,p) level. First-order hyperpolarizability, HOMO and LUMO energies were calculated at same level of theory. The calculated molecular geometry has been compared with the X-ray data. The observed and calculated frequencies were found in good agreement. The obtained NBO data and second-order perturbation energy values to elucidate the Lewis and non-Lewis types of bonding structures in the purine tautomer N9H10, have indicated the presence of an intramolecular hyperconjucative interaction between lone pair N and N-C bond orbital.

  18. Tautomeric purine forms of 2-amino-6-chloropurine (N9H10 and N7H10): structures, vibrational assignments, NBO analysis, hyperpolarizability, HOMO-LUMO study using B3 based density functional calculations.

    PubMed

    Balachandran, V; Parimala, K

    2012-10-01

    Two purine tautomers of 2-amino-6-chloropurine (ACP), in labeled as N(9)H(10) and N(7)H(10), were investigated by vibrational spectroscopy and quantum chemical method. The FT-IR and FT-Raman spectra of ACP have been recorded in the regions 4000-400 cm(-1) and 3500-100 cm(-1), respectively. The measured spectra were interpreted by aid of a normal coordinate analysis following DFT full geometry optimization and vibrational frequency calculations at B3LYP/6-311++G(d,p) level. First-order hyperpolarizability, HOMO and LUMO energies were calculated at same level of theory. The calculated molecular geometry has been compared with the X-ray data. The observed and calculated frequencies were found in good agreement. The obtained NBO data and second-order perturbation energy values to elucidate the Lewis and non-Lewis types of bonding structures in the purine tautomer N(9)H(10), have indicated the presence of an intramolecular hyperconjucative interaction between lone pair N and N-C bond orbital.

  19. Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ion-pair RP-HPLC/UV for the quantification of adenine and guanine pools.

    PubMed

    Graven, Patricia; Tambalo, Margherita; Scapozza, Leonardo; Perozzo, Remo

    2014-06-01

    Human African Trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. Although trypanosomes are well-studied model organisms, only little is known about their adenine and guanine nucleotide pools. Besides being building blocks of RNA and DNA, these nucleotides are also important modulators of diverse biochemical cellular processes. Adenine nucleotides also play an important role in the regulation of metabolic energy. The energetic state of cells is evaluated by the energy charge which gives information about how much energy is available in form of high energy phosphate bonds of adenine nucleotides. A sensitive and reproducible ion-pair RP-HPLC/UV method was developed and optimized, allowing the quantification of guanine and adenine nucleosides/nucleotides in T. brucei. With this method, the purine levels and their respective ratios were investigated in trypanosomes during logarithmic, stationary and senescent growth phases. Results of this study showed that all adenine and guanine purines under investigation were in the low mM range. The energy charge was found to decrease from logarithmic to static and to senescent phase whereas AMP/ATP, ADP/ATP and GDP/GTP ratios increased in the same order. In addition, the AMP/ATP ratio varied as the square of the ADP/ATP ratio, indicating AMP to be the key energy sensor molecule in trypanosomes.

  20. Identification of agents that reduce renal hypoxia-reoxygenation injury using cell-based screening: purine nucleosides are alternative energy sources in LLC-PK1 cells during hypoxia.

    PubMed

    Szoleczky, Petra; Módis, Katalin; Nagy, Nóra; Dóri Tóth, Zoltán; DeWitt, Douglas; Szabó, Csaba; Gero, Domokos

    2012-01-01

    Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure.

  1. Rare 2-Substituted Purine Nucleosides

    DTIC Science & Technology

    1989-05-01

    acetonyinosine, AVS-002159) has been found to have very high activity (TI > 1000) against the Sandfly Fever Virus (Phlebovirus). Another compound (2...propenyl group is occurring at the slow step of the reaction, i.e. the transmetalation stage. Temperature appears to be an important factor in...exclusive formation of the 1-propenyl compound 79 is realized when the temperature is raised to 110 °C. Deprotection of 79 with trimethyIsilyl iodide in

  2. Photoaffinity approaches to determining the sequence selectivities of DNA-small molecule interactions: actinomycin D and ethidium.

    PubMed Central

    Marsch, G A; Graves, D E; Rill, R L

    1995-01-01

    The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperidine. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in terms of 'effective site occupations', which express the ability of a ligand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanking C. High reactivities were also observed for bases in the first (5') GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules. PMID:7739904

  3. Computer assisted multiplex sequencing

    SciTech Connect

    Church, G.M.

    1992-08-01

    The objectives of this project are automation and optimization of multiplex sequencing. This year we have integrated direct transfer electrophoresis, automated multiplex hybridizations and automated film reading and applied this toward sequencing of three contiguous E. coli cosmids. Primers for the directed dideoxy sequence walking and sequence confirmation steps were synthesized with a 15 base tag complimentary to an alkaline phosphatase conjugate. A higher throughput synthesis device is well along in testing as are new automated hybridization devices. We have developed software for automatically annotating ORFs and databases of precise termini of proteis and RNA.

  4. Pronounced matrix effect in YbMo{sub 2}Al{sub 4}-type Ca(Au{sub x}Zn{sub 2−x})Au{sub 4} (x=0.09–0.89)

    SciTech Connect

    Mishra, Trinath; Lin, Qisheng; Corbett, John D.

    2014-10-15

    Electron-poor polar intermetallics Ca(Au{sub x}Zn{sub 2−x})Au{sub 4} have been synthesized through fusion of stoichiometric metals in sealed tantalum tubes at 800 °C and annealing at 400 °C for one week. Single crystal X-ray diffraction analyses reveal that this phase belongs to the YbMo{sub 2}Al{sub 4}-type structure (I4/mmm, Pearson symbol tI14), a≈6.943–7.017 Å, c≈5.278–5.286 Å, Z=2, with homogeneous composition range of x=0.09(1)–0.89(1). The structure exhibits a three-dimensional framework of (Au{sub 8}){sub 1/2} featuring square and octagonal channels extending in c, in which Ca and the infinite linear chains of [(Au,Zn){sub 2}]{sub 1/2} are located, respectively. Mulliken population analyses demonstrate that Zn prefers to form the linear chains in the whole homogeneous composition range, consistent with experimental observations. Crystal orbital Hamilton population (COHP) analyses reveal that the channel-to-chain Au–Zn contact has strong bonding interactions regardless of its large interatomic distance (∼2.85 Å), a signature of pronounced matrix effect. The last mentioned effect in YbMo{sub 2}Al{sub 4}-type structures is expected in case the linear chains are defined by small size atoms. - Graphical abstract: Pronounced matrix effect incurred by Au–Au bonding within the gold substructure in Ca(Au{sub x}Zn{sub 2−x})Au{sub 4} results in an elongation of the channel-to-chain Au–Zn interatomic distance without weakening bonding interactions. - Highlights: • The complete solid solution of Ca(Au{sub x}Zn{sub 2−x})Au{sub 4} was accomplished. • Site preference was explained by “coloring” analyses. • Abnormally large Au–Zn distance but with strong bonding was observed. • Pronounced matrix effect incurred by Au–Au bonding in gold substructure was found.

  5. Sequences, Series, and Mathematica.

    ERIC Educational Resources Information Center

    Mathews, John H.

    1992-01-01

    Describes how the computer algebra system Mathematica can be used to enhance the teaching of the topics of sequences and series. Examines its capabilities to find exact, approximate, and graphically generated approximate solutions to problems from these topics and to understand proofs about sequences. (MDH)

  6. Can sequence determine function?

    PubMed Central

    Gerlt, John A; Babbitt, Patricia C

    2000-01-01

    The functional annotation of proteins identified in genome sequencing projects is based on similarities to homologs in the databases. As a result of the possible strategies for divergent evolution, homologous enzymes frequently do not catalyze the same reaction, and we conclude that assignment of function from sequence information alone should be viewed with some skepticism. PMID:11178260

  7. Lichenase and coding sequences

    DOEpatents

    Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

    2000-08-15

    The present invention provides a fungal lichenase, i.e., an endo-1,3-1,4-.beta.-D-glucanohydrolase, its coding sequence, recombinant DNA molecules comprising the lichenase coding sequences, recombinant host cells and methods for producing same. The present lichenase is from Orpinomyces PC-2.

  8. Biotools: Patenting DNA sequences

    SciTech Connect

    Yablonsky, M.D.; Hone, W.J.

    1995-07-01

    The decision, known as In re Deuel{sup 2}, rejects the PTO`s interpretation of a previous decision of the Federal Circuit and makes it more possible that a {open_quotes}nucleic acid of a particular sequence{close_quotes} - commonly known as a gene sequence - may be patentable. 15 refs.

  9. Sequences for Student Investigation

    ERIC Educational Resources Information Center

    Barton, Jeffrey; Feil, David; Lartigue, David; Mullins, Bernadette

    2004-01-01

    We describe two classes of sequences that give rise to accessible problems for undergraduate research. These problems may be understood with virtually no prerequisites and are well suited for computer-aided investigation. The first sequence is a variation of one introduced by Stephen Wolfram in connection with his study of cellular automata. The…

  10. Cosmetology: Scope and Sequence.

    ERIC Educational Resources Information Center

    Nashville - Davidson County Metropolitan Public Schools, TN.

    This scope and sequence guide, developed for a cosmetology vocational education program, represents an initial step in the development of a systemwide articulated curriculum sequence for all vocational programs within the Metropolitan Nashville Public School System. It was developed as a result of needs expressed by teachers, parents, and the…

  11. Agriculture: Scope and Sequence.

    ERIC Educational Resources Information Center

    Nashville - Davidson County Metropolitan Public Schools, TN.

    This guide, which was written as an initial step in the development of a systemwide articulated curriculum sequence for all vocational programs within the Metropolitan Nashville Public School System, outlines the suggested scope and sequence of a 3-year program in agriculture. The guide consists of a course description; general course objectives;…

  12. Pronounced site preference and mixed dinuclear dichalcogenide anions (SSe)2- in ternary lanthanum sulfide selenides - The solid solution LaS1.9-LaSe1.9

    NASA Astrophysics Data System (ADS)

    Bartsch, Christian; Doert, Thomas

    2012-04-01

    Ternary lanthanum sulfide selenides LaS1.9-xSex (0 < x < 1.9) were obtained by reaction of the elements in a potassium bromide flux. The ternary compounds adopt the CeSe1.9 structure type (tetragonal, space group P42/n, No. 86) and form a complete solid solution series without indications of ordered superstructures. The lattice parameters of the samples follow Vegard's rule and are found in the range of 915 ≤ a ≤ 943 pm and 1644 ≤ c ≤ 1703 pm. A pronounced ordering of the distribution of chalcogen atoms is observed which can be attributed to charge density and size effects. The existence of three different types of dichalcogenide anions, namely S22-, Se22- and mixed (SSe)2-, is evidenced by Raman spectroscopy.

  13. Variation in the composition of corals, fishes, sponges, echinoderms, ascidians, molluscs, foraminifera and macroalgae across a pronounced in-to-offshore environmental gradient in the Jakarta Bay-Thousand Islands coral reef complex.

    PubMed

    Cleary, D F R; Polónia, A R M; Renema, W; Hoeksema, B W; Rachello-Dolmen, P G; Moolenbeek, R G; Budiyanto, A; Yahmantoro; Tuti, Y; Giyanto; Draisma, S G A; Prud'homme van Reine, W F; Hariyanto, R; Gittenberger, A; Rikoh, M S; de Voogd, N J

    2016-09-30

    Substrate cover, water quality parameters and assemblages of corals, fishes, sponges, echinoderms, ascidians, molluscs, benthic foraminifera and macroalgae were sampled across a pronounced environmental gradient in the Jakarta Bay-Thousand Islands reef complex. Inshore sites mainly consisted of sand, rubble and turf algae with elevated temperature, dissolved oxygen, pH and chlorophyll concentrations and depauperate assemblages of all taxa. Live coral cover was very low inshore and mainly consisted of sparse massive coral heads and a few encrusting species. Faunal assemblages were more speciose and compositionally distinct mid- and offshore compared to inshore. There were, however, small-scale differences among taxa. Certain midshore sites, for example, housed assemblages resembling those typical of the inshore environment but this differed depending on the taxon. Substrate, water quality and spatial variables together explained from 31% (molluscs) to 72% (foraminifera) of the variation in composition. In general, satellite-derived parameters outperformed locally measured parameters.

  14. Effect of dietary Quebracho tannin extract on feed intake, digestibility, excretion of urinary purine derivatives and milk production in dairy cows.

    PubMed

    Henke, Anika; Dickhoefer, Uta; Westreicher-Kristen, Edwin; Knappstein, Karin; Molkentin, Joachim; Hasler, Mario; Susenbeth, Andreas

    2017-02-01

    The aim of this study was to evaluate the effects of dietary Quebracho tannin extract (QTE) on feed intake, apparent total tract digestibility (ATTD), excretion of urinary purine derivatives (PD) and milk composition and yield in dairy cows. Fifty Holstein cows were divided into two groups. To reach a similar performance of both groups, cows were divided according to their milk yield, body weight, days in milk and number of lactations at the start of the experiment averaging 33.2 ± 8.2 kg/d, 637 ± 58 kg, 114 ± 73 d and 2.3 ± 1.6 lactations, respectively. The cows were fed a basal diet as total mixed ration containing on dry matter (DM) basis 34% grass silage, 32% maize silage and 34% concentrate feeds. Three dietary treatments were tested, the control (CON, basal diet without QTE), QTE15 (basal diet with QTE at 15 g/kg DM) and QTE30 (basal diet with QTE at 30 g/kg DM). Two treatments were arranged along six periods each 21 d (13 d adaptation phase and 8 d sampling phase). The ATTD of DM and organic matter were reduced only in Diet QTE30, whereas both QTE treatments reduced ATTD of fibre and nitrogen (N), indicating that QTE impaired rumen fermentation. Nevertheless, feed intake was unaffected by QTE. In Diet CON, urinary N excretion accounted for 29.8% of N intake and decreased in treatments QTE15 and QTE30 to 27.5% and 17.9%, respectively. Daily faecal N excretion increased in treatments CON, QTE15 and QTE30 from 211 to 237 and 273 g/d, respectively, which amounted to 39.0%, 42.4% and 51.7% of the N intake, respectively. Hence, QTE shifted N excretion from urine to faeces, whereas the proportion of ingested N appearing in milk was not affected by QTE (average 30.7% of N intake). Daily PD excretion as indicator for microbial crude protein (CP) flow at the duodenum decreased in treatment QTE30 compared with Diet CON from 413 to 280 mmol/d. The ratios of total PD to creatinine suggest that urinary PD excretion was already lower when feeding Diet

  15. Sequencing the maize genome.

    PubMed

    Martienssen, Robert A; Rabinowicz, Pablo D; O'Shaughnessy, Andrew; McCombie, W Richard

    2004-04-01

    Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.

  16. Sequence History Update Tool

    NASA Technical Reports Server (NTRS)

    Khanampompan, Teerapat; Gladden, Roy; Fisher, Forest; DelGuercio, Chris

    2008-01-01

    The Sequence History Update Tool performs Web-based sequence statistics archiving for Mars Reconnaissance Orbiter (MRO). Using a single UNIX command, the software takes advantage of sequencing conventions to automatically extract the needed statistics from multiple files. This information is then used to populate a PHP database, which is then seamlessly formatted into a dynamic Web page. This tool replaces a previous tedious and error-prone process of manually editing HTML code to construct a Web-based table. Because the tool manages all of the statistics gathering and file delivery to and from multiple data sources spread across multiple servers, there is also a considerable time and effort savings. With the use of The Sequence History Update Tool what previously took minutes is now done in less than 30 seconds, and now provides a more accurate archival record of the sequence commanding for MRO.

  17. Recognition of 5'-YpG-3' sequences by coupled stacking/hydrogen bonding interactions with amino acid residues.

    PubMed

    Lamoureux, Jason S; Maynes, Jason T; Glover, J N Mark

    2004-01-09

    The combined biochemical and structural study of hundreds of protein-DNA complexes has indicated that sequence-specific interactions are mediated by two mechanisms termed direct and indirect readout. Direct readout involves direct interactions between the protein and base-specific atoms exposed in the major and minor grooves of DNA. For indirect readout, the protein recognizes DNA by sensing conformational variations in the structure dependent on nucleotide sequence, typically through interactions with the phosphodiester backbone. Based on our recent structure of Ndt80 bound to DNA in conjunction with a search of the existing PDB database, we propose a new method of sequence-specific recognition that utilizes both direct and indirect readout. In this mode, a single amino acid side-chain recognizes two consecutive base-pairs. The 3'-base is recognized by canonical direct readout, while the 5'-base is recognized through a variation of indirect readout, whereby the conformational flexibility of the particular dinucleotide step, namely a 5'-pyrimidine-purine-3' step, facilitates its recognition by the amino acid via cation-pi interactions. In most cases, this mode of DNA recognition helps explain the sequence specificity of the protein for its target DNA.

  18. Next-Generation Sequencing.

    PubMed

    Le Gallo, Matthieu; Lozy, Fred; Bell, Daphne W

    2017-01-01

    Endometrial cancers are the most frequently diagnosed gynecological malignancy and were expected to be the seventh leading cause of cancer death among American women in 2015. The majority of endometrial cancers are of serous or endometrioid histology. Most human tumors, including endometrial tumors, are driven by the acquisition of pathogenic mutations in cancer genes. Thus, the identification of somatic mutations within tumor genomes is an entry point toward cancer gene discovery. However, efforts to pinpoint somatic mutations in human cancers have, until recently, relied on high-throughput sequencing of single genes or gene families using Sanger sequencing. Although this approach has been fruitful, the cost and throughput of Sanger sequencing generally prohibits systematic sequencing of the ~22,000 genes that make up the exome. The recent development of next-generation sequencing technologies changed this paradigm by providing the capability to rapidly sequence exomes, transcriptomes, and genomes at relatively low cost. Remarkably, the application of this technology to catalog the mutational landscapes of endometrial tumor exomes, transcriptomes, and genomes has revealed, for the first time, that serous and endometrioid endometrial cancers can be classified into four distinct molecular subgroups. In this chapter, we overview the characteristic genomic features of each subgroup and discuss the known and putative cancer genes that have emerged from next-generation sequencing of endometrial carcinomas.

  19. HIV Sequence Compendium 2015

    SciTech Connect

    Foley, Brian Thomas; Leitner, Thomas Kenneth; Apetrei, Cristian; Hahn, Beatrice; Mizrachi, Ilene; Mullins, James; Rambaut, Andrew; Wolinsky, Steven; Korber, Bette Tina Marie

    2015-10-05

    This compendium is an annual printed summary of the data contained in the HIV sequence database. We try to present a judicious selection of the data in such a way that it is of maximum utility to HIV researchers. Each of the alignments attempts to display the genetic variability within the different species, groups and subtypes of the virus. This compendium contains sequences published before January 1, 2015. Hence, though it is published in 2015 and called the 2015 Compendium, its contents correspond to the 2014 curated alignments on our website. The number of sequences in the HIV database is still increasing. In total, at the end of 2014, there were 624,121 sequences in the HIV Sequence Database, an increase of 7% since the previous year. This is the first year that the number of new sequences added to the database has decreased compared to the previous year. The number of near complete genomes (>7000 nucleotides) increased to 5834 by end of 2014. However, as in previous years, the compendium alignments contain only a fraction of these. A more complete version of all alignments is available on our website, http://www.hiv.lanl.gov/ content/sequence/NEWALIGN/align.html As always, we are open to complaints and suggestions for improvement. Inquiries and comments regarding the compendium should be addressed to seq-info@lanl.gov.

  20. Personalized Course Sequence Recommendations

    NASA Astrophysics Data System (ADS)

    Xu, Jie; Xing, Tianwei; van der Schaar, Mihaela

    2016-10-01

    Given the variability in student learning it is becoming increasingly important to tailor courses as well as course sequences to student needs. This paper presents a systematic methodology for offering personalized course sequence recommendations to students. First, a forward-search backward-induction algorithm is developed that can optimally select course sequences to decrease the time required for a student to graduate. The algorithm accounts for prerequisite requirements (typically present in higher level education) and course availability. Second, using the tools of multi-armed bandits, an algorithm is developed that can optimally recommend a course sequence that both reduces the time to graduate while also increasing the overall GPA of the student. The algorithm dynamically learns how students with different contextual backgrounds perform for given course sequences and then recommends an optimal course sequence for new students. Using real-world student data from the UCLA Mechanical and Aerospace Engineering department, we illustrate how the proposed algorithms outperform other methods that do not include student contextual information when making course sequence recommendations.

  1. Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.

    PubMed

    Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R

    2000-04-14

    There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

  2. Phylogenetic Trees From Sequences

    NASA Astrophysics Data System (ADS)

    Ryvkin, Paul; Wang, Li-San

    In this chapter, we review important concepts and approaches for phylogeny reconstruction from sequence data.We first cover some basic definitions and properties of phylogenetics, and briefly explain how scientists model sequence evolution and measure sequence divergence. We then discuss three major approaches for phylogenetic reconstruction: distance-based phylogenetic reconstruction, maximum parsimony, and maximum likelihood. In the third part of the chapter, we review how multiple phylogenies are compared by consensus methods and how to assess confidence using bootstrapping. At the end of the chapter are two sections that list popular software packages and additional reading.

  3. Automatic Command Sequence Generation

    NASA Technical Reports Server (NTRS)

    Fisher, Forest; Gladded, Roy; Khanampompan, Teerapat

    2007-01-01

    Automatic Sequence Generator (Autogen) Version 3.0 software automatically generates command sequences for the Mars Reconnaissance Orbiter (MRO) and several other JPL spacecraft operated by the multi-mission support team. Autogen uses standard JPL sequencing tools like APGEN, ASP, SEQGEN, and the DOM database to automate the generation of uplink command products, Spacecraft Command Message Format (SCMF) files, and the corresponding ground command products, DSN Keywords Files (DKF). Autogen supports all the major multi-mission mission phases including the cruise, aerobraking, mapping/science, and relay mission phases. Autogen is a Perl script, which functions within the mission operations UNIX environment. It consists of two parts: a set of model files and the autogen Perl script. Autogen encodes the behaviors of the system into a model and encodes algorithms for context sensitive customizations of the modeled behaviors. The model includes knowledge of different mission phases and how the resultant command products must differ for these phases. The executable software portion of Autogen, automates the setup and use of APGEN for constructing a spacecraft activity sequence file (SASF). The setup includes file retrieval through the DOM (Distributed Object Manager), an object database used to store project files. This step retrieves all the needed input files for generating the command products. Depending on the mission phase, Autogen also uses the ASP (Automated Sequence Processor) and SEQGEN to generate the command product sent to the spacecraft. Autogen also provides the means for customizing sequences through the use of configuration files. By automating the majority of the sequencing generation process, Autogen eliminates many sequence generation errors commonly introduced by manually constructing spacecraft command sequences. Through the layering of commands into the sequence by a series of scheduling algorithms, users are able to rapidly and reliably construct the

  4. Toward nanoscale genome sequencing.

    PubMed

    Ryan, Declan; Rahimi, Maryam; Lund, John; Mehta, Ranjana; Parviz, Babak A

    2007-09-01

    This article reports on the state-of-the-art technologies that sequence DNA using miniaturized devices. The article considers the miniaturization of existing technologies for sequencing DNA and the opportunities for cost reduction that 'on-chip' devices can deliver. The ability to construct nano-scale structures and perform measurements using novel nano-scale effects has provided new opportunities to identify nucleotides directly using physical, and not chemical, methods. The challenges that these technologies need to overcome to provide a US$1000-genome sequencing technology are also presented.

  5. Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

    PubMed Central

    Foster, Patricia L.; Lee, Heewook; Popodi, Ellen; Townes, Jesse P.; Tang, Haixu

    2015-01-01

    A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10−3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold. PMID:26460006

  6. Efficient sequence-directed psoralen targeting using pseudocomplementary Peptide nucleic acids.

    PubMed

    Kim, Ki-Hyun; Fan, Xue-Jun; Nielsen, Peter E

    2007-01-01

    A pair of decameric pseudocomplementary PNAs which bind to their mixed purine-pyrimidine sequence target in duplex DNA by double duplex invasion has been synthesized with a derivative of 8-methoxypsoralen conjugated to one of the PNAs. It is shown that this pair of psoralen-conjugated pseudocomplementary PNA oligomers, which target a site in the pBluescriptKS+ vector, upon irradiation with long-wavelength UV light (UVA) with high efficiency and specificity form photoadducts to an adjacent 5'-TA site, and more than 50% of these adducts are DNA interstrand cross-links. Transcription elongation by T7 or T3 RNA polymerase is specifically arrested at the psoralen cross-linking site, yielding more than 90% arrested product. These results emphasize the potential of pseudocomplementary PNA oligomers for highly specific gene targeting, in particular, with respect to sequence-directed psoralen photomodification of double-stranded DNA. Thus, such psoralen-PNA conjugates could be very useful in a range of biology and drug discovery applications.

  7. DNA sequences encoding osteoinductive products

    SciTech Connect

    Wang, E.A.; Wozney, J.M.; Rosen, V.

    1991-05-07

    This patent describes an isolated DNA sequence encoding an osteoinductive protein the DNA sequence comprising a coding sequence. It comprises: nucleotide No.1 through nucleotide No.387, nucleotide No.356 through nucleotide No.1543, nucleotide $402 through nucleotide No.1626, naturally occurring allelic sequences and equivalent degenerative codon sequences and sequences which hybridize to any of sequences under stringent hybridization conditions; and encode a protein characterized by the ability to induce the formation of bone and/or cartilage.

  8. Compact rotary sequencer

    NASA Technical Reports Server (NTRS)

    Appleberry, W. T.

    1980-01-01

    Rotary sequencer is assembled from conventional planetary differential gearset and latching mechanism utilizing inputs and outputs which are coaxial. Applications include automated production-line equipment in home appliances and in vehicles.

  9. Authentication of byte sequences

    SciTech Connect

    Stearns, S.D.

    1991-06-01

    Algorithms for the authentication of byte sequences are described. The algorithms are designed to authenticate data in the Storage, Retrieval, Analysis, and Display (SRAD) Test Data Archive of the Radiation Effects and Testing Directorate (9100) at Sandia National Laboratories, and may be used in similar situations where authentication of stored data is required. The algorithms use a well-known error detection method called the Cyclic Redundancy Check (CRC). When a byte sequence is authenticated and stored, CRC bytes are generated and attached to the end of the sequence. When the authenticated data is retrieved, the authentication check consists of processing the entire sequence, including the CRC bytes, and checking for a remainder of zero. The error detection properties of the CRC are extensive and result in a reliable authentication of SRAD data.

  10. Advances in sequence analysis.

    PubMed

    Califano, A

    2001-06-01

    In its early days, the entire field of computational biology revolved almost entirely around biological sequence analysis. Over the past few years, however, a number of new non-sequence-based areas of investigation have become mainstream, from the analysis of gene expression data from microarrays, to whole-genome association discovery, and to the reverse engineering of gene regulatory pathways. Nonetheless, with the completion of private and public efforts to map the human genome, as well as those of other organisms, sequence data continue to be a veritable mother lode of valuable biological information that can be mined in a variety of contexts. Furthermore, the integration of sequence data with a variety of alternative information is providing valuable and fundamentally new insight into biological processes, as well as an array of new computational methodologies for the analysis of biological data.

  11. HIV Sequence Compendium 2010

    SciTech Connect

    Kuiken, Carla; Foley, Brian; Leitner, Thomas; Apetrei, Christian; Hahn, Beatrice; Mizrachi, Ilene; Mullins, James; Rambaut, Andrew; Wolinsky, Steven; Korber, Bette

    2010-12-31

    This compendium is an annual printed summary of the data contained in the HIV sequence database. In these compendia we try to present a judicious selection of the data in such a way that it is of maximum utility to HIV researchers. Each of the alignments attempts to display the genetic variability within the different species, groups and subtypes of the virus. This compendium contains sequences published before January 1, 2010. Hence, though it is called the 2010 Compendium, its contents correspond to the 2009 curated alignments on our website. The number of sequences in the HIV database is still increasing exponentially. In total, at the time of printing, there were 339,306 sequences in the HIV Sequence Database, an increase of 45% since last year. The number of near complete genomes (>7000 nucleotides) increased to 2576 by end of 2009, reflecting a smaller increase than in previous years. However, as in previous years, the compendium alignments contain only a small fraction of these. Included in the alignments are a small number of sequences representing each of the subtypes and the more prevalent circulating recombinant forms (CRFs) such as 01 and 02, as well as a few outgroup sequences (group O and N and SIV-CPZ). Of the rarer CRFs we included one representative each. A more complete version of all alignments is available on our website, http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html. Reprints are available from our website in the form of both HTML and PDF files. As always, we are open to complaints and suggestions for improvement. Inquiries and comments regarding the compendium should be addressed to seq-info@lanl.gov.

  12. Efficient procedure for isolating methylated catechins from green tea and effective simultaneous analysis of ten catechins, three purine alkaloids, and gallic acid in tea by high-performance liquid chromatography with diode array detection.

    PubMed

    Hu, Bing; Wang, Lin; Zhou, Bei; Zhang, Xin; Sun, Yi; Ye, Hong; Zhao, Liyan; Hu, Qiuhui; Wang, Guoxiang; Zeng, Xiaoxiong

    2009-04-10

    Monomers of (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3''Me) and (-)-3-O-methyl epicatechin gallate (ECG3'Me) (purity, >97%) were successfully prepared from extract of green tea by two-time separation with Toyopearl HW-40S column chromatography eluted by 80% ethanol. In addition, monomers of (-)-catechin (C), (-)-gallocatechin (GC), (-)-gallocatechin gallate (GCG), and (-)-catechin gallate (CG) (purity, >98%) were prepared from EC, EGC, EGCG, and ECG by heat-epimerization and semi-preparative HPLC chromatography. With the prepared catechin standards, an effective and simultaneous HPLC method for the analysis of gallic acid, tea catechins, and purine alkaloids in tea was developed in the present study. Using an ODS-100Z C(18) reversed-phase column, fourteen compounds were rapidly separated within 15min by a linear gradient elution of formic acid solution (pH 2.5) and methanol. A 2.5-7-fold reduction in HPLC analysis time was obtained from existing analytical methods (40-105min) for gallic acid, tea catechins including O-methylated catechins and epimers of epicatechins, as well as purine alkaloids. Detection limits were generally on the order of 0.1-1.0ng for most components at the applied wavelength of 280nm. Method replication generally resulted in intraday and interday peak area variation of <6% for most tested components in green, Oolong, black, and pu-erh teas. Recovery rates were generally within the range of 92-106% with RSDs less than 4.39%. Therefore, advancement has been readily achievable with commonly used chromatography equipments in the present study, which will facilitate the analytical, clinical, and other studies of tea catechins.

  13. Development, validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase.

    PubMed

    Cattaneo, Giulia; Ubiali, Daniela; Calleri, Enrica; Rabuffetti, Marco; Höfner, Georg C; Wanner, Klaus T; De Moraes, Marcela C; Martinelli, Leonardo K B; Santos, Diógenes Santiago; Speranza, Giovanna; Massolini, Gabriella

    2016-11-02

    Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC 2.4.2.1) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The enzymatic assay (v = 0.5 mL, enzyme<0.2 μg/well, T = 37 °C) was performed with an overall time of about 15 min/plate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met the criteria of the CDER guidance of FDA. Kinetic parameters were in agreement with those reported in literature (HsPNP KM = 0.150 ± 0.020 mM vs 0.133 ± 0.015 mM; MtbPNP KM = 0.060 ± 0.009 mM vs 0.040 ± 0.003 mM for Ino), thus demonstrating the reliability of the newly developed enzymatic assay. Preliminary inhibition assays confirmed the effects reported for Acyclovir (Acv) and Formycin A (FA) against HsPNP and MtbPNP. The validated enzymatic assay was applied to the evaluation of a set of 8-halo-, 8-amino-, 8-O-alkyl-substituted purine ribonucleosides synthesized on purpose as potential inhibitors against MtbPNP. The assayed 8-substituted ribonucleosides did not exert a significant inhibitory effect against the tested enzymes up to 1 mM.

  14. Pairwise Sequence Alignment Library

    SciTech Connect

    Jeff Daily, PNNL

    2015-05-20

    Vector extensions, such as SSE, have been part of the x86 CPU since the 1990s, with applications in graphics, signal processing, and scientific applications. Although many algorithms and applications can naturally benefit from automatic vectorization techniques, there are still many that are difficult to vectorize due to their dependence on irregular data structures, dense branch operations, or data dependencies. Sequence alignment, one of the most widely used operations in bioinformatics workflows, has a computational footprint that features complex data dependencies. The trend of widening vector registers adversely affects the state-of-the-art sequence alignment algorithm based on striped data layouts. Therefore, a novel SIMD implementation of a parallel scan-based sequence alignment algorithm that can better exploit wider SIMD units was implemented as part of the Parallel Sequence Alignment Library (parasail). Parasail features: Reference implementations of all known vectorized sequence alignment approaches. Implementations of Smith Waterman (SW), semi-global (SG), and Needleman Wunsch (NW) sequence alignment algorithms. Implementations across all modern CPU instruction sets including AVX2 and KNC. Language interfaces for C/C++ and Python.

  15. Characterization of the Mutagenic Spectrum of 4-Nitroquinoline 1-Oxide (4-NQO) in Aspergillus nidulans by Whole Genome Sequencing

    PubMed Central

    Downes, Damien J.; Chonofsky, Mark; Tan, Kaeling; Pfannenstiel, Brandon T.; Reck-Peterson, Samara L.; Todd, Richard B.

    2014-01-01

    4-Nitroquinoline 1-oxide (4-NQO) is a highly carcinogenic chemical that induces mutations in bacteria, fungi, and animals through the formation of bulky purine adducts. 4-NQO has been used as a mutagen for genetic screens and in both the study of DNA damage and DNA repair. In the model eukaryote Aspergillus nidulans, 4-NQO−based genetic screens have been used to study diverse processes, including gene regulation, mitosis, metabolism, organelle transport, and septation. Early work during the 1970s using bacterial and yeast mutation tester strains concluded that 4-NQO was a guanine-specific mutagen. However, these strains were limited in their ability to determine full mutagenic potential, as they could not identify mutations at multiple sites, unlinked suppressor mutations, or G:C to C:G transversions. We have now used a whole genome resequencing approach with mutant strains generated from two independent genetic screens to determine the full mutagenic spectrum of 4-NQO in A. nidulans. Analysis of 3994 mutations from 38 mutant strains reveals that 4-NQO induces substitutions in both guanine and adenine residues, although with a 19-fold preference for guanine. We found no association between mutation load and mutagen dose and observed no sequence bias in the residues flanking the mutated purine base. The mutations were distributed randomly throughout most of the genome. Our data provide new evidence that 4-NQO can potentially target all base pairs. Furthermore, we predict that current practices for 4-NQO−induced mutagenesis are sufficient to reach gene saturation for genetic screens with feasible identification of causative mutations via whole genome resequencing. PMID:25352541

  16. DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

    SciTech Connect

    Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; Johnson, Reid C.; Leng, Fenfei

    2016-03-09

    The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.

  17. Stem-loop structures of the repetitive DNA sequences located at human centromeres

    SciTech Connect

    Gupta, G.; Garcia, A.E.; Ratliff, R.; Moyzis, R.K.; Catasti, P.; Hong, Lin; Yau, P.; Bradbury, E.M. |

    1993-09-01

    The presence of the highly conserved repetitive DNA sequences in the human centromeres argues for a special role of these sequences in their biological functions - most likely achieved by the formation of unusual structures. This prompted us to carry out quantitative one- and two-dimensional nuclear magnetic resonance (lD/2D NMR) spectroscopy to determine the structural properties of the human centromeric repeats, d(AATGG){sub n.d}(CCATT){sub n}. The studies on centromeric DNAs reveal that the complementary sequence, d(AATGG){sub n.d}(CCATT){sub n}, adopts the usual Watson-Crick B-DNA duplex and the pyrimidine-rich d(CCATT){sub n} strand is essentially a random coil. However, the purine-rich d(AATGG){sub n} strand is shown to adopt unusual stem-loop structures for repeat lengths, n=2,3,4, and 6. In addition to normal Watson-Crick A{center_dot}T pairs, the stem-loop structures are stabilized by mismatch A{center_dot}G and G{center_dot}G pairs in the stem and G-G-A stacking in the loop. Stem-loop structures of d(AATGG)n are independently verified by gel electrophoresis and nuclease digestion studies. Thermal melting studies show that the DNA repeats, d(AATGG){sub n}, are as stable as the corresponding Watson-Crick duplex d(AATGG){sub n.d}(CCATT){sub n}. Therefore, the sequence d(AATGG){sub n} can, indeed, nucleate a stem-loop structure at little free-energy cost and if, during mitosis, they are located on the chromosome surface they can provide specific recognition sites for kinetochore function.

  18. Program Synthesizes UML Sequ