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Sample records for pronounced purine sequence

  1. Draft Genome Sequence of Purine-Degrading Gottschalkia purinilyticum (Formerly Clostridium purinilyticum) WA1 (DSM 1384)

    PubMed Central

    Poehlein, Anja; Bengelsdorf, Frank R.; Schiel-Bengelsdorf, Bettina; Daniel, Rolf

    2015-01-01

    Here, we report the draft genome sequence of Gottschalkia purinilyticum (formerly Clostridium purinilyticum) WA1, an anaerobic bacterium specialized on degradation of purines (including adenine) and glycine, which uses the selenoprotein glycine reductase for substrate degradation. The genome consists of a single chromosome (3.40 Mb). PMID:26404607

  2. A Short Sequence within Two Purine-Rich Enhancers Determines 5′ Splice Site Specificity

    PubMed Central

    Elrick, Leslie L.; Humphrey, Mary Beth; Cooper, Thomas A.; Berget, Susan M.

    1998-01-01

    Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3′ splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5′ splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5′ splice site choice when placed between competing 5′ splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5′ splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5′ splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5′ splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3′ end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5′ splice site selectivity was different than that shown to be maximally important for activation of a 3′ splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in

  3. Pronounced population genetic differentiation in the rock bream Oplegnathus fasciatus inferred from mitochondrial DNA sequences.

    PubMed

    Xiao, Yongshuang; Li, Jun; Ren, Guijing; Ma, Daoyuan; Wang, Yanfeng; Xiao, ZhiZhong; Xu, Shihong

    2016-05-01

    The population genetic structure of the rock bream (Oplegnathus fasciatus) along the coastal waters of China was estimated based on three mtDNA fragments (D-loop, COI, and Cytb). A total of 112 polymorphic sites were checked, which defined 63 haplotypes. A pattern with high levels of haplotype diversity (hCOI = 0.886 ± 0.034, hCytb = 0.874 ± 0.023) and low levels of nucleotide diversity (лCOI = 0.009 ± 0.005, лCytb = 0.006 ± 0.003) was detected based on the COI and Cytb fragments, and high levels of genetic diversity (hD-loop = 0.995 ± 0.007, лD-loop = 0.021 ± 0.011) were detected from the mtDNA D-loop. The population genetic diversity of O. fasciatus in south China was significantly higher than those of north China. Three genealogical clades were checked in the O. fasciatus populations based on the NJ and MST analyses of mtDNA COI gene sequence, and the genetic distances among the clades ranged from 0.018 to 0.025. Significant population genetic differentiation was also checked based on the Fst (0.331, p = 0.000) and exact p (0.000) test analyses. No significant population differentiations were checked based on mtDNA D-loop and Cytb fragments. Using a variety of phylogenetic methods, coalescent reasoning, and molecular dating interpreted in conjunction with paleoclimatic and physiographic evidences, we inferred that the genetic make-up of extant populations of O. fasciatus was shaped by Pleistocene environmental impacts on the historical demography of this species. Coalescent analyses (neutrality tests, mismatch distribution analysis, and Bayesian skyline analyses) showed that the species along coastline of China has experienced population expansions originated in its most recent history at about 169-175 kya before present. PMID:25427804

  4. The Purine Bias of Coding Sequences is Determined by Physicochemical Constraints on Proteins

    PubMed Central

    de Leon, Miguel Ponce; de Miranda, Antonio Basilio; Alvarez-Valin, Fernando; Carels, Nicolas

    2014-01-01

    For this report, we analyzed protein secondary structures in relation to the statistics of three nucleotide codon positions. The purpose of this investigation was to find which properties of the ribosome, tRNA or protein level, could explain the purine bias (Rrr) as it is observed in coding DNA. We found that the Rrr pattern is the consequence of a regularity (the codon structure) resulting from physicochemical constraints on proteins and thermodynamic constraints on ribosomal machinery. The physicochemical constraints on proteins mainly come from the hydropathy and molecular weight (MW) of secondary structures as well as the energy cost of amino acid synthesis. These constraints appear through a network of statistical correlations, such as (i) the cost of amino acid synthesis, which is in favor of a higher level of guanine in the first codon position, (ii) the constructive contribution of hydropathy alternation in proteins, (iii) the spatial organization of secondary structure in proteins according to solvent accessibility, (iv) the spatial organization of secondary structure according to amino acid hydropathy, (v) the statistical correlation of MW with protein secondary structures and their overall hydropathy, (vi) the statistical correlation of thymine in the second codon position with hydropathy and the energy cost of amino acid synthesis, and (vii) the statistical correlation of adenine in the second codon position with amino acid complexity and the MW of secondary protein structures. Amino acid physicochemical properties and functional constraints on proteins constitute a code that is translated into a purine bias within the coding DNA via tRNAs. In that sense, the Rrr pattern within coding DNA is the effect of information transfer on nucleotide composition from protein to DNA by selection according to the codon positions. Thus, coding DNA structure and ribosomal machinery co-evolved to minimize the energy cost of protein coding given the functional

  5. Fast method of homology and purine-pyrimidine mutual relations between DNA sequences search.

    PubMed

    Korotkov, E V

    1994-01-01

    A new algorithm for scanning sequences is described. This algorithm uses the boolean operators AND and OR. The mutual information between the sequences is used as a measure of sequence interrelation. It allows evaluation of the probability of accidental sequence interrelation in a quantitative manner. The proposed algorithm was used for searching for MB1 repeats in human and other mammalian sequences. PMID:7841466

  6. Transferring the purine 2-amino group from guanines to adenines in DNA changes the sequence-specific binding of antibiotics.

    PubMed Central

    Bailly, C; Waring, M J

    1995-01-01

    The proposition that the 2-amino group of guanine plays a critical role in determining how antibiotics recognise their binding sites in DNA has been tested by relocating it, using tyrT DNA derivative molecules substituted with inosine plus 2,6-diaminopurine (DAP). Irrespective of their mode of interaction with DNA, such GC-specific antibiotics as actinomycin, echinomycin, mithramycin and chromomycin find new binding sites associated with DAP-containing sequences and are excluded from former canonical sites containing I.C base pairs. The converse is found to be the case for a group of normally AT-selective ligands which bind in the minor groove of the helix, such as netropsin: their preferred sites become shifted to IC-rich clusters. Thus the binding sites of all these antibiotics strictly follow the placement of the purine 2-amino group, which accordingly must serve as both a positive and negative effector. The footprinting profile of the 'threading' intercalator nogalamycin is potentiated in DAP plus inosine-substituted DNA but otherwise remains much the same as seen with natural DNA. The interaction of echinomycin with sites containing the TpDAP step in doubly substituted DNA appears much stronger than its interaction with CpG-containing sites in natural DNA. Images PMID:7731800

  7. Pur-1, a zinc-finger protein that binds to purine-rich sequences, transactivates an insulin promoter in heterologous cells.

    PubMed Central

    Kennedy, G C; Rutter, W J

    1992-01-01

    Purine-rich stretches of nucleotides (GAGA boxes) are often found just upstream of transcription start sites in many genes, including insulin. Mutational analysis suggests that the GAGA box plays an important role in transcription of the rat insulin I gene. We identify here at least four different proteins that bind specifically to the insulin GAGA box. Using a GAGA oligonucleotide, we have isolated a cDNA encoding a sequence-specific protein from a HIT (hamster insulinoma cell line) lambda gt11 library. This protein, which we designate Pur-1 (for purine binding), binds to the GAGA boxes of the rat insulin I and II genes and the human islet amyloid polypeptide gene. Pur-1 is a potent transactivator in both pancreatic and nonpancreatic cells. Furthermore, Pur-1 is able to activate an intact insulin promoter in HeLa cells, where it is normally inactive. Images PMID:1454839

  8. Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis.

    PubMed Central

    Watanabe, W; Sampei, G; Aiba, A; Mizobuchi, K

    1989-01-01

    It has been shown that the Escherichia coli purE locus specifying 5'-phosphoribosyl-5-amino-4-imidazole carboxylase in de novo purine nucleotide synthesis is divided into two cistrons. We cloned and determined a 2,449-nucleotide sequence including the purE locus. This sequence contains two overlapped open reading frames, ORF-18 and ORF-39, encoding proteins with molecular weights of 18,000 and 39,000, respectively. The purE mutations of CSH57A and DCSP22 were complemented by plasmids carrying ORF-18, while that of NK6051 was complemented by plasmids carrying ORF-39. Thus, the purE locus consists of two distinct genes, designated purE and purK for ORF-18 and ORF-39, respectively. These genes constitute a single operon. A highly conserved 16-nucleotide sequence, termed the PUR box, was found in the upstream region of purE by comparing the sequences of the purF and purMN operons. We also found three entire and one partial repetitive extragenic palindromic (REP) sequences in the downstream region of purK. Roles of the PUR box and REP sequences are discussed in relation to the genesis of the purEK operon. Images PMID:2644189

  9. 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.

  10. [Purine nucleoside phosphorylase].

    PubMed

    Pogosian, L G; Akopian, Zh I

    2013-01-01

    Purine nucleoside phosphorylase (PNP) is one of the most important enzymes of the purine metabolism, wich promotes the recycling of purine bases. Nowadays is the actual to search for effective inhibitors of this enzyme which is necessary for creation T-cell immunodeficient status of the organism in the organs and tissues transplantation, and chemotherapy of a number pathologies as well. For their successful practical application necessary to conduct in-depth and comprehensive study of the enzyme, namely a structure, functions, and an affinity of the reaction mechanism. In the review the contemporary achievements in the study of PNP from various biological objects are presented. New data describing the structure of PNP are summarised and analysed. The physiological role of the enzyme is discussed. The enzyme basic reaction mechanisms and actions are considered. The studies on enzyme physicochemical, kinetic, and catalytic research are presented. PMID:24479338

  11. Parallel-stranded duplex DNA containing blocks of trans purine-purine and purine-pyrimidine base pairs.

    PubMed Central

    Evertsz, E M; Rippe, K; Jovin, T M

    1994-01-01

    A 30 base pair parallel-stranded (ps) duplex ps-L1.L2 composed of two adjoined purine-purine and purine-pyrimidine sequence blocks has been characterized thermodynamically and spectroscopically. The 5'-terminal 15 residues in both strands ('left-half') consisted of the alternating d(GA)7G sequence that forms a ps homoduplex secondary structure stabilized by d(G.G) and d(A.A) base pairs. The 3'-terminal 15 positions of the sequence ('right-half') were combinations of A and T with complementary reverse Watson-Crick d(A.T) base pairing between the two strands. The characteristics of the full length duplex were compared to those of the constituent left and right halves in order to determine the compatibility of the two ps helical forms. The thermal denaturation curves and hyperchromicity profiles of all three duplexes determined by UV absorption spectroscopy were characteristic of ps-DNA, in accordance with previous studies. The thermodynamic properties of the 30 bp duplex corresponded within experimental error to the linear combination of the two 15-mers. Thus, the Tm and delta HvH of ps-L1.L2 in 10 mM MgCl2, derived from analyses according to a statistical mechanical formulation for the helix-coil transition, were 43 degrees C and 569 kJ mol-1, compared to 21 degrees C, 315 kJ mol-1 (ps-F5.F6) and 22 degrees C, 236 kJ mol-1 (ps-GA15). The UV absorption and CD spectra of ps-L1.L2 and the individual 15-mer ps motifs were also compared quantitatively. The sums of the two constituent native spectra (left+right halves) accurately matched that of the 30 bp duplex, with only small deviations in the 195-215 nm (CD) and 220-240 nm (absorption) regions. Based on analysis by native gel electrophoresis, the sequences studied formed duplex structures exclusively; there were no indications of higher order species. Chemical modification with diethyl pyrocarbonate showed no hyperreactivity of the junctional bases, indicating a smooth transition between the two parallel

  12. Basis for the control of purine biosynthesis by purine ribonucleotides.

    PubMed Central

    Itakura, M; Sabina, R L; Heald, P W; Holmes, E W

    1981-01-01

    An animal model was used to determine the basis for the increase in purine biosynthesis that results from hepatic depletion of purine nucleotides, such as seen in patients with type I glycogen storage disease or following fructose administration. Mice were injected intravenously with glucose or fructose, 2.5 mg/g of body weight, and the animals were killed at 0, 3, and 30 min following carbohydrate infusion. Fructose, but not glucose, administration led to a threefold increase in [14C]glycine incorporation into hepatic purine nucleotides documenting an increase in the rate of purine biosynthesis in the liver of fructose-treated animals. In the fructose, but not the glucose-treated animals, there was a reduction in the hepatic content of purine nucleotides that are inhibitory for amidophosphoribosyltransferase, the enzyme that catalyzes the first reaction unique to the pathway of purine biosynthesis. PP-ribose-P, an important metabolite in the control of purine biosynthesis, was increased 2,3-fold in liver following fructose, but not glucose administration. In conjunction with the decrease in inhibitory nucleotides and increase in PP-ribose-P 29% of amidophosphoribosyltransferase was shifted from the large inactive to the small active form of the enzyme. Results of these studies demonstrate that the end-products of the pathway, purine nucleotides, control the activity of the enzyme that catalyzes the first reaction leading to purine nucleotide synthesis either through a direct effect of purine nucleotides on the enzyme, through an indirect effect of the change in nucleotides on PP-ribose-P synthesis, or a combination of these effects. The resultant changes in amidophosphoribosyltransferase conformation and activity provide a basis for understanding the increase in purine biosynthesis that results from hepatic depletion of purine nucleotides. PMID:6162862

  13. The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae.

    PubMed Central

    Suárez, T; de Queiroz, M V; Oestreicher, N; Scazzocchio, C

    1995-01-01

    The uaY gene codes for a transcriptional activator mediating the induction of a number of unlinked genes involved in purine utilization in Aspergillus nidulans. Here we present the complete genomic and cDNA nucleotide sequence of this gene. The gene contains two introns. The derived polypeptide of 1060 residues contains a typical zinc binuclear cluster domain and shows a number of similarities with the PPR1 regulatory gene of Saccharomyces cerevisiae. These similarities are most striking in the putative linker and dimerization regions following the zinc cluster. Gel-shift and DNase I footprinting experiments have been carried out for three genes subject to UaY-mediated induction. The binding sequence is 5'-TCGG-6X-CCGA, which is identical to the proposed PPR1 binding sites. Nevertheless, the identity of the base immediately 3' of the 5'-TCGG sequence clearly affects the affinity of the site. The site upstream of the uapA gene has been shown to be active in vivo. Binding to this site has been analysed by a number of interference techniques. There is an interesting chemical similarity between the co-inducer of the purine utilization pathway (uric acid) and that of the genes of the pyrimidine biosynthetic pathway (dihydroorotic acid) and we show that dihydroorotic acid can act as a poor inducer of at least one activity under UaY control. These striking similarities, together with the unique pattern of regulation of pyrimidine biosynthesis in S. cerevisiae, suggest that PPR1 evolved through recruitment into the pyrimidine biosynthetic pathway of an ancestral gene related to uaY. Images PMID:7729421

  14. Metabolism and ecology of purine alkaloids.

    PubMed

    Anaya, Ana Luisa; Cruz-Ortega, Rocio; Waller, George R

    2006-01-01

    In this review, the biosynthesis, catabolism, ecological significance, and modes of action of purine alkaloids particularly, caffeine, theobromine and theophylline in plants are discussed. In the biosynthesis of caffeine, progress has been made in enzymology, the amino acid sequence of the enzymes, and in the genes encoding N-methyltransferases. In addition, caffeine-deficient plants have been produced. The ecology of purine alkaloids has not proved to be particularly promising. However, advances have been made in insecticidal and allelopathic fields, and in the role of microorganisms play in the changes that these compounds undergo in the soil. Caffeine inhibits cell plate formation during telophase throughout the development of coffee plants and other species. PMID:16720319

  15. Purine Salvage Pathways among Borrelia Species▿

    PubMed Central

    Pettersson, Jonas; Schrumpf, Merry E.; Raffel, Sandra J.; Porcella, Stephen F.; Guyard, Cyril; Lawrence, Kevin; Gherardini, Frank C.; Schwan, Tom G.

    2007-01-01

    Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood. PMID:17502392

  16. Evidence from CD spectra that d(purine).r(pyrimidine) and r(purine).d(pyrimidine) hybrids are in different structural classes.

    PubMed Central

    Hung, S H; Yu, Q; Gray, D M; Ratliff, R L

    1994-01-01

    CD spectra and difference CD spectra of four d(oligopurine).r(oligopyrimidine) and four r(oligopurine).d(oligopyrimidine) hybrid duplexes containing mixed A.T(U) and G.C base pairs were compared with the spectra of four DNA.DNA and four RNA.RNA oligomer duplexes of similar repeating sequences. The 16 duplexes were formed by mixing oligomers that were 24 nucleotides long. The buffer was 0.05 M Na+ (phosphate), pH 7.0. DNA.DNA and RNA.RNA oligomer duplexes were used as reference B-form and A-form structures. We found that the CD spectra of d(purine).r(pyrimidine) and r(purine).d(pyrimidine) hybrid duplexes were different from the CD spectra of either DNA.DNA or RNA.RNA duplexes. The data suggested that these hybrids have intermediate structures between A-form RNA and B-form DNA structures. The CD spectra of d(purine).r(pyrimidine) and r(purine).d(pyrimidine) hybrid duplexes were different from each other, but the hybrids in each class had consistent CD spectra as indicated by nearest-neighbor comparisons. Thus, it appeared that the two types of hybrids belonged to different structural classes. The negative 210 nm band found in difference CD spectra was correlated with the presence of an r(purine) strand in the hybrid duplexes. The melting temperatures (Tm values) of these hybrids were compared with the Tm values of the DNA.DNA and RNA.RNA duplexes. The order of the thermal stability was: RNA.RNA duplex > r(purine).d(pyrimidine) hybrid > DNA.DNA duplex > d(purine).r(pyrimidine) hybrid, when comparing analogous sequences. PMID:7937162

  17. T.C.G triplet in an antiparallel purine.purine.pyrimidine DNA triplex. Conformational studies by NMR.

    PubMed

    Dittrich, K; Gu, J; Tinder, R; Hogan, M; Gao, X

    1994-04-12

    The antiparallel purine.purine.pyrimidine DNA triplex, RRY6, which contains a T.C.G inverted triplet in the center of the sequence, was examined by proton and phosphorous two-dimensional NMR spectroscopy. The local conformation of the T.C.G triplet (T4.C11.G18) and the effect of this triplet on the global helical structure were analyzed in detail. The formation of the T.C.G triplet is confirmed by a set of cross-strand NOEs, including unusual cross-strand NOEs between the third strand and the pyrimidine strand as opposed to the purine strand of the duplex. NMR data suggest that the T.C.G triplet may be present in an equilibrium between a non-hydrogen-bonded form and a T(O4)-C(NH2) hydrogen-bonded form and that there is a distortion of the in-plane alignment of the three bases. The flanking G.G.C base triplets are well-defined on the 5'-side of T4, but somewhat interrupted on the 3'-side of T4. The effect of the third strand binding on the Watson-Crick duplex was probed by an NMR study of the free duplex RY6. NMR parameters are affected mostly around the T.C.G inversion site. The perturbations extend to at least two adjacent base triplets on either side. The binding of the third purine strand and the accommodation of a central T.C.G inversion in RRY6 does not require a readjustment in sugar pucker, which remains in the range of C2'-endo. 31P resonances of RRY6 distribute over a range of 2.2 ppm. The H-P coupling patterns of the third strand differ from those of the duplex. General spectral patterns defined by the marker protons of the RRY and YRY triplexes are compared. PMID:8155628

  18. Purine Catabolism in Plants 1

    PubMed Central

    Guranowski, Andrzej

    1982-01-01

    Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite. Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The Km value computed for inosine is 65 micromolar. Among the inosine analogs tested, the following nucleosides are substrates for the lupin inosine nucleosidase: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (Vmax/Km) towards adenosine is 48 times lower than that towards inosine. In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawełkiewicz 1978 Planta 139: 245-247), the inosine nucleosidase is present in both lupin seeds and seedlings. PMID:16662492

  19. Riboswitch structure: an internal residue mimicking the purine ligand

    PubMed Central

    Delfosse, Vanessa; Bouchard, Patricia; Bonneau, Eric; Dagenais, Pierre; Lemay, Jean-François; Lafontaine, Daniel A.; Legault, Pascale

    2010-01-01

    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. PMID:20022916

  20. 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.

  1. Prebiotic syntheses of purines and pyrimidines

    NASA Astrophysics Data System (ADS)

    Basile, B.; Lazcano, A.; Oró, J.

    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-disubstituted 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.

  2. Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis.

    PubMed

    Zhao, Hong; Chiaro, Christopher R; Zhang, Limin; Smith, Philip B; Chan, Chung Yu; Pedley, Anthony M; Pugh, Raymond J; French, Jarrod B; Patterson, Andrew D; Benkovic, Stephen J

    2015-03-13

    Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome. PMID:25605736

  3. Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis*♦

    PubMed Central

    Zhao, Hong; Chiaro, Christopher R.; Zhang, Limin; Smith, Philip B.; Chan, Chung Yu; Pedley, Anthony M.; Pugh, Raymond J.; French, Jarrod B.; Patterson, Andrew D.; Benkovic, Stephen J.

    2015-01-01

    Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome. PMID:25605736

  4. 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.

  5. Role of long purine stretches in controlling the expression of genes associated with neurological disorders.

    PubMed

    Singh, Himanshu Narayan; Rajeswari, Moganty R

    2015-11-10

    Purine repeat sequences present in the human genome are known to act as hotspots for mutations leading to chromosomal imbalances. It is established that large purine repeats (PRs) form stable DNA triplex structure which can inhibit gene expression. Friedreich's ataxia (FRDA), the autosomal neurodegenerative disorder is the only human disease known so far, where a large purine (GAA) repeat in the FXN gene is known to inhibit the expression of frataxin protein. We explored the hidden purine repeats (PRn with n ≥ 200) if any, in the human genome to find out how they are associated with neurological disorders. The results showed 28 PRs, which are mostly restricted to the intronic regions. Interestingly, the transcriptome expression analysis of PR-carrying genes (PR-genes) revealed that most of them are down-regulated in neurological disorders (autism, Alzheimer's disease, schizophrenia, epilepsy, mental retardation, Parkinson's disease, brain tumor) as compared to that in healthy controls. The altered gene expression in brain disorders can be interpreted in terms of a possible expansion of purine repeats leading to formation of very stable DNA-triplex and/or alleviation of the repair enzymes and/or other unknown cellular factors. Interactome analysis identified four PR-genes in signaling pathways whose dysregulation is correlated directly with pathogenesis: GRK5 and KLK6 in Alzheimer's disease; FGF14 in craniosynostosis, mental retardation and FLT1 in neuroferritinopathy. By virtue of being mutational hotspots and their ability to form DNA-triplex, purine repeats in genome disturb the genome integrity and interfere with the transcriptional regulation. However, validation of the disease linkage of PR-genes can be validated using knock-out techniques. PMID:26149656

  6. [BLOOD AND CEREBROSPINAL FLUID PURINES IN PREGNANT].

    PubMed

    Oreshnikov, E V; Oreshnikov, S F

    2015-01-01

    The research includes 88 pregnant women, that had their purine basis and malondialdehyde in water thermocoagulate extract of venous blood and cerebrospinal fluid examined (along with common standards clinical-laboratory tests) before the spinal anesthesia for the caesarian section was provided It was detected that preeclampsy and HELLP-syndine feature the increased adenine guanine hypoxantine and uric acid levels in cerebrospinal fluid, as well as increased concentrations of blood malondyaldehyde (higher than upper normal level), accompany with the increased hemotaencephalic barrier permeability for adenine, guanine and hypoxantine. It's demonstrated that level of guanine in blood serum can be used as a prognostic factor of spinal anesthesia quality in obstetrics. It is supposed to examine purine levels in pregnant women not only in blood but also in cere brospinal fluid. PMID:26596029

  7. Targeting Purine and Pyrimidine Metabolism in Human Apicomplexan Parasites

    PubMed Central

    Hyde, John E.

    2009-01-01

    Synthesis de novo, acquisition by salvage and interconversion of purines and pyrimidines represent the fundamental requirements for their eventual assembly into nucleic acids as nucleotides and the deployment of their derivatives in other biochemical pathways. A small number of drugs targeted to nucleotide metabolism, by virtue of their effect on folate biosynthesis and recycling, have been successfully used against apicomplexan parasites such as Plasmodium and Toxoplasma for many years, although resistance is now a major problem in the prevention and treatment of malaria. Many targets not involving folate metabolism have also been explored at the experimental level. However, the unravelling of the genome sequences of these eukaryotic unicellular organisms, together with increasingly sophisticated molecular analyses, opens up possibilities of introducing new drugs that could interfere with these processes. This review examines the status of established drugs of this type and the potential for further exploiting the vulnerability of apicomplexan human pathogens to inhibition of this key area of metabolism. PMID:17266529

  8. Purine and pyrimidine excretion in psoriasis

    PubMed Central

    Simmonds, H. A.; Bowyer, A.

    1974-01-01

    1 Urinary purine excretion has been investigated in two healthy controls and two patients with psoriasis, one a hyperuricaemic, one a normouricaemic. No difference was detected between the patients and controls. Therapy with allopurinol effectively lowered blood and urinary uric acid levels and produced a deficit in total urinary oxypurine excretion in both controls and patients with psoriasis. The concomitant increase in xanthine excretion was greater than the increase in hypoxanthine excretion and xanthine/hypoxanthine ratios (average 0.70 and 1.0 prior to therapy) were increased by allopurinol to an average of 3.0 and 3.8 respectively in the two groups. Allopurinol also reduced the excretion of 8-hydroxy-7-methyl guanine but no effect on the excretion levels of other minor purine bases was noted. 2 Allopurinol was metabolized similarly by both patients and controls, 84% of the administered allopurinol being accounted for as urinary metabolites. 74% of the drug in the urine was excreted as oxipurinol, 26% as unchanged allopurinol plus allopurinol riboside, the remainder being oxipurinol riboside. 3 Pseudouridine excretion in 25 healthy controls was 86.5 ± 17.8 mg/24 hours. Pseudouridine excretion was not excessive in the patients with psoriasis and was not altered by allopurinol therapy. 4 No abnormality or difference in purine or pyrimidine excretion in either patient was detected prior to or during therapy which could be related to the epidermal lesion. PMID:22454896

  9. 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. PMID:26809224

  10. 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.

  11. Purines in neurite growth and astroglia activation.

    PubMed

    Heine, Claudia; Sygnecka, Katja; Franke, Heike

    2016-05-01

    The mammalian nervous system is a complex, functional network of neurons, consisting of local and long-range connections. Neuronal growth is highly coordinated by a variety of extracellular and intracellular signaling molecules. Purines turned out to be an essential component of these processes. Here, we review the current knowledge about the involvement of purinergic signaling in the regulation of neuronal development. We particularly focus on its role in neuritogenesis: the formation and extension of neurites. In the course of maturation mammals generally lose their ability to regenerate the central nervous system (CNS) e.g. after traumatic brain injury; although, spontaneous regeneration still occurs in the peripheral nervous system (PNS). Thus, it is crucial to translate the knowledge about CNS development and PNS regeneration into novel approaches to enable neurons of the mature CNS to regenerate. In this context we give a general overview of growth-inhibitory and growth-stimulatory factors and mechanisms involved in neurite growth. With regard to neuronal growth, astrocytes are an important cell population. They provide structural and metabolic support to neurons and actively participate in brain signaling. Astrocytes respond to injury with beneficial or detrimental reactions with regard to axonal growth. In this review we present the current knowledge of purines in these glial functions. Moreover, we discuss organotypic brain slice co-cultures as a model which retains neuron-glia interactions, and further presents at once a model for CNS development and regeneration. In summary, the purinergic system is a pivotal factor in neuronal development and in the response to injury. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'. PMID:26498067

  12. 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

  13. Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

    SciTech Connect

    Pilch, D.S.; Shafer, R.H. ); Levenson, C. )

    1991-06-25

    The authors have investigated the structure and physical chemistry of the d(C{sub 3}T{sub 4}C{sub 3}){center dot}2(d(G{sub 3}A{sub 4}G{sub 3})) triple helix by polyacrylamide gel electrophoresis (PAGE), {sup 1}H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl{sub 2} at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur{center dot}pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the puring strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 C, depending on the DNA concentration. This marked enhancement in stability, coupled with the lack of an acidic pH requirement, suggests that pur-pur-pyr triplexes are appealing choices for use in applications involving oligonucleotide targeting of duplex DNA in vitro and in vivo.

  14. 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.

  15. Arginylation regulates purine nucleotide biosynthesis by enhancing the activity of phosphoribosyl pyrophosphate synthase.

    PubMed

    Zhang, Fangliang; Patel, Devang M; Colavita, Kristen; Rodionova, Irina; Buckley, Brian; Scott, David A; Kumar, Akhilesh; Shabalina, Svetlana A; Saha, Sougata; Chernov, Mikhail; Osterman, Andrei L; Kashina, Anna

    2015-01-01

    Protein arginylation is an emerging post-translational modification that targets a number of metabolic enzymes; however, the mechanisms and downstream effects of this modification are unknown. Here we show that lack of arginylation renders cells vulnerable to purine nucleotide synthesis inhibitors and affects the related glycine and serine biosynthesis pathways. We show that the purine nucleotide biosynthesis enzyme PRPS2 is selectively arginylated, unlike its close homologue PRPS1, and that arginylation of PRPS2 directly facilitates its biological activity. Moreover, selective arginylation of PRPS2 but not PRPS1 is regulated through a coding sequence-dependent mechanism that combines elements of mRNA secondary structure with lysine residues encoded near the N-terminus of PRPS1. This mechanism promotes arginylation-specific degradation of PRPS1 and selective retention of arginylated PRPS2 in vivo. We therefore demonstrate that arginylation affects both the activity and stability of a major metabolic enzyme. PMID:26175007

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

    PubMed Central

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

    2012-01-01

    SUMMARY 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. PMID

  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. PMID:24923662

  18. Cobalamin inactivation decreases purine and methionine synthesis in cultured lymphoblasts.

    PubMed

    Boss, G R

    1985-07-01

    The megaloblastic anemia of cobalamin deficiency appears secondary to decreased methionine synthetase activity. Decreased activity of this enzyme should cause 5-methyltetrahydrofolate to accumulate intracellularly, and consequently, decrease purine and DNA synthesis; this is the basis of the "methylfolate trap" hypothesis of cobalamin deficiency. However, only some of the clinical and biochemical manifestations of cobalamin deficiency can be explained by the methylfolate trap. We investigated cobalamin deficiency by treating cultured human lymphoblasts with N2O since N2O inhibits methionine synthetase activity by inactivating cobalamin. We found that 4 h of N2O exposure reduced rates of methionine synthesis by 89%. Rates of purine synthesis were not significantly reduced by N2O when folate and methionine were present at 100 microM in the medium; however, at the physiologic methionine concentration of 10 microM, N2O decreased rates of purine synthesis by 33 and 57% in the presence of 100 microM folate and in the absence of folate, respectively. The dependency of rates of purine synthesis on methionine availability would be expected in cells with restricted methionine synthetic capacity because methionine is the immediate precursor of S-adenosylmethionine, a potent inhibitor of 5-methyltetrahydrofolate synthesis; methionine serves as a source of formate for purine synthesis; and rates of purine synthesis are dependent on the intracellular availability of essential amino acids. We conclude that cobalamin inactivation decreases purine synthesis by both methylfolate trapping and reduction of intracellular methionine synthesis. PMID:2862163

  19. 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

  20. 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. PMID:19767584

  1. 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. PMID:24940702

  2. 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

  3. Synthetic strategies toward carbocyclic purine-pyrimidine hybrid nucleosides.

    PubMed

    Sadler, Joshua M; Mosley, Sylvester L; Dorgan, Kathleen M; Zhou, Zhaohui Sunny; Seley-Radtke, Katherine L

    2009-08-01

    The blending of key structural features from the purine and pyrimidine nucleobase scaffolds gives rise to a new class of hybrid nucleosides. The purine-pyrimidine hybrid nucleosides can be viewed as either N-3 ribosylated purines or 5,6-disubstituted pyrimidines, thus recognition by both purine- and pyrimidine-metabolizing enzymes is possible. Given the increasing reports of the development of resistance in many enzymatic systems, a drug that could be recognized by more than one enzyme could prove highly advantageous in overcoming resistance mechanisms related to binding site mutations. In that regard, the design, synthesis and results of preliminary biological activity for a series of carbocyclic uracil derivatives with either a fused imidazole or thiazole ring are presented herein. PMID:19592260

  4. Synthetic Strategies Toward Carbocyclic Purine-Pyrimidine Hybrid Nucleosides

    PubMed Central

    Sadler, Joshua M.; Mosley, Sylvester L.; Dorgan, Kathleen M.; Zhou, Zhaohui Sunny; Seley-Radtke, Katherine L.

    2009-01-01

    The blending of key structural features from the purine and pyrimidine nucleobase scaffolds gives rise to a new class of hybrid nucleosides. The purine-pyrimidine hybrid nucleosides can be viewed as either N-3 ribosylated purines or 5,6-disubstituted pyrimidines, thus recognition by both purine- and pyrimidine-metabolizing enzymes is possible. Given the increasing reports of the development of resistance in many enzymatic systems, a drug that could be recognized by more than one enzyme could prove highly advantageous in overcoming resistance mechanisms related to binding site mutations. In that regard, the design, synthesis and results of preliminary biological activity for a series of carbocyclic uracil derivatives with either a fused imidazole or thiazole ring are presented herein. PMID:19592260

  5. Purines and pyrimidines in sediments from lake erie.

    PubMed

    Van Der Velden, W; Schwartz, A W

    1974-08-23

    Quantitative analyses of purines and pyrimidines in sequential sections of cores from the central and eastern basins of Lake Erie show steeply increasing concentrations in the youngest sediments. This may be related to increased loading of nutrients and recent cultural eutrophication of the lake. The purine and pyrimidine distributions suggest the operation of a specific degradative process for uracil at an extremely early stage in, or prior to, sediment formation. PMID:17736373

  6. Regulation of Purine Metabolism in Intact Leaves of Coffea arabica.

    PubMed Central

    Nazario, G. M.; Lovatt, C. J.

    1993-01-01

    The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions. PMID:12232012

  7. 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.

  8. People with Easier to Pronounce Names Promote Truthiness of Claims

    PubMed Central

    Newman, Eryn J.; Sanson, Mevagh; Miller, Emily K.; Quigley-McBride, Adele; Foster, Jeffrey L.; Bernstein, Daniel M.; Garry, Maryanne

    2014-01-01

    When people make judgments about the truth of a claim, related but nonprobative information rapidly leads them to believe the claim–an effect called “truthiness” [1]. Would the pronounceability of others’ names also influence the truthiness of claims attributed to them? We replicated previous work by asking subjects to evaluate people’s names on a positive dimension, and extended that work by asking subjects to rate those names on negative dimensions. Then we addressed a novel theoretical issue by asking subjects to read that same list of names, and judge the truth of claims attributed to them. Across all experiments, easily pronounced names trumped difficult names. Moreover, the effect of pronounceability produced truthiness for claims attributed to those names. Our findings are a new instantiation of truthiness, and extend research on the truth effect as well as persuasion by showing that subjective, tangential properties such as ease of processing can matter when people evaluate information attributed to a source. PMID:24586368

  9. Purines and sensory neuropeptides in human asthma.

    PubMed

    Karlsson, J A

    1987-01-01

    Mediators acting on different cells in the lung may produce features of asthma such as bronchoconstriction, plasma leakage from the tracheobronchial microcirculation and mucus secretion. The clinical effectiveness of anticholinergic agents has stimulated the search for mediators other than acetyolcholine and the hope that specific antagonists would improve asthma therapy. The purine, nucleoside adenosine, produces certain asthma-like signs such as bronchoconstriction in asthmatics. Studies with theophylline and nonadenosine-blocking bronchodilator xanthines have, however, demonstrated that adenosine is unlikely to be an asthma mediator, although it may still possess significant extrapulmonary actions. Sensory nerves within the lung show immunoreactivity to a wide variety of peptides, including substance P and other tachykinins. Tachykinins produce bronchoconstriction and plasma extravasation in guinea-pig and rat lungs. In asthmatic subjects, nebulized neurokinin A reduces specific airways conductance. Inhalation of capsaicin, which presumably acts through stimulation of chemosensitive afferent C-fibres, produces cough and a transient upper airway constriction. Elucidation of a role in asthma must await the development of a clinically useful tachykinin antagonist. Accumulating data seems to indicate that asthma pathology is caused by released substances acting in conjunction on target cells in the lung. Functional antagonism, rather than inhibition of a single mediator, thus appears to be essential for clinically effective antiasthma drugs. PMID:2822185

  10. 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.

  11. A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani.

    PubMed

    Martin, Jessica L; Yates, Phillip A; Boitz, Jan M; Koop, Dennis R; Fulwiler, Audrey L; Cassera, Maria Belen; Ullman, Buddy; Carter, Nicola S

    2016-07-01

    Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non-permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine-containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long-term survival of Leishmania in a purine-scarce environment. PMID:27062185

  12. Polyamine effects on purine-purine-pyrimidine triple helix formation by phosphodiester and phosphorothioate oligodeoxyribonucleotides.

    PubMed Central

    Musso, M; Van Dyke, M W

    1995-01-01

    Utilization of oligodeoxyribonucleotides to inhibit specific gene transcription in vivo (antigene strategy) requires the efficient formation of triple helices under physiological conditions. However, pyrimidine-motif triplexes are not favored at physiological pH, and physiological concentrations of potassium cations hamper purine-motif triplex formation. Here we investigated the effects of polyamines on promoting triplex formation by G/T-rich oligodeoxyribonucleotides containing either phosphodiester or a diastereomeric mixture of phosphorothioate linkages. Compared with Mg2+, equimolar concentrations of polyamines greatly facilitated purine-motif triplex formation with the following order of effectiveness: spermine > spermidine > putrescine. At low polyamine concentrations, phosphorothioate oligonucleotides were better at triplex formation than the corresponding phosphodiester oligonucleotides. Kinetic studies indicated that polyamines facilitated triplex formation by increasing the rate of oligonucleotide-duplex DNA association. However, triplex accumulation with either oligonucleotide was still low under physiological conditions (140 mM K+, 10 mM Mg2+, 1 mM spermine). The inhibitory effects of K+ could be partially overcome with high concentrations of Mg2+ or spermine, with phosphodiester oligonucleotides being better able to form triplexes than phosphorothioates under these conditions. Images PMID:7610062

  13. 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.

  14. Transmural distribution of extracellular purines in isolated guinea pig heart.

    PubMed Central

    Zhu, Q Y; Headrick, J P; Berne, R M

    1991-01-01

    The purine adenosine appears to be involved in regulation of coronary vascular tone. Little is known concerning the levels and distribution of adenosine and related purines in the extracellular fluid of the heart. We have measured epicardial and endocardial levels of adenosine, inosine, hypoxanthine, AMP, and IMP in isolated constant flow perfused guinea pig hearts by using a recently developed technique with porous nylon sampling discs. Venous effluent purine levels were also measured. Concentrations of all purines measured, excluding IMP, were significantly higher in endocardial fluid samples than in epicardial fluid samples (P less than 0.05). Conversely, IMP levels were significantly lower in endocardial than in epicardial samples. The magnitude of the endocardial/epicardial ratios for adenosine, inosine, hypoxanthine, AMP, and IMP were approximately 12:1, 4:1, 5:1, 4:1, and 1:2, respectively. To assess cellular damage, lactate dehydrogenase activity was measured in all fluid samples and was not significantly different in endocardial and epicardial fluid. These data support the existence of significant transmural gradients for extracellular purine levels in crystalloid perfused guinea pig hearts. Transmural differences in vasoactive adenosine levels may be partially due to the greater endocardial oxygen consumption and metabolism and may be involved in maintaining relatively high subendocardial blood flows in the face of high intramyocardial pressures. Images PMID:1988961

  15. Double functionalization of carbon nanotubes with purine and pyrimidine derivatives.

    PubMed

    Singh, Prabhpreet; Ménard-Moyon, Cécilia; Battigelli, Alessia; Toma, Francesca Maria; Raya, Jesus; Kumar, Jitendra; Nidamanuri, Nagapradeep; Verma, Sandeep; Bianco, Alberto

    2013-07-01

    Herein, we have developed a synthetic strategy for the covalent double functionalization of single-walled carbon nanotubes (SWCNTs) with a combination of purine-pyrimidine and purine-purine nucleobase systems. The nucleobases were introduced on the sidewall of oxidized SWCNTs through 1,3-dipolar cycloaddition and by amidation of the carboxylic acids located at the tips and defect sites of the nanotubes. The new nanohybrids were characterized by transmission electron microscopy, thermogravimetric analysis, FTIR and Raman spectroscopy, magic-angle spinning NMR spectroscopy, and Kaiser test. The nucleobase/SWCNT conjugates can be envisaged for the modulation of the interactions with nucleic acids by means of base pairing, thereby opening new possibilities in the development of DNA/CNT nanobioconjugates. PMID:23703975

  16. Phylogenetic Analysis and Comparative Genomics of Purine Riboswitch Distribution in Prokaryotes

    PubMed Central

    Singh, Payal; Sengupta, Supratim

    2012-01-01

    Riboswitches are regulatory RNA that control gene expression by undergoing conformational changes on ligand binding. Using phylogenetic analysis and comparative genomics we have been able to identify the class of genes/operons regulated by the purine riboswitch and obtain a high-resolution map of purine riboswitch distribution across all bacterial groups. In the process, we are able to explain the absence of purine riboswitches upstream to specific genes in certain genomes. We also identify the point of origin of various purine riboswitches and argue that not all purine riboswitches are of primordial origin, and that some purine riboswitches must have originated after the divergence of certain Firmicute orders in the course of evolution. Our study also reveals the role of horizontal transfer events in accounting for the presence of purine riboswitches in some gammaproteobacterial species. Our work provides significant insights into the origin, distribution and regulatory role of purine riboswitches in prokaryotes. PMID:23170063

  17. Acquired Hemochromatosis with Pronounced Pigment Deposition of the Upper Eyelids

    PubMed Central

    Morrison, Brian; Hu, Shasa

    2013-01-01

    Hemochromatosis may be classified into two groups: primary (hereditary) or secondary (acquired). The acquired type most commonly occurs after massive intake of iron supplements or blood transfusions and is also known as transfusional iron overload. In the past, hemochromatosis was usually recognized at an advanced stage by the classic triad of hyperpigmentation, diabetes mellitus (“bronze diabetes”), and hepatic cirrhosis. Cutaneous hyperpigmentation is present in 70 percent of patients due to two different mechanisms: (1) hemosiderin deposition resulting in diffuse, slate-gray darkening and (2) increased production of melanin in the epidermis. A 47-year-old woman who receives regular transfusions due to low iron and chronic, unresolving anemia and who subsequently developed pronounced hyperpigmentation of the upper eyelids is described. The presentation, diagnosis, pathogenesis, and treatment options of hyperpigmentation due to secondary hemochromatosis are discussed. PMID:24155994

  18. Inborn errors of purine metabolism: clinical update and therapies.

    PubMed

    Balasubramaniam, Shanti; Duley, John A; Christodoulou, John

    2014-09-01

    Inborn errors of purine metabolism exhibit broad neurological, immunological, haematological and renal manifestations. Limited awareness of the phenotypic spectrum, the recent descriptions of newer disorders and considerable genetic heterogeneity, have contributed to long diagnostic odysseys for affected individuals. These enzymes are widely but not ubiquitously distributed in human tissues and are crucial for synthesis of essential nucleotides, such as ATP, which form the basis of DNA and RNA, oxidative phosphorylation, signal transduction and a range of molecular synthetic processes. Depletion of nucleotides or accumulation of toxic intermediates contributes to the pathogenesis of these disorders. Maintenance of cellular nucleotides depends on the three aspects of metabolism of purines (and related pyrimidines): de novo synthesis, catabolism and recycling of these metabolites. At present, treatments for the clinically significant defects of the purine pathway are restricted: purine 5'-nucleotidase deficiency with uridine; familial juvenile hyperuricaemic nephropathy (FJHN), adenine phosphoribosyl transferase (APRT) deficiency, hypoxanthine phosphoribosyl transferase (HPRT) deficiency and phosphoribosyl-pyrophosphate synthetase superactivity (PRPS) with allopurinol; adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies have been treated by bone marrow transplantation (BMT), and ADA deficiency with enzyme replacement with polyethylene glycol (PEG)-ADA, or erythrocyte-encapsulated ADA; myeloadenylate deaminase (MADA) and adenylosuccinate lyase (ADSL) deficiencies have had trials of oral ribose; PRPS, HPRT and adenosine kinase (ADK) deficiencies with S-adenosylmethionine; and molybdenum cofactor deficiency of complementation group A (MOCODA) with cyclic pyranopterin monophosphate (cPMP). In this review we describe the known inborn errors of purine metabolism, their phenotypic presentations, established diagnostic methodology and recognised

  19. Beyond Crystallography: Investigating the Conformational Dynamics of the Purine Riboswitch

    NASA Astrophysics Data System (ADS)

    Stoddard, Colby D.; Batey, Robert T.

    Riboswitches are structured elements located in the 5'-untranslated regions of numerous bacterial mRNAs that serve to regulate gene expression via their ability to specifically bind metabolites. The purine riboswitch ligand-binding domain has emerged as an important model system for investigating the relationship between RNA structure and function. Directed by NMR and crystallographically generated structures of this RNA, a variety of biophysical and biochemical techniques have been utilized to understand its dynamic nature. In this review, we describe these various approaches and what they reveal about the purine riboswitch.

  20. The Purine-Utilizing Bacterium Clostridium acidurici 9a: A Genome-Guided Metabolic Reconsideration

    PubMed Central

    Hartwich, Katrin; Poehlein, Anja; Daniel, Rolf

    2012-01-01

    Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists of one chromosome (3,105,335 bp) and one small circular plasmid (2,913 bp). The lack of candidate genes encoding glycine reductase indicates that C. acidurici 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of C. acidurici 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as C. acetobutylicum, C. saccharolyticum, and C. beijerinckii. The only amino acid that can be degraded by C. acidurici is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by C. acidurici but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of C. acidurici towards bacitracin, acriflavine and azaleucine was experimentally confirmed. PMID:23240052

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. Pronounced kidney hypoxia precedes albuminuria in type 1 diabetic mice.

    PubMed

    Franzén, Stephanie; Pihl, Liselotte; Khan, Nadeem; Gustafsson, Håkan; Palm, Fredrik

    2016-05-01

    Intrarenal tissue hypoxia has been proposed as a unifying mechanism for the development of chronic kidney disease, including diabetic nephropathy. However, hypoxia has to be present before the onset of kidney disease to be the causal mechanism. To establish whether hypoxia precedes the onset of diabetic nephropathy, we implemented a minimally invasive electron paramagnetic resonance oximetry technique using implanted oxygen sensing probes for repetitive measurements of in vivo kidney tissue oxygen tensions in mice. Kidney cortex oxygen tensions were measured before and up to 15 days after the induction of insulinopenic diabetes in male mice and compared with normoglycemic controls. On day 16, urinary albumin excretions and conscious glomerular filtration rates were determined to define the temporal relationship between intrarenal hypoxia and disease development. Diabetic mice developed pronounced intrarenal hypoxia 3 days after the induction of diabetes, which persisted throughout the study period. On day 16, diabetic mice had glomerular hyperfiltration, but normal urinary albumin excretion. In conclusion, intrarenal tissue hypoxia in diabetes precedes albuminuria thereby being a plausible cause for the onset and progression of diabetic nephropathy. PMID:26936871

  6. 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.

  7. Copper-catalyzed synthesis of purine-fused polycyclics.

    PubMed

    Qu, Gui-Rong; Liang, Lei; Niu, Hong-Ying; Rao, Wei-Hao; Guo, Hai-Ming; Fossey, John S

    2012-09-01

    A novel protocol for a Cu-catalyzed direct C((sp(2)))-H activation/intramolecular amination reaction of 6-anilinopurine nucleosides has been developed. This approach provides a new access to a variety of multiheterocyclic compounds from purine compounds via Cu-catalyzed intramolecular N-H bond tautomerism which are endowed with fluorescence. PMID:22900616

  8. 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. PMID:21880467

  9. 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. PMID:24856135

  10. Solution structure of ligands involved in purine salvage pathway.

    PubMed

    Karnawat, Vishakha; Puranik, Mrinalini

    2015-12-01

    Analogues of intermediates involved in the purine salvage pathway can be exploited as potential drug molecules against enzymes of protozoan parasites. To develop such analogues we need knowledge of the solution structures, predominant tautomer at physiological pH and protonation-state of the corresponding natural ligand. In this regard, we have employed ultraviolet resonance Raman spectroscopy (UVRR) in combination with density functional theory (DFT) to study the solution structures of two relatively unexplored intermediates, 6-phosphoryl IMP (6-pIMP) and succinyl adenosine-5'-monophosphate (sAMP), of purine salvage pathway. These molecules are intermediates in a two step enzymatic process that converts inosine-5'-monpophosphate (IMP) to adenosine-5'-monophosphate (AMP). Experimental data on the molecular structure of these ligands is lacking. We report UVRR spectra of these two ligands, obtained at an excitation wavelength of 260 nm. Using isotope induced shifts and DFT calculations we assigned observed spectra to computed normal modes. We find that sAMP exists as neutral species at physiological pH and the predominant tautomer in solution bears proton at N10 position of purine ring. Though transient in solution, 6-pIMP is captured in the enzyme-bound form. This work provides the structural information of these ligands in solution state at physiological pH. We further compare these structures with the structures of AMP and IMP. Despite the presence of similar purine rings in AMP and sAMP, their UVRR spectra are found to be very different. Similarly, though the purine ring in 6-pIMP resembles that of IMP, UVRR spectra of the two molecules are distinct. These differences in the vibrational spectra provide direct information on the effects of exocyclic groups on the skeletal structures of these molecules. Our results identify key bands in the vibrational spectra of these ligands which may serve as markers of hydrogen bonding interactions upon binding to the active

  11. Cloning, expression and preliminary crystallographic studies of the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni.

    PubMed

    Pereira, Humberto M; Cleasby, Anne; Pena S, Sérgio D J; Franco G, Glória R; Garratt, Richard C

    2003-06-01

    The parasite Schistosoma mansoni, unlike its mammalian hosts, lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. The gene encoding one enzyme of this pathway, purine nucleoside phosphorylase from S. mansoni (SmPNP) was identified, fully sequenced and cloned into the bacterial expression vector pMAL c2G to produce a protein in fusion with maltose-binding protein. The recombinant fusion protein was expressed at high levels and was purified in a single step by amylose resin affinity chromatography. After factor Xa cleavage, SmPNP was purified using a cation-exchange column and crystallized by hanging-drop vapour diffusion using polyethylene glycol 1500 as precipitant in the presence of 20% glycerol in acetate buffer. The use of the non-detergent sulfobetaine 195 (NDSB 195) as an additive had a marked effect on the size of the resulting crystals. Two data sets were obtained, one from a crystal grown in the absence of NDSB 195 and one from a crystal grown in its presence. The crystals are isomorphous and belong to the space group P2(1)2(1)2(1). It is intended to use the structures in the discovery and development of specific inhibitors of SmPNP. PMID:12777786

  12. Polypurine sequences within a downstream exon function as a splicing enhancer

    SciTech Connect

    Tanaka, Kenji; Watakabe, Akiya; Shimura, Yoshiro

    1994-02-01

    We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin {mu} gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin {mu} gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection. 50 refs., 7 figs., 2 tabs.

  13. 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.

  14. Purine Synthesis and Catabolism in Soybean Seedlings 1

    PubMed Central

    Polayes, Deborah A.; Schubert, Karel R.

    1984-01-01

    The ureides, allantoin and allantoic acid, are the major nitrogenous substances transported within the xylem of N2-fixing soybeans (Glycine max L. Merr. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobium or grown in the presence of 10 millimolar nitrate. The patterns of activity for uricase and allantoinase, enzymes involved in ureide synthesis, were positively correlated with the accumulation of ureides in the roots and cotyledons. Allopurinol and azaserine inhibited ureide production in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4-day-old seedlings with [14C]serine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3- and 4-day-old roots was probably the cotyledons. The inhibition of ureide accumulation by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with [14C]serine suggested that the roots had acquired the capability to synthesize ureides via de novo synthesis of purines. These data indicate that both de novo purine synthesis and nucleotide breakdown are involved in the production of ureides in young soybean seedlings. PMID:16663743

  15. Experimental and theoretical dipole moments of purines in their ground and lowest excited singlet states

    NASA Astrophysics Data System (ADS)

    Aaron, Jean-Jacques; Diabou Gaye, Mame; Párkányi, Cyril; Cho, Nam Sook; Von Szentpály, László

    1987-01-01

    The ground-state dipole moments of seven biologically important purines (purine, 6-chloropurine, 6-mercaptopurine, hypoxanthine, theobromine, theophylline and caffeine) were determined at 25°C in acetic acid (all the above compounds with the exception of purine) and in ethyl acetate (purine, theophylline and caffeine). Because of its low solubility, it was not possible to measure the dipole moment of uric acid. The first excited singlet-state dipole moments were obtained on the basis of the Bakhshiev and Chamma—Viallet equations using the variation of the Stokes shift with the solvent dielectric constant-refractive index term. The theoretical dipole moments for all the purines listed above and including uric acid were calculated by combining the use of the PPP (π-LCI-SCF-MO) method for the π-contribution to the overall dipole moment with the σ-contribution obtained as a vector sum of the σbond moments and group moments. The experimental and theoretical values were compared with the data available in the literature for some of the purines under study. For several purines, the calculations were carried out for different tautomeric forms. Excited singlet-state dipole moments are smaller than the ground-state values by 0.8 to 2.2 Debye units for all purines under study with the exception of 6-chloropurine. The effects of the structure upon the ground- and excited-state dipole moments of the purines are discussed.

  16. 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. PMID:19353717

  17. 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.

  18. Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: cellular models of de novo purine biosynthesis deficiency disorders.

    PubMed

    Duval, Nathan; Luhrs, Kyleen; Wilkinson, Terry G; Baresova, Veronika; Skopova, Vaclava; Kmoch, Stanislav; Vacano, Guido N; Zikanova, Marie; Patterson, David

    2013-03-01

    Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic

  19. 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.

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

    PubMed Central

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

    2014-01-01

    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 P. 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. PMID:25424653

  1. 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.

  2. 40 CFR 721.4685 - Substituted purine metal salt (generic name).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4685 Substituted purine metal salt (generic name). (a) Chemical... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted purine metal salt...

  3. 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. PMID:25424653

  4. 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.

  5. 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

  6. Purine derivative excretion in dairy cows: endogenous excretion and the effect of exogenous nucleic acid supply.

    PubMed

    Gonzalez-Ronquillo, M; Balcells, J; Guada, J A; Vicente, F

    2003-04-01

    An experiment was conducted with dairy cows to study the partitioning of excreted purine derivatives between urine and milk and to quantify the endogenous contribution following the isotopic labeling of microbial purine bases. Three lactating cows in their second lactation that had been cannulated in the rumen and the duodenum were fed a mixed diet (48:52, roughage/concentrate ratio) distributed in equal fractions every 2 h, and duodenal flow of purine bases was determined by the dual-phase marker system. Nitrogen-15 was infused continuously into the rumen to label microbial purine bases, and the endogenous fraction was determined from the isotopic dilution in urinary purine derivatives. Urinary and milk recovery of duodenal purine bases were estimated at early (wk 10) and late (wk 33) lactation by the duodenal infusion of incremental doses (75 and 150 mmol purine bases/d) of RNA from Torula yeast. Each period was 6 d, with RNA being infused during the last 4 d, followed by measurement of the flow of purine bases to the duodenum. The isotope dilution of purine derivatives in urine samples confirmed the presence of an endogenous fraction (512 +/- 36.43 micromol/W0.75 or 56.86 mmol/d) amounting to 26 +/- 3.8% of total renal excretion. Total excretion of purine derivatives in urine plus milk was linearly related to the duodenal input of purine bases, but the slopes differed (P < 0.005) between lactation stages resulting in a lower equimolar recovery in early (y = 58.86 (+/-3.89) +0.56 (+/-0.0164) x; r = 0.90) than late lactation (y = 58.86 (+/-3.89) + 0.70 (+/-0.046) x; r = 0.80). Excretion of purine derivatives through milk represented a minimum fraction of total excretion but responded significantly to the duodenal input of purine bases. No differences between lactation stages were detected, and variations in milk yield did modify significantly the amount of purine derivatives excreted through the milk. PMID:12741553

  7. [Uric acid and purine plasma levels as plausible markers for placental dysfunction in pre-eclampsia].

    PubMed

    Escudero, Carlos; Bertoglia, Patricio; Muñoz, Felipe; Roberts, James M

    2013-07-01

    Uric acid is the final metabolite of purine break down, such as ATP, ADP, AMP, adenosine, inosine and hypoxanthine. The metabolite has been used broadly as a renal failure marker, as well as a risk factor for maternal and neonatal morbidity during pre-eclamptic pregnancies. High purine levels are observed in pre-eclamptic pregnancies, but the sources of these purines are unknown. However, there is evidence that pre-eclampsia (mainly severe pre-eclampsia) is associated with an increased release of cellular fragments (or microparticles) from the placenta to the maternal circulation. These in fact could be the substrate for purine metabolism. Considering this background, we propose that purines and uric acid are part of the same physiopathological phenomenon in pre-eclampsia (i.e., placental dysfunction) and could become biomarkers for placental dysfunction and postnatal adverse events. PMID:24356738

  8. Chemoselective Multicomponent One-Pot Assembly of Purine Precursors in Water

    PubMed Central

    2010-01-01

    The recent development of a sequential, high-yielding route to activated pyrimidine nucleotides, under conditions thought to be prebiotic, is an encouraging step toward the greater goal of a plausible prebiotic pathway to RNA and the potential for an RNA world. However, this synthesis has led to a disparity in the methodology available for stepwise construction of the canonical pyrimidine and purine nucleotides. To address this problem, and further explore prebiotically accessible chemical systems, we have developed a high-yielding, aqueous, one-pot, multicomponent reaction that tethers masked-sugar moieties to prebiotically plausible purine precursors. A pH-dependent three-component reaction system has been discovered that utilizes key nucleotide synthons 2-aminooxazole and 5-aminoimidazoles, which allows the first divergent purine/pyrimidine synthesis to be proposed. Due to regiospecific aminoimidazole tethering, the pathway allows N9 purination only, thus suggesting the first prebiotically plausible mechanism for regiospecific N9 purination. PMID:21043502

  9. Interaction of purine bases and nucleosides with serum albumin

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Michnik, A.

    1997-06-01

    The proton NMR spectra of alkyl derivatives of adenine and adenosine have been studied. High-resolution (400 MHz) proton spectra were recorded at 300 K at increasing concentrations of serum albumin. The dependence of the chemical shifts and the line width of the individual spectral lines on the protein concentration provides some detailed information about the nature of the complexes between the purine derivatives and albumin. Comparison of data for the methylated and non-methylated purine bases and nucleosides indicates the formation of non-specific complexes with serum albumin. However, the presence of the ethyl group in 8-ethyl-9 N-methyladenine means that in the adenine derivative-serum albumin complex the ethyl chain preserves its dominant role in binding. An advantage of our model is that the π-π interaction between the adenine ring and the amino acids of the protein can be replaced by hydrophobic interaction in the case of complexation of the ethyl adenine derivative.

  10. Antiparasitic chemotherapy: tinkering with the purine salvage pathway.

    PubMed

    Datta, Alok Kumar; Datta, Rupak; Sen, Banibrata

    2008-01-01

    Distinguishable differences between infectine organisms and their respective hosts with respect to metabolism and macromolecular structure provide scopes for detailed characterization of target proteins and/or macromolecules as the focus for the development of selective inhibitors. In order to develop a rational approach to antiparasitic chemotherapy, finding differences in the biochemical pathways of the parasite with respect to the host it infects is therefore of primary importance. Like most parasitic protozoan, the genus Leishmania is an obligate auxotroph of purines and hence for requirement of purine bases depends on its own purine salvage pathways. Among various purine acquisition routes used by the parasite, the pathway involved in assimilation of adenosine nucleotide is unique and differs significantly in the extracellular form of the parasite (promastigotes) from its corresponding intracellular form (amastigotes). Adenosine kinase (AdK) is the gateway enzyme of this pathway and displays stage-specific activity pattern. Therefore, understanding the catalytic mechanism of the enzyme, its structural complexities and mode of its regulation have emerged as one of the major areas of investigation. This review, in general, discusses possible strategies to validate several purine salvage enzymes as targets for chemotherapeutic manipulation with special reference to adenosine kinase of Leishmania donovani. Systemic endotheliosis, commonly known as Kala-azar in India, is caused by the parasitic protozoon Leishmania donovani. The spread of leishmaniases follows the distribution of these vectors in the temperate, tropical and subtropical regions of the world leading to loss of thousands of human lives.' WHO has declared leishmaniasis among one of the six major diseases namely leishmaniasis, malaria, amoebiasis, filariasis, Chagas disease and schistosomiasis in its Special Programme for Research and Training in Tropical Diseases. Strategies for better prophylaxis and

  11. 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

  12. 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. PMID:25990537

  13. The Drosophila melanogaster ade5 gene encodes a bifunctional enzyme for two steps in the de novo purine synthesis pathway.

    PubMed Central

    O'Donnell, A F; Tiong, S; Nash, D; Clark, D V

    2000-01-01

    Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively. In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC. We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E. coli purC purine auxotroph. This cDNA encodes AIRc yet is unable to complement an E. coli purE mutant, suggesting functional differences between Drosophila and E. coli AIRc. In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis. In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies. Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated. Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated. Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs. PMID:10757766

  14. 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...

  15. Placental Hypomethylation Is More Pronounced in Genomic Loci Devoid of Retroelements

    PubMed Central

    Chatterjee, Aniruddha; Macaulay, Erin C.; Rodger, Euan J.; Stockwell, Peter A.; Parry, Matthew F.; Roberts, Hester E.; Slatter, Tania L.; Hung, Noelyn A.; Devenish, Celia J.; Morison, Ian M.

    2016-01-01

    The human placenta is hypomethylated compared to somatic tissues. However, the degree and specificity of placental hypomethylation across the genome is unclear. We assessed genome-wide methylation of the human placenta and compared it to that of the neutrophil, a representative homogeneous somatic cell. We observed global hypomethylation in placenta (relative reduction of 22%) compared to neutrophils. Placental hypomethylation was pronounced in intergenic regions and gene bodies, while the unmethylated state of the promoter remained conserved in both tissues. For every class of repeat elements, the placenta showed lower methylation but the degree of hypomethylation differed substantially between these classes. However, some retroelements, especially the evolutionarily younger Alu elements, retained high levels of placental methylation. Surprisingly, nonretrotransposon-containing sequences showed a greater degree of placental hypomethylation than retrotransposons in every genomic element (intergenic, introns, and exons) except promoters. The differentially methylated fragments (DMFs) in placenta and neutrophils were enriched in gene-poor and CpG-poor regions. The placentally hypomethylated DMFs were enriched in genomic regions that are usually inactive, whereas hypermethylated DMFs were enriched in active regions. Hypomethylation of the human placenta is not specific to retroelements, indicating that the evolutionary advantages of placental hypomethylation go beyond those provided by expression of retrotransposons and retrogenes. PMID:27172225

  16. Placental Hypomethylation Is More Pronounced in Genomic Loci Devoid of Retroelements.

    PubMed

    Chatterjee, Aniruddha; Macaulay, Erin C; Rodger, Euan J; Stockwell, Peter A; Parry, Matthew F; Roberts, Hester E; Slatter, Tania L; Hung, Noelyn A; Devenish, Celia J; Morison, Ian M

    2016-01-01

    The human placenta is hypomethylated compared to somatic tissues. However, the degree and specificity of placental hypomethylation across the genome is unclear. We assessed genome-wide methylation of the human placenta and compared it to that of the neutrophil, a representative homogeneous somatic cell. We observed global hypomethylation in placenta (relative reduction of 22%) compared to neutrophils. Placental hypomethylation was pronounced in intergenic regions and gene bodies, while the unmethylated state of the promoter remained conserved in both tissues. For every class of repeat elements, the placenta showed lower methylation but the degree of hypomethylation differed substantially between these classes. However, some retroelements, especially the evolutionarily younger Alu elements, retained high levels of placental methylation. Surprisingly, nonretrotransposon-containing sequences showed a greater degree of placental hypomethylation than retrotransposons in every genomic element (intergenic, introns, and exons) except promoters. The differentially methylated fragments (DMFs) in placenta and neutrophils were enriched in gene-poor and CpG-poor regions. The placentally hypomethylated DMFs were enriched in genomic regions that are usually inactive, whereas hypermethylated DMFs were enriched in active regions. Hypomethylation of the human placenta is not specific to retroelements, indicating that the evolutionary advantages of placental hypomethylation go beyond those provided by expression of retrotransposons and retrogenes. PMID:27172225

  17. A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

    PubMed Central

    Hastings, M L; Wilson, C M; Munroe, S H

    2001-01-01

    The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA. PMID:11421362

  18. 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

  19. 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

  20. Purine nucleoside modulation of functions of human lymphocytes.

    PubMed

    Priebe, T; Platsoucas, C D; Seki, H; Fox, F E; Nelson, J A

    1990-09-01

    The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by

  1. Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

    PubMed

    Gregus, Zoltán; Németi, Balázs

    2002-11-01

    The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP

  2. High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni

    PubMed Central

    Cameron, Andrew; Huynh, Steven; Scott, Nichollas E.; Frirdich, Emilisa; Apel, Dmitry; Foster, Leonard J.; Parker, Craig T.

    2015-01-01

    ABSTRACT Phenotypic variation is prevalent in the zoonotic pathogen Campylobacter jejuni, the leading agent of enterocolitis in the developed world. Heterogeneity enhances the survival and adaptive malleability of bacterial populations because variable phenotypes may allow some cells to be protected against future stress. Exposure to hyperosmotic stress previously revealed prevalent differences in growth between C. jejuni strain 81-176 colonies due to resistant or sensitive phenotypes, and these isolated colonies continued to produce progeny with differential phenotypes. In this study, whole-genome sequencing of isolated colonies identified allelic variants of two purine biosynthesis genes, purF and apt, encoding phosphoribosyltransferases that utilize a shared substrate. Genetic analyses determined that purF was essential for fitness, while apt was critical. Traditional and high-depth amplicon-sequencing analyses confirmed extensive intrapopulation genetic variation of purF and apt that resulted in viable strains bearing alleles with in-frame insertion duplications, deletions, or missense polymorphisms. Different purF and apt alleles were associated with various stress survival capabilities under several niche-relevant conditions and contributed to differential intracellular survival in an epithelial cell infection model. Amplicon sequencing revealed that intracellular survival selected for stress-fit purF and apt alleles, as did exposure to oxygen and hyperosmotic stress. Putative protein recognition direct repeat sequences were identified in purF and apt, and a DNA-protein affinity screen captured a predicted exonuclease that promoted the global spontaneous mutation rate. This work illustrates the adaptive properties of high-frequency genetic variation in two housekeeping genes, which influences C. jejuni survival under stress and promotes its success as a pathogen. PMID:26419875

  3. Sequestration-Mediated Downregulation of de Novo Purine Biosynthesis by AMPK.

    PubMed

    Schmitt, Danielle L; Cheng, Yun-Ju; Park, Junyong; An, Songon

    2016-07-15

    Dynamic partitioning of de novo purine biosynthetic enzymes into multienzyme compartments, purinosomes, has been associated with increased flux of de novo purine biosynthesis in human cells. However, we do not know of a mechanism by which de novo purine biosynthesis would be downregulated in cells. We have investigated the functional role of AMP-activated protein kinase (AMPK) in the regulation of de novo purine biosynthesis because of its regulatory action on lipid and carbohydrate biosynthetic pathways. Using pharmacological AMPK activators, we have monitored subcellular localizations of six pathway enzymes tagged with green fluorescent proteins under time-lapse fluorescence single-cell microscopy. We revealed that only one out of six pathway enzymes, formylglycinamidine ribonucleotide synthase (FGAMS), formed spatially distinct cytoplasmic granules after treatment with AMPK activators, indicating the formation of single-enzyme self-assemblies. In addition, subsequent biophysical studies using fluorescence recovery after photobleaching showed that the diffusion kinetics of FGAMS were slower when it localized inside the self-assemblies than within the purinosomes. Importantly, high-performance liquid chromatographic studies revealed that the formation of AMPK-promoted FGAMS self-assembly caused the reduction of purine metabolites in HeLa cells, indicating the downregulation of de novo purine biosynthesis. Collectively, we demonstrate here that the spatial sequestration of FGAMS by AMPK is a mechanism by which de novo purine biosynthesis is downregulated in human cells. PMID:27128383

  4. [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. PMID:27420623

  5. Probing the reactivity of singlet oxygen with purines

    PubMed Central

    Dumont, Elise; Grüber, Raymond; Bignon, Emmanuelle; Morell, Christophe; Moreau, Yohann; Monari, Antonio; Ravanat, Jean-Luc

    2016-01-01

    The reaction of singlet molecular oxygen with purine DNA bases is investigated by computational means. We support the formation of a transient endoperoxide for guanine and by classical molecular dynamics simulations we demonstrate that the formation of this adduct does not affect the B-helicity. We thus identify the guanine endoperoxide as a key intermediate, confirming a low-temperature nuclear magnetic resonance proof of its existence, and we delineate its degradation pathway, tracing back the preferential formation of 8-oxoguanine versus spiro-derivates in B-DNA. Finally, the latter oxidized 8-oxodGuo product exhibits an almost barrierless reaction profile, and hence is found, coherently with experience, to be much more reactive than guanine itself. On the contrary, in agreement with experimental observations, singlet-oxygen reactivity onto adenine is kinetically blocked by a higher energy transition state. PMID:26656495

  6. Basal Ganglia Dopamine Loss Due to Defect in Purine Recycling

    PubMed Central

    Egami, Kiyoshi; Yitta, Silaja; Kasim, Suhail; Lewers, J. Chris; Roberts, Rosalinda C.; Lehar, Mohamed; Jinnah, H. A.

    2007-01-01

    Several rare inherited disorders have provided valuable experiments of nature highlighting specific biological processes of particular importance to the survival or function of midbrain dopamine neurons. In both humans and mice, deficiency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) is associated with profound loss of striatal dopamine, with relative preservation of other neurotransmitters. In the current studies of knockout mice, no morphological signs of abnormal development or degeneration were found in an exhaustive battery that included stereological and morphometric measures of midbrain dopamine neurons, electron microscopic studies of striatal axons and terminals, and stains for degeneration or gliosis. A novel culture model involving HPRT-deficient dopaminergic neurons also exhibited significant loss of dopamine without a morphological correlate. These results suggest dopamine loss in HPRT deficiency has a biochemical rather than anatomical basis, and imply purine recycling to be a biochemical process of particular importance to the function of dopaminergic neurons. PMID:17374562

  7. Probing the reactivity of singlet oxygen with purines.

    PubMed

    Dumont, Elise; Grüber, Raymond; Bignon, Emmanuelle; Morell, Christophe; Moreau, Yohann; Monari, Antonio; Ravanat, Jean-Luc

    2016-01-01

    The reaction of singlet molecular oxygen with purine DNA bases is investigated by computational means. We support the formation of a transient endoperoxide for guanine and by classical molecular dynamics simulations we demonstrate that the formation of this adduct does not affect the B-helicity. We thus identify the guanine endoperoxide as a key intermediate, confirming a low-temperature nuclear magnetic resonance proof of its existence, and we delineate its degradation pathway, tracing back the preferential formation of 8-oxoguanine versus spiro-derivates in B-DNA. Finally, the latter oxidized 8-oxodGuo product exhibits an almost barrierless reaction profile, and hence is found, coherently with experience, to be much more reactive than guanine itself. On the contrary, in agreement with experimental observations, singlet-oxygen reactivity onto adenine is kinetically blocked by a higher energy transition state. PMID:26656495

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

    PubMed Central

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

    2005-01-01

    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. PMID:16511068

  9. Novel developments in metabolic disorders of purine and pyrimidine metabolism and therapeutic applications of their analogs.

    PubMed

    Torres, Rosa J; Peters, Godefridus J; Puig, Juan G

    2014-01-01

    The biennial 15th symposium on Purine and Pyrimidine metabolism was held in Madrid, June 2013 (PP13). During the meeting, several novel developments on the diagnosis, pathophysiology, and treatment of several inborn errors of purine and pyrimidine metabolism were presented. These ranged from new drugs for gout to enzyme replacement therapies for mitochondrial diseases. A relatively novel aspect in this meeting was the interest in purine and pyrimidine metabolism in nonmammalian systems, such as parasites, mycoplasms, and bacteria. Development of novel analogs for parasite infections, cardiovascular diseases, inflammatory diseases, and cancer were also discussed. PMID:24940665

  10. Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors.

    PubMed Central

    Ealick, S E; Babu, Y S; Bugg, C E; Erion, M D; Guida, W C; Montgomery, J A; Secrist, J A

    1991-01-01

    Competitive inhibitors of the salvage pathway enzyme purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) have been designed by using the three-dimensional structure of the enzyme as determined by x-ray crystallography. The process was an iterative one that utilized interactive computer graphics, Monte Carlo-based conformational searching, energy minimization, and x-ray crystallography. The proposed compounds were synthesized and tested by an in vitro assay. Among the compounds designed and synthesized are the most potent competitive inhibitors of purine nucleoside phosphorylase thus far reported. Images PMID:1763067

  11. Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: cellular models of de novo purine biosynthesis deficiency disorders

    PubMed Central

    Wilkinson, Terry G.; Baresova, Veronika; Skopova, Vaclava; Kmoch, Stanislav; Vacano, Guido N.; Zikanova, Marie; Patterson, David

    2014-01-01

    Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, renal stones, combined immunodeficiency, and autistic features. The pathogenetic mechanism is unknown for any of these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis

  12. Purines are required at the 5' ends of newly initiated RNAs for optimal RNA polymerase III gene expression.

    PubMed Central

    Zecherle, G N; Whelen, S; Hall, B D

    1996-01-01

    We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the +1 and +2 nucleotides of pre-tRNA Tyr, had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5' ends of the nascent transcripts of many genes act to stabilize the early ternary complex. PMID:8816494

  13. 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

  14. Transition State Analogues of Purine Nucleoside Phosphorylase: the Work of Vernon L. Schramm

    PubMed Central

    Kresge, Nicole; Simoni, Robert D.; Hill, Robert L.

    2010-01-01

    Transition State Analogue Inhibitors of Purine Nucleoside Phosphorylase from Plasmodium falciparum (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219–3225) Purine-less Death in Plasmodium falciparum Induced by Immucillin-H, a Transition State Analogue of Purine Nucleoside Phosphorylase (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Schramm, V. L., and Kim, K. (2002) J. Biol. Chem. 277, 3226–3231) Achieving the Ultimate Physiological Goal in Transition State Analogue Inhibitors for Purine Nucleoside Phosphorylase (Lewandowicz, A., Tyler, P. C., Evans, G. B., Furneaux, R. H., and Schramm, V. L. (2003) J. Biol. Chem. 278, 31465–31468)

  15. 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.

  16. mTORC1 Induces Purine Synthesis Through Control of the Mitochondrial Tetrahydrofolate Cycle

    PubMed Central

    Ricoult, Stéphane J.H.; Asara, John M.; Manning, Brendan D.

    2016-01-01

    In response to growth signals, mTOR complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by ATF4, which was activated by mTORC1 independent of its canonical induction downstream of eIF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals. PMID:26912861

  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. Borrelia burgdorferi Harbors a Transport System Essential for Purine Salvage and Mammalian Infection

    PubMed Central

    Jain, Sunny; Sutchu, Selina; Rosa, Patricia A.; Byram, Rebecca

    2012-01-01

    Borrelia burgdorferi is the tick-borne bacterium that causes the multistage inflammatory disease Lyme disease. B. burgdorferi has a reduced genome and lacks the enzymes required for de novo synthesis of purines for synthesis of RNA and DNA. Therefore, this obligate pathogen is dependent upon the tick vector and mammalian host environments for salvage of purine bases for nucleic acid biosynthesis. This pathway is vital for B. burgdorferi survival throughout its infectious cycle, as key enzymes in the purine salvage pathway are essential for the ability of the spirochete to infect mice and critical for spirochete replication in the tick. The transport of preformed purines into the spirochete is the first step in the purine salvage pathway and may represent a novel therapeutic target and/or means to deliver antispirochete molecules to the pathogen. However, the transport systems critical for purine salvage by B. burgdorferi have yet to be identified. Herein, we demonstrate that the genes bbb22 and bbb23, present on B. burgdorferi's essential plasmid circular plasmid 26 (cp26), encode key purine transport proteins. BBB22 and/or BBB23 is essential for hypoxanthine transport and contributes to the transport of adenine and guanine. Furthermore, B. burgdorferi lacking bbb22-23 was noninfectious in mice up to a dose of 1 × 107 spirochetes. Together, our data establish that bbb22-23 encode purine permeases critical for B. burgdorferi mammalian infectivity, suggesting that this transport system may serve as a novel antimicrobial target for the treatment of Lyme disease. PMID:22710875

  19. The Role of Gene Duplication in the Evolution of Purine Nucleotide Salvage Pathways

    NASA Astrophysics Data System (ADS)

    Becerra, Arturo; Lazcano, Antonio

    1998-10-01

    Purine nucleotides are formed de novo by a widespread biochemical route that may be of monophyletic origin, or are synthesized from preformed purine bases and nucleosides through different salvage pathways. Three monophyletic sets of purine salvage enzymes, each of which catalyzes mechanistically similar reactions, can be identified: (a) adenine-, xanthine-, hypoxanthine- and guanine-phosphoribosyltransferases, which are all homologous among themselves, as well as to nucleoside phosphorylases; (b) adenine deaminase, adenosine deaminase, and adenosine monophophate deaminase; and (c) guanine reductase and inosine monophosphate dehydrogenase. These homologies support the idea that substrate specificity is the outcome of gene duplication, and that the purine nucleotide salvage pathways were assembled by a patchwork process that probably took place before the divergence of the three cell domains (Bacteria, Archaea, and Eucarya). Based on the ability of adenine PRTase to catalyze the condensation of PRPP with 4-aminoimidazole-5-carboxamide (AICA), a simpler scheme of purine nucleotide biosynthesis is presented. This hypothetical route requires the prior evolution of PRPP biosynthesis. Since it has been argued that PRPP, nucleosides, and nucleotides are susceptible to hydrolysis, they are very unlikely prebiotic compounds. If this is the case, it implies that many purine salvage pathways appeared only after the evolution of phosphorylated sugar biosynthetic pathways made ribosides available.

  20. 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. PMID:23347082

  1. Deprotonated purine dissociation: experiments, computations, and astrobiological implications.

    PubMed

    Cole, Callie A; Wang, Zhe-Chen; Snow, Theodore P; Bierbaum, Veronica M

    2015-01-15

    A central focus of astrobiology is the determination of abiotic formation routes to important biomolecules. The dissociation mechanisms of these molecules lend valuable insights into their synthesis pathways. Because of the detection of organic anions in the interstellar medium (ISM), it is imperative to study their role in these syntheses. This work aims to experimentally and computationally examine deprotonated adenine and guanine dissociation in an effort to illuminate potential anionic precursors to purine formation. Collision-induced dissociation (CID) products and their branching fractions are experimentally measured using an ion trap mass spectrometer. Deprotonated guanine dissociates primarily by deammoniation (97%) with minor losses of carbodiimide (HNCNH) and/or cyanamide (NH2CN), and isocyanic acid (HNCO). Deprotonated adenine fragments by loss of hydrogen cyanide and/or isocyanide (HCN/HNC; 90%) and carbodiimide (HNCNH) and/or cyanamide (NH2CN; 10%). Tandem mass spectrometry (MS(n)) experiments reveal that deprotonated guanine fragments lose additional HCN and CO, while deprotonated adenine fragments successively lose HNC and HCN. Every neutral fragment observed in this study has been detected in the ISM, highlighting the potential for nucleobases such as these to form in such environments. Lastly, the acidity of abundant fragment ions is experimentally bracketed. Theoretical calculations at the B3LYP/6-311++G(d,p) level of theory are performed to delineate the mechanisms of dissociation and analyze the energies of reactants, intermediates, transition states, and products of these CID processes. PMID:25559322

  2. 6-(2-Methoxy­benzyl­amino)purine

    PubMed Central

    Trávníček, Zdeněk; Matiková-Maľarová, Miroslava; Mikulík, Jiří

    2008-01-01

    The title compound, C13H13N5O, consists of discrete mol­ecules connected by N—H⋯N hydrogen bonds to form infinite chains, with N⋯N separations of 3.0379 (15) and 2.8853 (15) Å. The benzene and purine ring systems make a dihedral angle of 77.58 (3)°. The crystal structure is further stabilized by intra­molecular N⋯O inter­actions [2.9541 (12) Å] and inter­molecular C—H⋯C and C⋯C contacts [3.304 (2), 3.368 (2), 3.667 (2), 3.618 (2) and 3.512 (2) Å] which arrange the mol­ecules into graphite-like layers. The inter­layer separations are 3.248 and 3.256 Å. PMID:21202313

  3. Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.

    PubMed

    Kyoung, Minjoung; Russell, Sarah J; Kohnhorst, Casey L; Esemoto, Nopondo N; An, Songon

    2015-01-27

    Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly. PMID:25540829

  4. Learning to Read: Developing Processes for Recognizing, Understanding and Pronouncing Written Words

    ERIC Educational Resources Information Center

    Stuart, Morag

    2006-01-01

    Major theories of how skilled readers recognize, understand and pronounce written words include processes for phonological recoding (i.e., translating segments of print to their corresponding segments of sound) and processes by which direct access is achieved from printed words to their meanings. If these are the processes employed in skilled…

  5. Autoimmune Dysregulation and Purine Metabolism in Adenosine Deaminase Deficiency

    PubMed Central

    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. PMID:22969765

  6. Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs.

    PubMed Central

    Saxild, H H; Nygaard, P

    1987-01-01

    Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively. PMID:3110131

  7. 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.

  8. Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

    PubMed

    Donaldson, Teraya M; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C; Kim, Kami

    2014-01-01

    Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket. PMID:24416224

  9. 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. PMID:26355499

  10. 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

  11. 39 CFR 211.3 - Executive orders and other executive pronouncements; circulars, bulletins, and other issuances of...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... pronouncements; circulars, bulletins, and other issuances of the Office of Management and Budget. 211.3 Section... REGULATIONS § 211.3 Executive orders and other executive pronouncements; circulars, bulletins, and other... executive orders, and other executive pronouncements and certain circulars, bulletins, and other...

  12. 39 CFR 211.3 - Executive orders and other executive pronouncements; circulars, bulletins, and other issuances of...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... pronouncements; circulars, bulletins, and other issuances of the Office of Management and Budget. 211.3 Section... REGULATIONS § 211.3 Executive orders and other executive pronouncements; circulars, bulletins, and other... executive orders, and other executive pronouncements and certain circulars, bulletins, and other...

  13. Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na(+)-nucleoside cotransporter.

    PubMed

    Che, M; Ortiz, D F; Arias, I M

    1995-06-01

    We previously characterized a purine-specific Na(+)-nucleoside cotransport system in bile canalicular membrane. The function of this transport system may be related to conserving nucleosides and preventing cholestasis. We report here the isolation of a cDNA encoding a Na(+)-dependent nucleoside transporter from rat liver using an expression cloning strategy. The substrate specificities and kinetic characteristics of the cloned cotransporter are consistent with the properties of the Na(+)-dependent, purine-selective nucleoside transporter in bile canalicular membranes. The nucleotide sequence predicts a protein of 659 amino acids (72 kDa) with 14 putative membrane-spanning domains. Northern blot analysis showed that the transcripts are present in liver and several other tissues. Data base searches indicate significant sequence similarity to the pyrimidine-selective nucleoside transporter (cNT1) of rat jejunum. Although these two subtypes of Na(+)-nucleoside cotransporter have different substrate specificities and tissue localizations, they are members of a single gene family. PMID:7775409

  14. 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

  15. Preliminary crystallographic studies of purine nucleoside phosphorylase from the cariogenic pathogen Streptococcus mutans

    PubMed Central

    Hou, Qiao-Ming; Liu, Xiang; Brostromer, Erik; Li, Lan-Fen; Su, Xiao-Dong

    2009-01-01

    The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and α-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6 Å resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1 Å. PMID:20054131

  16. AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization.

    PubMed

    Asby, Daniel J; Cuda, Francesco; Beyaert, Maxime; Houghton, Franchesca D; Cagampang, Felino R; Tavassoli, Ali

    2015-07-23

    5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders. PMID:26144885

  17. Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs

    PubMed Central

    Gilbert, Sunny D.; Reyes, Francis E.; Edwards, Andrea L.; Batey, Robert T.

    2009-01-01

    SUMMARY Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity. PMID:19523903

  18. 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).

  19. Pronounced palatal and mandibular tori observed in a patient with chronic phenytoin therapy: a case report.

    PubMed

    Sasaki, H; Ikedo, D; Kataoka, M; Kido, J; Kitamura, S; Nagata, T

    1999-04-01

    Phenytoin, an anticonvulsant drug for epileptic patients, has many adverse effects, including calvarial thickening and coarsening of the facial features. Previous studies have demonstrated that phenytoin has an anabolic action on bone cells. This report describes pronounced palatal and mandibular tori found in a 45-year-old Japanese man undergoing chronic phenytoin therapy. The tori were extremely large, lobular, and symmetrical. A palatal torus appeared along the middle of the hard palate and mandibular tori consisted of 2 pairs of nodular masses extensively filling the lingual floor of the oral cavity. Pronounced osseous outgrowth occurred for the duration of a dose-increase of phenytoin from 1985 to 1997. His parents did not have any palatal or mandibular tori. These facts suggest that these unusual tori may have been the result of chronic phenytoin therapy, rather than association with the familial background. PMID:10328658

  20. 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

    2016-05-01

    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. PMID:27101306

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

    PubMed

    Endo, T; Uratani, B; Freese, E

    1983-07-01

    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

  2. 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

  3. Anti‐flavivirus Activity of Different Tritylated Pyrimidine and Purine Nucleoside Analogues

    PubMed Central

    Serpi, Michaela; Slusarczyk, Magdalena; Ferrari, Valentina; Pertusati, Fabrizio; Meneghesso, Silvia; Derudas, Marco; Farleigh, Laura; Zanetta, Paola; Bugert, Joachim

    2016-01-01

    Abstract A series of tritylated and dimethoxytritylated analogues of selected pyrimidine and purine nucleosides were synthesized and evaluated for their in vitro inhibitory activity against two important members of the genus Flavivirus in the Flaviviridae family, the yellow fever (YFV) and dengue viruses (DENV). Among all compounds tested, the 5′‐O‐tritylated and the 5′‐O‐dimethoxytritylated 5‐fluorouridine derivatives exerted potency against YFV. Interestingly in the series of purine analogues, the 5′O, N‐bis‐tritylated fludarabine derivative revealed strong inhibitory activity against DENV at μm concentrations, however significantly weaker potency against YFV. PMID:27551659

  4. 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.

  5. Anti-flavivirus Activity of Different Tritylated Pyrimidine and Purine Nucleoside Analogues.

    PubMed

    McGuigan, Christopher; Serpi, Michaela; Slusarczyk, Magdalena; Ferrari, Valentina; Pertusati, Fabrizio; Meneghesso, Silvia; Derudas, Marco; Farleigh, Laura; Zanetta, Paola; Bugert, Joachim

    2016-06-01

    A series of tritylated and dimethoxytritylated analogues of selected pyrimidine and purine nucleosides were synthesized and evaluated for their in vitro inhibitory activity against two important members of the genus Flavivirus in the Flaviviridae family, the yellow fever (YFV) and dengue viruses (DENV). Among all compounds tested, the 5'-O-tritylated and the 5'-O-dimethoxytritylated 5-fluorouridine derivatives exerted potency against YFV. Interestingly in the series of purine analogues, the 5'O, N-bis-tritylated fludarabine derivative revealed strong inhibitory activity against DENV at μm concentrations, however significantly weaker potency against YFV. PMID:27551659

  6. Copper-catalyzed intramolecular cyclization of N-propargyl-adenine: synthesis of purine-fused tricyclics.

    PubMed

    Li, Ren-Long; Liang, Lei; Xie, Ming-Sheng; Qu, Gui-Rong; Niu, Hong-Ying; Guo, Hai-Ming

    2014-04-18

    A novel protocol to construct fluorescent purine-fused tricyclic products via intramolecular cyclization of N-propargyl-adenine has been developed. With CuBr as the catalyst, a series of purine-fused tricyclic products were obtained in good to excellent yields (19 examples, 75-89% yields). When R2 was a hydrogen atom in N-propargyl-adenines, the reactions only afforded the endocyclic double bond products. When R2 was an aryl group, the electron-donating groups favored the endocyclic double bond products, while the electron-withdrawing groups favored the exocyclic double bond products. PMID:24678722

  7. Influence of prime lexicality, frequency, and pronounceability on the masked onset priming effect.

    PubMed

    Dimitropoulou, Maria; Duñabeitia, Jon Andoni; Carreiras, Manuel

    2010-09-01

    The present study investigates the origins of the masked onset priming effect (MOPE). There are two alternative interpretations that account for most of the evidence reported on the MOPE, so far. The speech planning account (SP) identifies the locus of the MOPE in the preparation of the speech response. In contrast, the dual-route theory proposes that the effect arises as a result of the processing of the prime by the nonlexical route. In a series of masked onset priming word naming experiments we test the validity of these accounts by manipulating the primes' frequency, their lexical status, and pronounceability. We found consistent MOPEs of similar magnitude with high- and low-frequency prime words as well as with pronounceable nonwords. Contrarily, when primes consisted of unpronounceable consonantal strings the effect disappeared, suggesting that pronounceability of the prime is a prerequisite for the emergence of the MOPE. These results are in accordance with the predictions of the SP account. The pattern of effects obtained in the present study further defines the origins of the MOPE. PMID:20221948

  8. 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

  9. Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element-Binding Protein B.

    PubMed

    Rumora, Amy E; Ferris, Lauren A; Wheeler, Tamar R; Kelm, Robert J

    2016-05-17

    Myofibroblast differentiation is characterized by an increased level of expression of cytoskeletal smooth muscle α-actin. In human and murine fibroblasts, the gene encoding smooth muscle α-actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5' promoter-enhancer in response to environmental cues signaling tissue repair and remodeling. Purine-rich element-binding protein B (Purβ) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5' polypurine sequence containing an inverted MCAT cis element required for gene activation. In this study, we evaluated the chemical basis of nucleoprotein complex formation between the Purβ repressor and the purine-rich strand of the MCAT element in the mouse Acta2 promoter. Quantitative single-stranded DNA (ssDNA) binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent suggested that the assembly of a high-affinity nucleoprotein complex is driven by a combination of electrostatic and hydrophobic interactions. Consistent with the results of pH titration analysis, site-directed mutagenesis revealed several basic amino acid residues in the intermolecular (R267) and intramolecular (K82 and R159) subdomains that are essential for Purβ transcriptional repressor function in Acta2 promoter-reporter assays. In keeping with their diminished Acta2 repressor activity in fibroblasts, purified Purβ variants containing an R267A mutation exhibited reduced binding affinity for purine-rich ssDNA. Moreover, certain double and triple-point mutants were also defective in binding to the Acta2 corepressor protein, Y-box-binding protein 1. Collectively, these findings establish the repertoire of noncovalent interactions that account for the unique structural and functional properties of Purβ. PMID:27064749

  10. Structure-activity relationships and molecular studies of novel arylpiperazinylalkyl purine-2,4-diones and purine-2,4,8-triones with antidepressant and anxiolytic-like activity.

    PubMed

    Zagórska, Agnieszka; Kołaczkowski, Marcin; Bucki, Adam; Siwek, Agata; Kazek, Grzegorz; Satała, Grzegorz; Bojarski, Andrzej J; Partyka, Anna; Wesołowska, Anna; Pawłowski, Maciej

    2015-06-01

    A novel series of arylpiperazinylalkyl purine-2,4-diones (4-27) and purine-2,4,8-triones (31-38) was synthesized and tested to evaluated their affinity for the serotoninergic (5-HT1A, 5-HT6, 5-HT7) and dopaminergic (D2) receptors. Compounds with purine-2,4-dione nucleus generally had affinity values higher than the corresponding purine-2,4,8-trione compounds. A spectrum of receptor activities was observed for compounds with a substituent at the 7-position of the imidazo[2,1-f]purine-2,4-dione system and some potent 5-HT1A (18, 25), 5-HT7 (14) and mixed 5-HT1A/5-HT7 (8, 9) receptor ligands with additional affinity for dopamine D2 receptors (15) has been identified. Moreover, docking studies proved that a substituent at the 7-position of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione could be essential for receptor affinity and selectivity, especially towards 5-HT1A and 5-HT7. The results of the preliminary pharmacological in vivo studies of selected derivatives of 1,3-dimethyl-(1H,8H)-imidazo[2,1-f]purine-2,4-dione, including 9 as a potential anxiolytic, 8 and 15 as potential antidepressants, and 18 and 25 as potential antidepressant and anxiolytic agents. PMID:25965777

  11. A First Microwave-Assisted Synthesis of a New Class of Purine and Guanine Thioglycoside Analogs.

    PubMed

    Elgemeie, Galal; Abu-Zaied, Mamdouh; Hebishy, Ali; Abbas, Nermen; Hamed, Mai

    2016-09-01

    A first microwave-assisted synthesis of a new class of novel purine thioglycoside analogs from readily available starting materials has been described. The key step of this protocol is the formation of sodium pyrazolo[1,5-a]pyrimidine-7-thiolate and 7-mercaptopyrazolo[1,5-a]pyrimidine derivatives via condensation of 5-amino-1H-pyrazoles with sodium 2,2-dicyanoethene-1,1-bis(thiolate) salts or 2-(dimercaptomethylene)malononitrile, respectively, under microwave irradiation, followed by coupling with halo sugars to give the corresponding purine thioglycoside analogs. The obtained purines and purines thioglycosides derivatives were evaluated in vitro against lung (A549), colon (HCT116), liver (HEPG2), and prostate (PC3) cancer cell lines. Some of these compounds (5b, 5d, 5f, and 9a-d) exhibited little potency toward the four cell lines. On the other hand, compound 5a elicited higher cytotoxicity on both prostate (PC3) and colon (HCT116), respectively, while it was found moderate on lung (A549), and inactive on liver (HEPG2). Moreover, compound 5c was found moderate with LC50 values 52.0-88.9 μM for almost all the cell lines. PMID:27556784

  12. Analysis of purine metabolites in maternal serum for evaluating the risk of gestosis.

    PubMed

    Senyavina, N V; Khaustova, S A; Grebennik, T K; Pavlovich, S V

    2013-09-01

    Metabolome analysis of the serum from pregnant patients aimed at detection of low-molecular-weight biomarkers of gestation process disorders indicated a relationship between the metabolic profile of maternal serum and risk of gestosis. In women with pre-eclampsia or preterm delivery, analysis of serum purine metabolites revealed changes in the metabolite concentrations, associated with pregnancy complications. PMID:24288739

  13. Stressed-Out HSCs Turn Up p38α and Purine to Proliferate.

    PubMed

    Essers, Marieke A G

    2016-08-01

    Changes in cellular metabolism drive hematopoietic stem cell (HSC) behavior during homeostasis, although whether they control HSC behavior during stress conditions is unclear. In this issue of Cell Stem Cell, Karigane et al. (2016) identify a p38α-dependent pathway that alters purine metabolism in HSCs during stress hematopoiesis, promoting hematopoietic recovery. PMID:27494667

  14. mTORC1 induces purine synthesis through control of the mitochondrial tetrahydrofolate cycle.

    PubMed

    Ben-Sahra, Issam; Hoxhaj, Gerta; Ricoult, Stéphane J H; Asara, John M; Manning, Brendan D

    2016-02-12

    In response to growth signals, mechanistic target of rapamycin complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by activating transcription factor 4 (ATF4), which was activated by mTORC1 independent of its canonical induction downstream of eukaryotic initiation factor 2α eIF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals. PMID:26912861

  15. Structure and electronic spectra of purine-methyl viologen charge transfer complexes.

    PubMed

    Jalilov, Almaz S; Patwardhan, Sameer; Singh, Arunoday; Simeon, Tomekia; Sarjeant, Amy A; Schatz, George C; Lewis, Frederick D

    2014-01-01

    The structure and properties of the electron donor-acceptor complexes formed between methyl viologen and purine nucleosides and nucleotides in water and the solid state have been investigated using a combination of experimental and theoretical methods. Solution studies were performed using UV-vis and (1)H NMR spectroscopy. Theoretical calculations were performed within the framework of density functional theory (DFT). Energy decomposition analysis indicates that dispersion and induction (charge-transfer) interactions dominate the total binding energy, whereas electrostatic interactions are largely repulsive. The appearance of charge transfer bands in the absorption spectra of the complexes are well-described by time-dependent DFT and are further explained in terms of the redox properties of purine monomers and solvation effects. Crystal structures are reported for complexes of methyl viologen with the purines 2'-deoxyguanosine 3'-monophosphate (DAD'DAD' type) and 7-deazaguanosine (DAD'ADAD' type). Comparison of the structures determined in the solid state and by theoretical methods in solution provides valuable insights into the nature of charge-transfer interactions involving purine bases as electron donors. PMID:24294996

  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. 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.

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

    PubMed

    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

  19. 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

  20. Molecular Dissection of a Borrelia burgdorferi In Vivo Essential Purine Transport System

    PubMed Central

    Jain, Sunny; Showman, Adrienne C.

    2015-01-01

    The Lyme disease spirochete Borrelia burgdorferi is dependent on purine salvage from the host environment for survival. The genes bbb22 and bbb23 encode purine permeases that are essential for B. burgdorferi mouse infectivity. We now demonstrate the unique contributions of each of these genes to purine transport and murine infection. The affinities of spirochetes carrying bbb22 alone for hypoxanthine and adenine were similar to those of spirochetes carrying both genes. Spirochetes carrying bbb22 alone were able to achieve wild-type levels of adenine saturation but not hypoxanthine saturation, suggesting that maximal hypoxanthine uptake requires the presence of bbb23. Moreover, the purine transport activity conferred by bbb22 was dependent on an additional distal transcriptional start site located within the bbb23 open reading frame. The initial rates of uptake of hypoxanthine and adenine by spirochetes carrying bbb23 alone were below the level of detection. However, these spirochetes demonstrated a measurable increase in hypoxanthine uptake over a 30-min time course. Our findings indicate that bbb22-dependent adenine transport is essential for B. burgdorferi survival in mice. The bbb23 gene was dispensable for B. burgdorferi mouse infectivity, yet its presence was required along with that of bbb22 for B. burgdorferi to achieve maximal spirochete loads in infected mouse tissues. These data demonstrate that both genes, bbb22 and bbb23, are critical for B. burgdorferi to achieve wild-type infection of mice and that the differences in the capabilities of the two transporters may reflect distinct purine salvage needs that the spirochete encounters throughout its natural infectious cycle. PMID:25776752

  1. Diseases associated with pronounced eosinophilia: a study of 105 dogs in Sweden.

    PubMed

    Lilliehöök, I; Gunnarsson, L; Zakrisson, G; Tvedten, H

    2000-06-01

    Records of 105 dogs with pronounced eosinophilia (>2.2 x 10(9) eosinophils/litre) were evaluated in a retrospective study to determine diseases associated with the abnormality in dogs in Sweden. Inflammatory disease in organs with large epithelial surfaces, such as the gut, lungs or skin, was found in 36 per cent of the dogs. A further one-quarter of the 105 cases were placed in the 'miscellaneous' category, which comprised various diseases found at low frequency. The most well defined diagnosis was pulmonary infiltrates with eosinophils in 12 per cent of the dogs. A further 11 per cent had parasitic disease caused by either sarcoptic mange or nasal mite. No atopic dog was found and rottweilers were over-represented in most disease groups. Pronounced eosinophilia, in many cases transient, seems to be associated with a variety of disorders in dogs. In the present study, rottweilers appeared to be more prone to a high eosinophil response than other breeds. PMID:10879402

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

    NASA Astrophysics Data System (ADS)

    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 He4 films (n=2) by Garcia and Chan [Phys. Rev. Lett. 83, 1187 (1999), 10.1103/PhysRevLett.83.1187] and in Monte Carlo simulations for the three-dimensional Ising model (n =1) by O. Vasilyev et al. [Europhys. Lett. 80, 60009 (2007), 10.1209/0295-5075/80/60009]. 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.

  3. 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. PMID:20661644

  4. Equivalence class formation as a function of the pronounceability of the sample stimulus.

    PubMed

    Mandell, C; Sheen, V

    1994-06-01

    The role of naming in stimulus equivalence was studied by varying the pronounceability of sample stimulus pseudowords. Experiment 1 compared three conditions: in the first, sample stimuli consisted of phonologically correct pseudowords; in the second, sample stimuli consisted of phonologically incorrect words; in the last, sample stimuli consisted of punctuation marks. Subjects exposed to pronounceable stimuli demonstrated equivalence class formation more quickly and with fewer errors than did other subjects. In Experiment 2, subjects were trained in equivalence-class formation using only non-phonological sample stimuli. Half the subjects were exposed to a pretraining procedure in which they read a list of non-phonological pseudowords aloud. Remaining subjects transcribed the same list of pseudowords - a procedure which equated exposure to the pseudowords, but did not necessarily encourage subjects to name them. Subjects who were pretrained with the read-aloud task made significantly fewer errors than those who transcribed the words. These data are consistent with the theory that equivalence class formation is mediated by verbal behavior. PMID:24925111

  5. Sequence-dependent dynamics of duplex DNA: the applicability of a dinucleotide model.

    PubMed Central

    Okonogi, T M; Alley, S C; Reese, A W; Hopkins, P B; Robinson, B H

    2002-01-01

    The short-time (submicrosecond) bending dynamics of duplex DNA were measured to determine the effect of sequence on dynamics. All measurements were obtained from a single site on duplex DNA, using a single, site-specific modified base containing a rigidly tethered, electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of single-step sequence-dependent bending force constants, determined from the mean squared amplitude of bending relative to the end-to-end vector using the modified weakly bending rod model. The bending dynamics at a single site are a function of the sequence of the nucleotides constituting the duplex DNA. We developed and examined several dinucleotide-based models for flexibility. The models indicate that the dominant feature of the dynamics is best explained in terms of purine- and pyrimidine-type steps, although distinction is made among all 10 unique steps: It was found that purine-purine steps (which are the same as pyrimidine-pyrimidine steps) were near average in flexibility, but the pyrimidine-purine steps (5' to 3') were nearly twice as flexible, whereas purine-pyrimidine steps were more than half as flexible as average DNA. Therefore, the range of stepwise flexibility is approximately fourfold and is characterized by both the type of base pair step (pyrimidine/purine combination) and the identity of the bases within the pair (G, A, T, or C). All of the four models considered here underscore the complexity of the dependence of dynamics on DNA sequence with certain sequences not satisfactorily explainable in terms of any dinucleotide model. These findings provide a quantitative basis for interpreting the dynamics and kinetics of DNA-sequence-dependent biological processes, including protein recognition and chromatin packaging. PMID:12496111

  6. 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. PMID:27107949

  7. Excessive chemotherapy-related granulocytopenia in a child with non-Hodgkin's lymphoma and a congenital abnormality of purine salvage.

    PubMed

    Blatt, J

    1990-01-01

    A girl with non-Hodgkin's lymphoma and immunodeficiency based on absence of the purine salvage pathway enzyme purine nucleoside phosphorylase experienced profound neutropenia while receiving combination chemotherapy with cyclophosphamide, vincristine, methotrexate, and prednisone (COMP). Neutropenia was most severe following courses that included either systemic or intrathecal methotrexate, even in the face of major dose reductions. Delays in the development of neutropenia-during periods of leucovorin administration also implicate methotrexate as the primary responsible agent. This case suggests that certain immunodeficiency states predispose patients to extensive chemotherapy-induced myelosuppression and supports the concept that purine salvage is a clinically important mechanism for modulating methotrexate toxicity. PMID:2113161

  8. Correction of point mutations at the endogenous locus of the dihydrofolate reductase gene using repair-PolyPurine Reverse Hoogsteen hairpins in mammalian cells.

    PubMed

    Solé, Anna; Ciudad, Carlos J; Chasin, Lawrence A; Noé, Véronique

    2016-06-15

    Correction of point mutations that lead to aberrant transcripts, often with pathological consequences, has been the focus of considerable research. In this work, repair-PPRHs are shown to be a new powerful tool for gene correction. A repair-PPRH consists of a PolyPurine Reverse Hoogsteen hairpin core bearing an extension sequence at one end, homologous to the DNA strand to be repaired but containing the wild type nucleotide instead of the mutation. Previously, we had corrected a single-point mutation with repair-PPRHs using a mutated version of a dihydrofolate reductase (dhfr) minigene. To further evaluate the utility of these molecules, different repair-PPRHs were designed to correct insertions, deletions, substitutions and a double substitution present in a collection of mutants at the endogenous locus of the dhfr gene, the product of which is the target of the chemotherapeutic agent methotrexate. We also describe an approach to use when the point mutation is far away from the homopyrimidine target domain. This strategy consists in designing Long-Distance- and Short-Distance-Repair-PPRHs where the PPRH core is bound to the repair tail by a five-thymidine linker. Surviving colonies in a DHFR selective medium, lacking glycine and sources of purines and thymidine, were analyzed by DNA sequencing, and by mRNA, protein and enzymatic measurements, confirming that all the dhfr mutants had been corrected. These results show that repair-PPRHs can be effective tools to accomplish a permanent correction of point mutations in the DNA sequence of mutant mammalian cells. PMID:27063945

  9. The Fiber Contractility and Cytoskeleton Losses in Space are Less Pronounced in Mongolian Gerbils

    NASA Astrophysics Data System (ADS)

    Lipets, E. N.; Ponomareva, E. V.; Ogneva, I. V.; Vikhliantsev, I. M.; Karaduleva, E. V.; Kartashkina, N. L.; Kuznetsov, S. L.; Podlubnaia, Z. A.; Shenkman, B. S.

    2008-06-01

    This work was purposed on the comparison of space flight effects on m. soleus and m. tibialis anterior of Mongolian gerbils. The animals have been flown onboard biosatellite Foton-M3 for 12 days. Contractile properties of single skinned muscle fibers were studied. It was revealed that diameter of m. soleus skinned fibers and maximal isometric tension were decreased by 19.7% and 21.8% respectively. The Ca-sensitivity reduction wasn't significant, that was in accordance with absence of changes of titin and nebulin relative content in soleus and minor manifestations in slow-to-fast fiber ratio (9%, p<0.05). There weren't observed significant changes of the same parameters in m. tibialis anterior. Ultimately the fiber contractility and cytoskeleton losses in space are less pronounced in Mongolian gerbils than in rats.

  10. Computational design of donor-bridge-acceptor systems exhibiting pronounced quantum interference effects.

    PubMed

    Gorczak, Natalie; Renaud, Nicolas; Galan, Elena; Eelkema, Rienk; Siebbeles, Laurens D A; Grozema, Ferdinand C

    2016-03-01

    Quantum interference is a well-known phenomenon that dictates charge transport properties of single molecule junctions. However, reports on quantum interference in donor-bridge-acceptor molecules are scarce. This might be due to the difficulties in meeting the conditions for the presence of quantum interference in a donor-bridge-acceptor system. The electronic coupling between the donor, bridge, and acceptor moieties must be weak in order to ensure localised initial and final states for charge transfer. Yet, it must be strong enough to allow all bridge orbitals to mediate charge transfer. We present the computational route to the design of a donor-bridge-acceptor molecule that features the right balance between these contradicting requirements and exhibits pronounced interference effects. PMID:26878200

  11. Hypouricemic effects of novel concentrative nucleoside transporter 2 inhibitors through suppressing intestinal absorption of purine nucleosides.

    PubMed

    Hiratochi, Masahiro; Tatani, Kazuya; Shimizu, Kazuo; Kuramochi, Yu; Kikuchi, Norihiko; Kamada, Noboru; Itoh, Fumiaki; Isaji, Masayuki

    2012-09-01

    We have developed concentrative nucleoside transporter 2 (CNT2) inhibitors as a novel pharmacological approach for improving hyperuricemia by inhibiting intestinal absorption of purines. Dietary purine nucleosides are absorbed in the small intestines by CNTs expressed in the apical membrane. In humans, the absorbed purine nucleosides are rapidly degraded to their final end product, uric acid, by xanthine oxidase. Based on the expression profile of human CNTs in digestive tract tissues, we established a working hypothesis that mainly CNT2 contributes to the intestinal absorption of purine nucleosides. In order to confirm this possibility, we developed CNT2 inhibitors and found that (2R,3R,4S,5R)-2-(6-amino-8-{[3'-(3-aminopropoxy)-biphenyl-4-ylmethyl]-amino}-9H-purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (KGO-2142) and 1-[3-(5-{[1-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-benzimidazol-2-ylamino]-methyl}-2-ethoxyphenoxy)-propyl]-piperidine-4-carboxylic acid amide (KGO-2173) were inhibitory. These CNT2 inhibitors had potent inhibitory activity against inosine uptake via human CNT2, but they did not potently interfere with nucleoside uptake via human CNT1, CNT3 or equilibrative nucleoside transporters (ENTs) in vitro. After oral administration of KGO-2173 along with [(14)C]-inosine, KGO-2173 significantly decreased the urinary excretion of radioactivity at 6 and 24h in rats. Since dietary purine nucleosides are not utilized in the body and are excreted into the urine rapidly, this decrease in radioactivity in the urine represented the inhibitory activity of KGO-2173 toward the absorption of [(14)C]-inosine in the small intestines. KGO-2142 almost completely inhibited dietary RNA-induced hyperuricemia and the increase in urinary excretion of uric acid in cebus monkeys. These novel CNT2 inhibitors, KGO-2142 and KGO-2173, could be useful therapeutic options for the treatment of hyperuricemia. PMID:22709993

  12. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine.

    PubMed

    Ritzel, M W; Yao, S Y; Ng, A M; Mackey, J R; Cass, C E; Young, J D

    1998-01-01

    Two Na(+)-dependent nucleoside transporters implicated in adenosine and uridine transport in mammalian cells are distinguished functionally on the basis of substrate specificity: CNT1 is selective for pyrimidine nucleosides but also transports adenosine; CNT2 (also termed SPNT) is selective for purine nucleosides but also transports uridine. Both proteins belong to a gene family that includes the NupC proton/nucleoside symporter of E. coli. cDNAs encoding members of the CNT family have been isolated from rat tissues (jejunum, brain, liver; rCNT1 and rCNT2/SPNT) and, most recently, human kidney (hCNT1 and hSPNT1). Here, the molecular cloning and functional characterization of a CNT2/SPNT-type transporter from human small intestine are described. The encoded 658-residue protein (hCNT2 in the nomenclature) had the same predicted amino acid sequence as human kidney hSPNT1, except for a polymorphism at residue 75 (Arg substituted by Ser), and was 83 and 72% identical to rCNT2 and hCNT1, respectively. Sequence differences between hCNT2 and rCNT2 were greatest at the N-terminus. In Xenopus oocytes, recombinant hCNT2 exhibited the functional characteristics of a Na(+)-dependent nucleoside transporter with selectivity for adenosine, other purine nucleosides and uridine (adenosine and uridine K(m) app values 8 and 40 microM, respectively). hCNT2 transcripts were found in kidney and small intestine but, unlike rCNT2, were not detected in liver. Deoxyadenosine, which undergoes net renal secretion in humans, was less readily transported than adenosine. hCNT2 also mediated small, but significant, fluxes of the antiviral purine nucleoside analogue 2',3'-dideoxyinosine. hCNT2 is, therefore potentially involved in both the intestinal absorption and renal handling of purine nucleosides (including adenosine), uridine and purine nucleoside drugs. The gene encoding hCNT2 was mapped to chromosome 15q15. PMID:10087507

  13. Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation.

    PubMed

    Paramasivam, Manikandan; Cogoi, Susanna; Filichev, Vyacheslav V; Bomholt, Niels; Pedersen, Erik B; Xodo, Luigi E

    2008-06-01

    Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA-TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA-TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA-TFOs were found to abrogate the formation of a DNA-protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA-TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome. PMID:18456705

  14. Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis.

    PubMed

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

    2014-07-17

    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 mammalian target of rapamycin 2 and IκB kinase regulate Akt activity and Akt association and phosphorylation of transketolase (TKT), a key enzyme of the nonoxidative pentose phosphate pathway (PPP). Akt phosphorylates TKT on Thr382, markedly enhancing enzyme activity and increasing carbon flow through the nonoxidative PPP, thereby increasing purine synthesis. Mice fed a lysine-deficient diet for 2 days show decreased Akt activity, TKT activity, and purine synthesis in multiple organs. These results provide a mechanism whereby Akt coordinates amino acid availability with glucose utilization, purine synthesis, and RNA and DNA synthesis. PMID:24981175

  15. The purinosome, a multi-protein complex involved in the de novo biosynthesis of purines in humans

    PubMed Central

    Zhao, Hong; French, Jarrod B.; Fang, Ye; Benkovic, Stephen J.

    2013-01-01

    Purine nucleotides are ubiquitous molecules that play vital roles in all kingdoms of life, not only as components of nucleic acids, but also participating in signaling and energy storage. Cellular pools of purines are maintained by the tight control of several complementary and sometimes competing processes including de novo biosynthesis, salvage and catabolism of nucleotides. While great strides have been made over the past sixty years in understanding the biosynthesis of purines, we are experiencing a renaissance in this field. In this feature article we discuss the most recent discoveries relating to purine biosynthesis, with particular emphasis upon the dynamic multi-protein complex called the purinosome. In particular we highlight advances made towards understanding the assembly, control and function of this protein complex and the attempts made to exploit this knowledge for drug discovery. PMID:23575936

  16. Sequence context effect for hMSH2-hMSH6 mismatch-dependent activation

    PubMed Central

    Mazurek, Anthony; Johnson, Christopher N.; Germann, Markus W.; Fishel, Richard

    2009-01-01

    Numerous DNA mismatches and lesions activate MutS homologue (MSH) ATPase activity that is essential for mismatch repair (MMR). We have found that a mismatch embedded in a nearest-neighbor sequence context containing symmetric 3′-purines (2 × 3′-purines) enhanced, whereas symmetric 3′-pyrimidines (2 × 3′-pyrimidines) reduced, hMSH2-hMSH6 ATPase activation. The 3′-purine/pyrimidine effect was most evident for G-containing mispairs. A similar trend pervaded mismatch binding (KD) and the melting of unbound oligonucleotides (Tm; ΔG). However, these latter measures did not accurately predict the hierarchy of MSH ATPase activation. NMR studies of imino proton lifetime, solvent accessibility, and NOE connectivity suggest that sequence contexts that provoke improved MSH-activation displayed enhanced localized DNA flexibility: a dynamic DNA signature that may account for the wide range of lesions that activate MSH functions. PMID:19237577

  17. Purine oversecretion in cultured murine lymphoma cells deficient in adenylosuccinate synthetase: genetic model for inherited hyperuricemia and gout.

    PubMed Central

    Ullman, B; Wormsted, M A; Cohen, M B; Martin, D W

    1982-01-01

    Alterations in several specific enzymes have been associated with increased rates of purine synthesis de novo in human and other mammalian cells. However, these recognized abnormalities in humans account for only a few percent of the clinical cases of hyperuricemia and gout. We have examined in detail the rates of purine production de novo and purine excretion by normal and by mutant (AU-100) murine lymphoma T cells (S49) 80% deficient in adenylosuccinate synthetase [IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4]. The intracellular ATP concentration of the mutant cells is slightly diminished, but their GTP is increased 50% and their IMP, four-fold. Compared to wild-type cells, the AU-100 cells excrete into the culture medium 30- to 50-fold greater amounts of purine metabolites consisting mainly of inosine. Moreover, the AU-100 cell line overproduces total purines. In an AU-100-derived cell line, AU-TG50B, deficient in adenylosuccinate synthetase and hypoxanthine/guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), purine nucleoside excretion is increased 50- to 100-fold, and de novo synthesis is even greater than that for AU-100 cells. The overexcretion of purine metabolites by the AU-100 cells seems to be due to the primary genetic deficiency of adenylosuccinate synthetase, a deficiency that requires the cell to increase intracellular IMP in an attempt to maintain ATP levels. As a consequence of elevated IMP pools, large amounts of inosine are secreted into the culture medium. We propose that a similar primary genetic defect may account for the excessive purine excretion in some patients with dominantly inherited hyperuricemia and gout. Images PMID:6957854

  18. Mass Modulation of Protein Dynamics Associated with Barrier Crossing in Purine Nucleoside Phosphorylase

    PubMed Central

    Antoniou, Dimitri; Ge, Xiaoxia; Schramm, Vern L.; Schwartz, Steven D.

    2012-01-01

    The role of protein dynamics on different time scales in enzyme catalysis remains an area of active debate. The connection between enzyme dynamics on the femtosecond time scale and transition state formation has been demonstrated in human purine nucleoside phosphorylase (PNP) through the study of a mass-altered enzyme. Isotopic substitution in human PNP (heavy PNP) decreased the rate of on-enzyme chemistry but did not alter either the transition state structure or steady-state kinetic parameters. Here we investigate the underlying atomic motions associated with altered barrier crossing probability for heavy PNP. Transition path sampling was employed to illuminate the molecular differences between barrier crossing in light and heavy enzymes. The mass effect is apparent in promoting vibrations that polarize the N-ribosidic bond, and that promote the stability of the purine leaving group. These motions facilitate barrier crossing. PMID:24496053

  19. Synthesis of novel substituted purine derivatives and identification of the cell death mechanism.

    PubMed

    Demir, Zeynep; Guven, Ebru Bilget; Ozbey, Suheyla; Kazak, Canan; Atalay, Rengul Cetin; Tuncbilek, Meral

    2015-01-01

    Novel 9-(substituted amino/piperazinoethyl)adenines (4-12), 6-(substituted piperazino/amino)purines (15-27), 9-(p-toluenesulfonyl/cyclopentyl/ethoxycarbonylmethyl)-6-(substituted amino/piperazino)purines (28-34, 36, 37, 38-41) were synthesized and evaluated initially for their cytotoxic activities on liver Huh7, breast T47D and colon HCT116 carcinoma cells. N(6)-(4-Trifluoromethylphenyl)piperazine derivative (17) and its 9-(p-toluene-sulfonyl)/9-cyclopentyl analogues (28, 36) had promising cytotoxic activities. Compounds 17, 28 and 36 were further analysed for their cytotoxicity in a panel of a liver cancer cell lines. The compound 36 had better cytotoxic activities (IC50 ≤ 1 μM) than the nucleobase 5-FU and nucleosides fludarabine, cladribine, and pentostatine on Huh7 cells. Cytotoxicity induced by 36 was later identified as senescence associated cell death by SA-β-Gal assay. PMID:25462277

  20. 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

  1. 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

  2. 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.

  3. Purine pathway implicated in mechanism of resistance to aspirin therapy: pharmacometabolomics-informed pharmacogenomics.

    PubMed

    Yerges-Armstrong, L M; Ellero-Simatos, S; Georgiades, A; Zhu, H; Lewis, J P; Horenstein, R B; Beitelshees, A L; Dane, A; Reijmers, T; Hankemeier, T; Fiehn, O; Shuldiner, A R; Kaddurah-Daouk, R

    2013-10-01

    Although aspirin is a well-established antiplatelet agent, the mechanisms of aspirin resistance remain poorly understood. Metabolomics allows for measurement of hundreds of small molecules in biological samples, enabling detailed mapping of pathways involved in drug response. We defined the metabolic signature of aspirin exposure in subjects from the Heredity and Phenotype Intervention Heart Study. Many metabolites, including known aspirin catabolites, changed on exposure to aspirin, and pathway enrichment analysis identified purine metabolism as significantly affected by drug exposure. Furthermore, purines were associated with aspirin response, and poor responders had higher postaspirin adenosine and inosine levels than did good responders (n = 76; both P < 4 × 10(-3)). Using our established "pharmacometabolomics-informed pharmacogenomics" approach, we identified genetic variants in adenosine kinase associated with aspirin response. Combining metabolomics and genomics allowed for more comprehensive interrogation of mechanisms of variation in aspirin response--an important step toward personalized treatment approaches for cardiovascular disease. PMID:23839601

  4. 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.

  5. A high-yielding, strictly regioselective prebiotic purine nucleoside formation pathway.

    PubMed

    Becker, Sidney; Thoma, Ines; Deutsch, Amrei; Gehrke, Tim; Mayer, Peter; Zipse, Hendrik; Carell, Thomas

    2016-05-13

    The origin of life is believed to have started with prebiotic molecules reacting along unidentified pathways to produce key molecules such as nucleosides. To date, a single prebiotic pathway to purine nucleosides had been proposed. It is considered to be inefficient due to missing regioselectivity and low yields. We report that the condensation of formamidopyrimidines (FaPys) with sugars provides the natural N-9 nucleosides with extreme regioselectivity and in good yields (60%). The FaPys are available from formic acid and aminopyrimidines, which are in turn available from prebiotic molecules that were also detected during the Rosetta comet mission. This nucleoside formation pathway can be fused to sugar-forming reactions to produce pentosides, providing a plausible scenario of how purine nucleosides may have formed under prebiotic conditions. PMID:27174989

  6. [Determination of individual purine and pyrimidine bases in carbohydrate-rich food].

    PubMed

    Lassek, E; Montag, A

    1987-05-01

    The following method was developed for the qualitative and quantitative determination of purine and pyrimidine bases in carbohydrate rich food: The bases were liberated from nucleic acids, nucleotides or nucleosides by acid hydrolysis in a pressure digestion vessel. A complete liberation without losses of purine bases occurs upon hydrolysis for 15 min at 240 degrees C with trifluoroacetic and formic acids (1+1; V + V), pyrimidine bases need 45 min at 240 degrees C. The products arising from side reactions (such as hydroxymethylfurfural from hexoses and furfural from pentoses) could be removed from the hydrolysate by extraction with dichlormethane. The liberated bases could be separated upon stepwise elution by cation exchange chromatography. They were detected and determined by UV-measurements, continuously monitoring at lambda = 260 nm, and integrating electronically. The evaluation was carried out by a method with internal standard. PMID:3604458

  7. 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.

  8. 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.

  9. 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. PMID:24530799

  10. 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

  11. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed Central

    Livneh, Z; Elad, D; Sperling, J

    1979-01-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  12. Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase.

    PubMed

    Miller, Danielle V; Brown, Anne M; Xu, Huimin; Bevan, David R; White, Robert H

    2016-06-01

    Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc. PMID:26990095

  13. 76 FR 80955 - Prospective Grant of Exclusive License: Use of Methanocarba Analogues of Purine and Pyrimidine...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-27

    ... (1'S,2R,3S,4'R,5'S)-4- (6-amino-2-chloro-9H-purin-9-yl)-1- bicycle hexane-2,3-diol) (MRS2339) to... is notice, in accordance with 35 U.S.C. 209(c)(1) and 37 CFR 404.7(a)(1)(i), that the National... law, will not be released under the Freedom of Information Act, 5 U.S.C. 552. Dated: December 20,...

  14. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed

    Livneh, Z; Elad, D; Sperling, J

    1979-11-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  15. Evidence for incorporation of intact dietary pyrimidine (but not purine) nucleosides into hepatic RNA.

    PubMed Central

    Berthold, H K; Crain, P F; Gouni, I; Reeds, P J; Klein, P D

    1995-01-01

    The absorption and metabolism of dietary nucleic acids have received less attention than those of other organic nutrients, largely because of methodological difficulties. We supplemented the rations of poultry and mice with the edible alga Spirulina platensis, which had been uniformly labeled with 13C by hydroponic culture in 13CO2. The rations were ingested by a hen for 4 wk and by four mice for 6 days; two mice were fed a normal diet and two were fed a nucleic acid-deficient diet. The animals were killed and nucleosides were isolated from hepatic RNA. The isotopic enrichment of all mass isotopomers of the nucleosides was analyzed by selected ion monitoring of the negative chemical ionization mass spectrum and the labeling pattern was deconvoluted by reference to the enrichment pattern of the tracer material. We found a distinct difference in the 13C enrichment pattern between pyrimidine and purine nucleosides; the isotopic enrichment of uniformly labeled [M + 9] isotopomers of pyrimidines exceeded that of purines [M + 10] by > 2 orders of magnitude in the avian nucleic acids and by 7- and 14-fold in the murine nucleic acids. The purines were more enriched in lower mass isotopomers, those less than [M + 3], than the pyrimidines. Our results suggest that large quantities of dietary pyrimidine nucleosides and almost no dietary purine nucleosides are incorporated into hepatic nucleic acids without hydrolytic removal of the ribose moiety. In addition, our results support a potential nutritional role for nucleosides and suggest that pyrimidines are conditionally essential organic nutrients. PMID:7479738

  16. 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. PMID:27127808

  17. Ab initio molecular orbital study of the structures of purine hydrates

    SciTech Connect

    Colson, A.O.; Sevilla, M.D.

    1996-03-14

    The structures of the isomers of purine hydrates [4(5)-hydroxy-5(4)-hydropurines] have been geometry optimized with ab initio quantum chemical methods at the 6-31G{sup *} basis set and with the semiempirical method PM3. These hydrates which can result from reduction of radical species formed by attack of hydroxyl radical at the 4,5 double bond in the purines, show significant geometrical distortion when compared to the natural bases. More specifically, the cis isomers adopt a `butterfly` conformation, while in the trans isomers, the pyrimidine and imidazole rings tilt opposite to each other. Our results predict the cis purine hydrate isomers are far more stable than the trans isomers by 10-18 kcal/mol at the 6-31G{sup *} level, whereas the 4-hydroxy-5-hydropurines are found to be slightly more energetically stable than the 5-hydroxy-4-hydropurines. The `butterfly` conformation of the cis isomers constitutes a bulky lesion which will result in a significant distortion of the DNA helix. 33 refs., 2 figs., 3 tabs.

  18. 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.

  19. [Degradation of purine nucleotides in patients with chronic obstruction to airflow].

    PubMed

    Mateos Antón, F; García Puig, J; Gómez Fernández, P; Ramos Hernández, T; López Jiménez, M

    1989-03-11

    The increase in hypoxanthine (Hx), xanthine (X), uric acid (VA) and total purines (TP) that may be found in several clinical conditions associated with tissue hypoxia has been attributed to an increase in adenine nucleotides degradation by a reduced ATP synthesis caused by oxygen deprivation. To test this hypothesis we have investigated the urinary excretion of Hx, X, VA, TP and radioactivity elimination after labeling the adenine nucleotides with adenine (8-14C) in 5 patients with chronic airflow obstruction (CAFO), in the basal state and after oxygen therapy (FiO2, 24%). The results were compared with those from 4 normal individuals. Patients with COFA showed an increase of the renal elimination of Hx, X, VA, TP and radioactivity, which was significantly different from the control group (p less than 0.05). Oxygen administration was associated with a significant reduction in the excretion of purines and radioactivity (p less than 0.01), which decreased to values similar to those found in normal individuals. These findings suggest that in patients with COFA and severe hypoxemia there is a marked increase in the degradation of adenine nucleotides. The normalization of the purine and radioactivity excretion after oxygen therapy points to a basic role of oxygen in the catabolism of adenine nucleotides. PMID:2716427

  20. Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe

    SciTech Connect

    Speiser, D.M.; Ortiz, D.F.; Kreppel, L.; Scheel, G.; McDonald, G.; Ow, D.W. Univ. of California, Berkeley )

    1992-12-01

    Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems. 41 refs., 8 figs., 2 tabs.

  1. Purine nucleoside phosphorylase from Schistosoma mansoni in complex with ribose-1-phosphate

    PubMed Central

    D’Muniz Pereira, Humberto; Oliva, Glaucius; Garratt, Richard Charles

    2011-01-01

    Schistosomes are blood flukes which cause schistosomiasis, a disease affecting approximately 200 million people worldwide. Along with several other important human parasites including trypanosomes and Plasmodium, schistosomes lack the de novo pathway for purine synthesis and depend exclusively on the salvage pathway for their purine requirements, making the latter an attractive target for drug development. Part of the pathway involves the conversion of inosine (or guanosine) into hypoxanthine (or guanine) together with ribose-1-phosphate (R1P) or vice versa. This inter-conversion is undertaken by the enzyme purine nucleoside phosphorylase (PNP) which has been used as the basis for the development of novel anti-malarials, conceptually validating this approach. It has been suggested that, during the reverse reaction, R1P binding to the enzyme would occur only as a consequence of conformational changes induced by hypoxanthine, thus making a binary PNP–R1P complex unlikely. Contradictory to this statement, a crystal structure of just such a binary complex involving the Schistosoma mansoni enzyme has been successfully obtained. The ligand shows an intricate hydrogen-bonding network in the phosphate and ribose binding sites and adds a further chapter to our knowledge which could be of value in the future development of selective inhibitors. PMID:21169694

  2. 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.

  3. 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. PMID:26946024

  4. Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

    PubMed Central

    Knox, Aaron J.; Graham, Christine; Bleskan, John; Brodsky, Gary; Patterson, David

    2009-01-01

    Mutations in several steps of de novo purine synthesis lead to human inborn errors of metabolism often characterized by mental retardation, hypotonia, sensorineural hearing loss, optic atrophy, and other features. In animals, the phosphoribosylglycinamide transformylase (GART) gene encodes a trifunctional protein carrying out 3 steps of de novo purine synthesis, phosphoribosylglycinamide synthase (GARS), phosphoribosylglycinamide transformylase (also abbreviated as GART), and phosphoribosylaminoimidazole synthetase (AIRS) and a smaller protein that contains only the GARS domain of GART as a functional protein. The GART gene is located on human chromosome 21 and is aberrantly regulated and overexpressed in individuals with Down syndrome (DS), and may be involved in the phenotype of DS. The GART activity of GART requires 10-formyltetrahydrofolate and has been a target for anti-cancer drugs. Thus, a considerable amount of information is available about GART, while less is known about the GARS and AIRS domains. Here we demonstrate that the amino acid residue glu75 is essential for the activity of the GARS enzyme and that the gly684 residue is essential for the activity of the AIRS enzyme by analysis of mutations in the Chinese hamster ovary (CHO-K1) cell that require purines for growth. We report the effects of these mutations on mRNA and protein content for GART and GARS. Further, we discuss the likely mechanisms by which mutations inactivating the GART protein might arise in CHO-K1 cells. PMID:19007868

  5. Pronounced Fixation, Strong Population Differentiation and Complex Population History in the Canary Islands Blue Tit Subspecies Complex

    PubMed Central

    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

  6. 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. PMID:27187616

  7. 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

  8. Strenuous exercise induces a hyperreactive rebalanced haemostatic state that is more pronounced in men.

    PubMed

    Huskens, Dana; Roest, Mark; Remijn, Jasper A; Konings, Joke; Kremers, Romy M W; Bloemen, Saartje; Schurgers, Evelien; Selmeczi, Anna; Kelchtermans, Hilde; van Meel, Rinaldo; Meex, Steven J; Kleinegris, Marie-Claire; de Groot, Philip G; Urbanus, Rolf T; Ninivaggi, Marisa; de Laat, Bas

    2016-06-01

    Physical exercise is recommended for a healthy lifestyle. Strenuous exercise, however, may trigger the haemostatic system, increasing the risk of vascular thrombotic events and the incidence of primary cardiac arrest. Our goal was to study the effects of strenuous exercise on risk factors of cardiovascular disease. Blood was collected from 92 healthy volunteers who participated in the amateur version of the pro-tour Amstel Gold cycling race, before and directly after the race. Thrombin generation showed a shortening of the lag time and time to peak and an increase of the velocity index. Interestingly, the endogenous thrombin potential measured in plasma decreased due to reduced prothrombin conversion. Platelet reactivity increased and this effect was stronger in men than in women. Lower fibrinogen and higher D-dimer levels after exercise indicated higher fibrin formation. On the other hand, fibrinolysis was also elevated as indicated by a shortening of the clot lysis time. Exercise activated the endothelium (von Willebrand factor (VWF) and active VWF levels were elevated) and the immune system (concentrations IL-6, IL-8, MCP-1, RANTES and PDGF increased). Additionally, an increased cardiac troponin T level was measured post-exercise. Strenuous exercise induces a temporary hyperreactive state in the body with enhanced pro- and anticoagulant responses. As strenuous exercise has a more pronounced effect on platelet function in male subjects, this gives a possible explanation for the higher incidence of sudden cardiac death during exercise compared to women. This trial is registered at www.clinicaltrials.gov as NCT02048462. PMID:26864794

  9. Resolution of pronounced painless weakness arising from radiculopathy and disk extrusion.

    PubMed

    Lipetz, Jason S; Misra, Neelam; Silber, Jeff S

    2005-07-01

    In this retrospective, consecutive case series, we report the nonsurgical and rehabilitation outcomes of consecutive patients who presented with pronounced painless weakness arising from disk extrusion. Seven consecutive patients who chose physiatric care were followed clinically, and strength return was monitored. Each presented with predominantly painless radiculopathy, functionally significant strength loss, and radiographic evidence of disk extrusion or sequestration. Each patient participated in a targeted strengthening program, and in some cases, transforaminal injection therapy was employed. Each patient demonstrated an eventual full functional recovery. In most cases, electrodiagnostic studies were performed and included a needle examination of the affected limb and compound muscle action potentials from the most clinically relevant and weakened limb muscle. The electrodiagnostic findings and, in particular, the quantitative compound muscle action potential data seemed to correlate with the timing of motor recovery. Patients with predominantly painless and significant weakness arising from disk extrusion can demonstrate successful rehabilitation outcomes. Despite a relative absence of pain, such patients can present with a more rapidly reversible neurapraxic type of weakness. The more quantitative compound muscle action potential data obtained through electrodiagnostic studies may offer the treating physician an additional means of characterizing the type of neuronal injury at play and the likelihood and timing of strength return. PMID:15973090

  10. Pronounced and prevalent intersexuality does not impede the 'Demon Shrimp' invasion.

    PubMed

    Green Etxabe, Amaia; Short, Stephen; Flood, Tim; Johns, Tim; Ford, Alex T

    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

  11. 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

  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

    2014-05-01

    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

  13. 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.

  14. 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

  15. The purine degradation pathway: possible role in paralytic shellfish toxin metabolism in the cyanobacterium Planktothrix sp. FP1.

    PubMed

    Pomati, F; Manarolla, G; Rossi, O; Vigetti, D; Rossetti, C

    2001-12-01

    The paralytic shellfish toxins (PSTs) are potent neurotoxic alkaloids and their major biological effect is due to the blockage of voltage-gated sodium channels in excitable cells. They have been recognised as an important health risk for humans, animals, and ecosystems worldwide. The metabolic pathways that lead to the production and the degradation of these toxic metabolites are still unknown. In this study, we investigated the possible link between PST accumulation and the activation of the metabolism that leads to purine degradation in the filamentous freshwater cyanobacterium Planktothrix sp. FP1. The purine catabolic pathway is related to the nitrogen microcycle in water environments, in which cyanobacteria use traces of purines and ureides as a nitrogen source for growth. Thus, the activity of allantoicase, a key inducible enzyme of this metabolism, was used as tool for assaying the activation of the purine degradation pathway. The enzyme and the pathway were induced by allantoic acid, the direct substrate of allantoicase, as well as by adenine and, to a lower degree, by urea, one of the main products of purine catabolism. Crude cell extract of Escherichia coli was also employed and showed the best induction of allantoicase activity. In culture, Planktothrix sp. FP1 showed a differential accumulation of PST in consequence of the induction with different substrates. The cyanobacterial culture induced with allantoic acid accumulated 61.7% more toxins in comparison with the control. On the other hand, the cultures induced with adenine, urea, and the E. coli extract showed low PST accumulation, respectively, 1%, 38%, and 5% of the total toxins content detected in the noninduced culture. A degradation pathway for the PSTs can be hypothesised: as suggested for purine alkaloids in higher plants, saxitoxin (STX) and derivatives may also be converted into xanthine, urea, and further to CO2 and NH4+ or recycled in the primary metabolism through the purine degradation

  16. The genomes of the South American opossum (Monodelphis domestica) and platypus (Ornithorhynchus anatinus) encode a more complete purine catabolic pathway than placental mammals.

    PubMed

    Keebaugh, Alaine C; Thomas, James W

    2009-09-01

    The end product of purine catabolism varies amongst vertebrates and is a consequence of independent gene inactivation events that have truncated the purine catabolic pathway. Mammals have traditionally been grouped into two classes based on their end product of purine catabolism: most mammals, whose end product is allantoin due to an ancient loss of allantoinase (ALLN), and the hominoids, whose end product is uric acid due to recent inactivations of urate oxidase (UOX). However little is known about purine catabolism in marsupials and monotremes. Here we report the results of a comparative genomics study designed to characterize the purine catabolic pathway in a marsupial, the South American opossum (Monodelphis domestica), and a monotreme, the platypus (Ornithorhynchus anatinus). We found that both genomes encode a more complete set of genes for purine catabolism than do eutherians and conclude that a near complete purine catabolic pathway was present in the common ancestor of all mammals, and that the loss of ALLN is specific to placental mammals. Our results therefore provide a revised history for gene loss in the purine catabolic pathway and suggest that marsupials and monotremes represent a third class of mammals with respect to their end products of purine catabolism. PMID:20161190

  17. [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.

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

    PubMed Central

    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

  19. 1,3,5-Triazine-based analogues of purine: from isosteres to privileged scaffolds in medicinal chemistry.

    PubMed

    Lim, Felicia Phei Lin; Dolzhenko, Anton V

    2014-10-01

    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. PMID:25105925

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

    PubMed

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

    2015-09-01

    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 human African trypanosomiasis. 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

  1. Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals

    PubMed Central

    Furukawa, Junji; Inoue, Katsuhisa; Maeda, Junya; Yasujima, Tomoya; Ohta, Kinya; Kanai, Yoshikatsu; Takada, Tappei; Matsuo, Hirotaka; Yuasa, Hiroaki

    2015-01-01

    The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na+ and H+, but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes. PMID:26455426

  2. Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals.

    PubMed

    Furukawa, Junji; Inoue, Katsuhisa; Maeda, Junya; Yasujima, Tomoya; Ohta, Kinya; Kanai, Yoshikatsu; Takada, Tappei; Matsuo, Hirotaka; Yuasa, Hiroaki

    2015-01-01

    The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na(+) and H(+), but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes. PMID:26455426

  3. 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

  4. Distally pronounced infantile spinal muscular atrophy with severe axonal and demyelinating neuropathy associated with the S230L mutation of SMN1.

    PubMed

    Rudnik-Schöneborn, Sabine; Barisić, Nina; Eggermann, Katja; Ortiz Brüchle, Nadina; Grđan, Petra; Zerres, Klaus

    2016-02-01

    Two Croatian siblings with atypical clinical findings in the presence of SMN1 gene mutations are reported. The girl presented with delayed motor development and weakness in hands and feet in her first year of life. She never stood or walked and developed scoliosis and joint contractures during childhood. Her hands and feet were non-functional when last seen at age 14 years. Her 4-year-old brother was more severely affected and had a clinical picture resembling infantile spinal muscular atrophy (SMA) type 1. He also showed unusual distally pronounced weakness and facial weakness. Both patients had no sensory deficits but gave evidence of a mixed axonal and demyelinating neuropathy with pronounced slowing in the distal nerve segments. Unexpectedly, both siblings showed a compound heterozygous SMN1 mutation (heterozygous deletion and missense mutation c.689C > T; p.S230L), thus confirming infantile SMA. In addition, next generation sequencing of 52 genes for hereditary neuropathies revealed a heterozygous missense mutation c.505T > C; p.Y169H in the SH3TC2 gene that was transmitted by the healthy father. Our observations widen the phenotypic consequences of SMN1 gene mutations and support the notion to look for additional genetic factors which may modify the clinical picture in atypical cases. PMID:26794302

  5. Hepatic and seric levels of purines in rats experimentally infected by Fasciola hepatica.

    PubMed

    Baldissera, Matheus D; Mendes, Ricardo E; Doleski, Pedro H; Bottari, Nathieli B; Casali, Emerson A; Moritz, Cesar Eduardo Jacintho; Cardoso, Valesca V; Henker, Luan C; Christ, Ricardo; Stedille, Fernanda A; Stefani, Lenita M; Da Silva, Aleksandro S

    2016-06-01

    The aim of this study was to evaluate hepatic and seric levels of purines, as well as their breakdown products in rats infected by Fasciola hepatica on days 15 and 87 post-infection (PI). Rats were divided into two groups: uninfected (n = 10) and infected (n = 20). On day 15 (n = 5 for uninfected group and n = 10 for infected group) and 87 PI (n = 5 for uninfected group and n = 10 for infected group), animals were euthanized for sampling to evaluate levels of purines by high-performance liquid chromatography. In serum, ATP increased (P < 0.05) and ADP decreased (P < 0.05) on days 15 and 87 PI, while AMP increased (P < 0.05) only on day 15 PI. Hypoxanthine levels increased (P < 0.05) on days 15 and 87 PI, while adenosine and xanthine levels decreased and increased (P < 0.05), respectively, on day 87 PI. No difference was observed regarding seric inosine and uric acid (P > 0.05). Hepatic ATP, adenosine, and uric acid levels decreased (P < 0.05) on days 15 and 87 PI. AMP levels decreased (P < 0.05) on day 87 PI, while xanthine levels increased (P < 0.05) on day 15 PI in the liver. Also in the liver, hypoxanthine levels increased (P < 0.05) on day 15 PI and decreased (P < 0.05) on day 87 PI. On the other hand, there was no difference on hepatic ADP and inosine levels (P > 0.05). Therefore, it is possible to conclude that F. hepatica infection can change purine levels, which may be associated with an inflammatory process, and these alterations may influence fasciolosis pathogenesis. PMID:26971323

  6. Targeting the parasite's DNA with methyltriazenyl purine analogs is a safe, selective, and efficacious antitrypanosomal strategy.

    PubMed

    Rodenko, Boris; Wanner, Martin J; Alkhaldi, Abdulsalam A M; Ebiloma, Godwin U; Barnes, Rebecca L; Kaiser, Marcel; Brun, Reto; McCulloch, Richard; Koomen, Gerrit-Jan; de Koning, Harry P

    2015-11-01

    The human and veterinary disease complex known as African trypanosomiasis continues to inflict significant global morbidity, mortality, and economic hardship. Drug resistance and toxic side effects of old drugs call for novel and unorthodox strategies for new and safe treatment options. We designed methyltriazenyl purine prodrugs to be rapidly and selectively internalized by the parasite, after which they disintegrate into a nontoxic and naturally occurring purine nucleobase, a simple triazene-stabilizing group, and the active toxin: a methyldiazonium cation capable of damaging DNA by alkylation. We identified 2-(3-acetyl-3-methyltriazen-1-yl)-6-hydroxypurine (compound 1) as a new lead compound, which showed submicromolar potency against Trypanosoma brucei, with a selectivity index of >500, and it demonstrated a curative effect in animal models of acute trypanosomiasis. We investigated the mechanism of action of this lead compound and showed that this molecule has significantly higher affinity for parasites over mammalian nucleobase transporters, and it does not show cross-resistance with current first-line drugs. Once selectively accumulated inside the parasite, the prodrug releases a DNA-damaging methyldiazonium cation. We propose that ensuing futile cycles of attempted mismatch repair then lead to G2/M phase arrest and eventually cell death, as evidenced by the reduced efficacy of this purine analog against a mismatch repair-deficient (MSH2(-/-)) trypanosome cell line. The observed absence of genotoxicity, hepatotoxicity, and cytotoxicity against mammalian cells revitalizes the idea of pursuing parasite-selective DNA alkylators as a safe chemotherapeutic option for the treatment of human and animal trypanosomiasis. PMID:26282430

  7. 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. PMID:26986520

  8. Targeting the Parasite's DNA with Methyltriazenyl Purine Analogs Is a Safe, Selective, and Efficacious Antitrypanosomal Strategy

    PubMed Central

    Wanner, Martin J.; Alkhaldi, Abdulsalam A. M.; Ebiloma, Godwin U.; Barnes, Rebecca L.; Kaiser, Marcel; Brun, Reto; McCulloch, Richard; Koomen, Gerrit-Jan

    2015-01-01

    The human and veterinary disease complex known as African trypanosomiasis continues to inflict significant global morbidity, mortality, and economic hardship. Drug resistance and toxic side effects of old drugs call for novel and unorthodox strategies for new and safe treatment options. We designed methyltriazenyl purine prodrugs to be rapidly and selectively internalized by the parasite, after which they disintegrate into a nontoxic and naturally occurring purine nucleobase, a simple triazene-stabilizing group, and the active toxin: a methyldiazonium cation capable of damaging DNA by alkylation. We identified 2-(3-acetyl-3-methyltriazen-1-yl)-6-hydroxypurine (compound 1) as a new lead compound, which showed submicromolar potency against Trypanosoma brucei, with a selectivity index of >500, and it demonstrated a curative effect in animal models of acute trypanosomiasis. We investigated the mechanism of action of this lead compound and showed that this molecule has significantly higher affinity for parasites over mammalian nucleobase transporters, and it does not show cross-resistance with current first-line drugs. Once selectively accumulated inside the parasite, the prodrug releases a DNA-damaging methyldiazonium cation. We propose that ensuing futile cycles of attempted mismatch repair then lead to G2/M phase arrest and eventually cell death, as evidenced by the reduced efficacy of this purine analog against a mismatch repair-deficient (MSH2−/−) trypanosome cell line. The observed absence of genotoxicity, hepatotoxicity, and cytotoxicity against mammalian cells revitalizes the idea of pursuing parasite-selective DNA alkylators as a safe chemotherapeutic option for the treatment of human and animal trypanosomiasis. PMID:26282430

  9. 2,6,9-Trisubstituted purines as CRK3 kinase inhibitors with antileishmanial activity in vitro.

    PubMed

    Řezníčková, Eva; Popa, Alexandr; Gucký, Tomáš; Zatloukal, Marek; Havlíček, Libor; Bazgier, Václav; Berka, Karel; Jorda, Radek; Popa, Igor; Nasereddin, Abdelmajeed; Jaffe, Charles L; Kryštof, Vladimír; Strnad, Miroslav

    2015-06-01

    Here we describe the leishmanicidal activities of a library of 2,6,9-trisubstituted purines that were screened for interaction with Cdc2-related protein kinase 3 (CRK3) and subsequently for activity against parasitic Leishmania species. The most active compound inhibited recombinant CRK3 with an IC50 value of 162 nM and was active against Leishmania major and Leishmania donovani at low micromolar concentrations in vitro. Its mode of binding to CRK3 was investigated by molecular docking using a homology model. PMID:25937014

  10. QSAR studies on benzodiazepine receptor binding of purines and amino acid derivatives.

    PubMed

    Saha, R N; Meera, J; Agrawal, N; Gupta, S P

    1991-01-01

    Quantitative structure-activity relationship (QSAR) studies are reported on the benzodiazepine receptor binding of a series of substituted 9-benzyl-6-dimethylamino-9H-purines and N-(indol-3-ylglyoxylyl)amino acid derivatives. The nitrogen of the five membered heterocyclic ring and the polar substituent in the aromatic ring, present in both series of compounds, form important centres in the binding interaction. We conclude that the receptor must possess a strong nucleophilic centre and a polar site, and that a hydrophobic pocket exists to accommodate hydrophobic moieties. PMID:1654919

  11. Inhibition and structure of Trichomonas vaginalis purine nucleoside phosphorylase with picomolar transition state analogues.

    PubMed

    Rinaldo-Matthis, Agnes; Wing, Corin; Ghanem, Mahmoud; Deng, Hua; Wu, Peng; Gupta, Arti; Tyler, Peter C; Evans, Gary B; Furneaux, Richard H; Almo, Steven C; Wang, Ching C; Schramm, Vern L

    2007-01-23

    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 state 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 Km/Kd 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 Km/Kd ratio of 203,300. The tight binding of DADMe-ImmA supports a late SN1 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 x ImmA x PO4 and TvPNP x DADMe-ImmA x PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 A ionic interaction between a PO4 oxygen and the N1' cation of the hydroxypyrrolidine and is weaker in the TvPNP x ImmA x PO4 structure at 3.5 A. However, the TvPNP x ImmA x PO4 structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP x DADMe-ImmA x PO4. 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-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP x ImmH x PO4, causing an unfavorable leaving-group interaction

  12. 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.

  13. Analysis of purine metabolic enzymes in human CD4 Leu 8- and CD4 Leu 8+ lymphocyte subpopulations.

    PubMed

    Fernandez-Mejia, C; Polmar, S H; Peralta-Zaragoza, O; Madrid-Marina, V

    1993-02-01

    1. Specific activities of adenosine deaminase, purine nucleoside phosphorylase, adenosine kinase, 5'-nucleotidase, S-adenosyl-L-homocysteine hydrolase, AMP deaminase, adenine phosphoribosyl transferase, and hypoxanthine phosphoribosyl transferase were analyzed in human CD4 T-lymphocyte subsets. 2. CD4 Leu 8- (helper/inducer) and CD4 Leu 8+ (suppressor/inducer) subpopulations were obtained by panning or fluorescence activated cell sorting techniques using specific monoclonal antibodies. 3. A 45% decrease of 5'-NT AMP activity in the CD4 Leu 8- cells (suppressor/inducer) compared with CD4 total cell population. 4. No statistical significant differences in enzyme activity were found between the subsets analyzed in other purine enzymes. 5. These results suggest that the distribution of purine metabolic enzymes is homogeneous in CD4 Leu 8- and CD4 Leu 8+ T-lymphocyte subpopulations. PMID:8444317

  14. Pronounced and extensive microtubule defects in a Saccharomyces cerevisiae DIS3 mutant.

    PubMed

    Smith, Sarah B; Kiss, Daniel L; Turk, Edward; Tartakoff, Alan M; Andrulis, Erik D

    2011-11-01

    Subunits of the RNA processing exosome assemble into structurally distinct protein complexes that function in disparate cellular compartments and RNA metabolic pathways. Here, in a genetic, cell biological and transcriptomic analysis, we examined the role of Dis3, an essential polypeptide with endo- and 3'→5' exo-ribonuclease activity, in cell cycle progression. We present several lines of evidence that perturbation of DIS3 affects microtubule (MT) localization and structure in Saccharomyces cerevisiae. Cells with a DIS3 mutant: (a) accumulate anaphase and pre-anaphase mitotic spindles; (b) exhibit spindles that are misorientated and displaced from the bud neck; (c) harbour elongated spindle-associated astral MTs; (d) have an increased G1 astral MT length and number; and (e) are hypersensitive to MT poisons. Mutations in the core exosome genes RRP4 and MTR3 and the exosome cofactor gene MTR4, but not other exosome subunit gene mutants, also elicit MT phenotypes. RNA deep sequencing analysis (RNA-seq) shows broad changes in the levels of cell cycle- and MT-related transcripts in mutant strains. Collectively, the data presented in this study suggest an evolutionarily conserved role for Dis3 in linking RNA metabolism, MTs and cell cycle progression. PMID:21919057

  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. 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. PMID:25728022

  17. Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase.

    PubMed

    Filgueira de Azevedo, Walter; dos Santos, Giovanni César; dos Santos, Denis Marangoni; Olivieri, Johnny Rizzieri; Canduri, Fernanda; Silva, Rafael Guimarães; Basso, Luiz Augusto; Renard, Gaby; da Fonseca, Isabel Osório; Mendes, Maria Anita; Palma, Mário Sérgio; Santos, Diógenes Santiago

    2003-10-01

    Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed. PMID:13679062

  18. 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

  19. The purine nucleotide cycle. A pathway for ammonia production in the rat kidney.

    PubMed Central

    Bogusky, R T; Lowenstein, L M; Lowenstein, J M

    1976-01-01

    Particle-free extracts prepared from kidney cortex of rat catalyze the formation of ammonia via the purine nucleotide cycle. The cycle generates ammonia and fumarate from aspartate, using catalytic amounts of inosine monophosphate, adenylosuccinate, and adenosine monophosphate. The specific activities of the enzymes of the cycle are 1.27+/-0.27 nmol/mg protein per min (SE) for adenoylosuccinate synthetase, 1.38+/-0.16 for adenylosuccinase, and 44.0+/-3.3 for AMP deaminase. Compared with controls, extracts prepared from kidneys of rats fed ammonium chloride for 2 days show a 60% increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity, and a greater and more rapid synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate. Extracts prepared from kidneys of rats fed a potassium-deficient diet show a twofold increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity. In such extracts the rate of synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate is also increased. These results show that the reactions of the purine nucleotide cycle are present and can operate in extracts of kidney cortex. The operational capacity of the cycle is accelerated by ammonium chloride feeding and potassium depletion, conditions known to increase renal ammonia excretion. Extracts of kidney cortex convert inosine monophosphate to uric acid. This is prevented by addition of allopurinol of 1-pyrophosphoryl ribose 5-phosphate to the reaction mixture. PMID:821968

  20. 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

  1. Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii.

    PubMed

    Kamel, M Y; Fahmy, A S; Ghazy, A H; Mohamed, M A

    1991-04-01

    Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56,000 - 58,000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of beta-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6-7 and 8-9. The enzyme was completely inactivated by thiol reagents and reactivated by excess beta-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism. PMID:1905141

  2. Structural Phylogenomics Reveals Gradual Evolutionary Replacement of Abiotic Chemistries by Protein Enzymes in Purine Metabolism

    PubMed Central

    Caetano-Anollés, Kelsey; Caetano-Anollés, Gustavo

    2013-01-01

    The origin of metabolism has been linked to abiotic chemistries that existed in our planet at the beginning of life. While plausible chemical pathways have been proposed, including the synthesis of nucleobases, ribose and ribonucleotides, the cooption of these reactions by modern enzymes remains shrouded in mystery. Here we study the emergence of purine metabolism. The ages of protein domains derived from a census of fold family structure in hundreds of genomes were mapped onto enzymes in metabolic diagrams. We find that the origin of the nucleotide interconversion pathway benefited most parsimoniously from the prebiotic formation of adenine nucleosides. In turn, pathways of nucleotide biosynthesis, catabolism and salvage originated ∼300 million years later by concerted enzymatic recruitments and gradual replacement of abiotic chemistries. Remarkably, this process led to the emergence of the fully enzymatic biosynthetic pathway ∼3 billion years ago, concurrently with the appearance of a functional ribosome. The simultaneous appearance of purine biosynthesis and the ribosome probably fulfilled the expanding matter-energy and processing needs of genomic information. PMID:23516625

  3. 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. PMID:22890959

  4. 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

  5. Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.

    PubMed

    de Moraes, Marcela Cristina; Cardoso, Carmen L; Cass, Quezia B

    2013-05-01

    The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol L (-1) and 164 ± 13.4 μmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives. PMID:23535739

  6. Survival of Purines and Pyrimidines Adsorbed on a Solid Surface in a High Radiation Field

    NASA Astrophysics Data System (ADS)

    Guzman-Marmolejo, A.; Ramos-Bernal, S.; Negrón-Mendoza, A.

    2009-12-01

    According to astronomical data, organic molecules are abundant in interstellar space. These molecules have arisen from non-equilibrium processes driven by the energy of photons and cosmic rays. The presence of dirty ices show that a rich low temperature solid phase chemistry takes place in such environments. These chemical evolution reactions have been assumed to proceed mainly within solid surfaces of interstellar dust particles, as well as on macrobodies. Among solid surfaces for chemical processes, alumino-silicates are widely distributed in terrestrial and extraterrestrial bodies, such as meteorites, and the Martian soil, which showed the presence of carbonates and clays. Therefore, alumino-silicates are considered a likely inorganic material to promote organic reactions that might have played a role in the survival of organic molecules adsorbed on their surfaces. It is also known that they have a high surface area and a high affinity for organic compounds. Purines and pyrimidines are important organic compounds due to their role in biological processes. Their synthesis and stability are of paramount importance in chemical evolution. In this work we propose a mechanism to account for the survival of purines and pyrimidines adsorbed in a solid surface in a high radiation field.

  7. Encapsulation of ruthenium nitrosylnitrate and DNA purines in nanostructured sol-gel silica matrices.

    PubMed

    Lopes, Luís M F; Garcia, Ana R; Fidalgo, Alexandra; Ilharco, Laura M

    2009-09-01

    The interactions between DNA purines (guanine and adenine) and the ruthenium complex Ru(NO)(NO(3))(3) were studied within nanostructured silica matrices prepared by a two-step sol-gel process. By infrared analysis in diffuse reflectance mode, it was proved that encapsulation induces a profound modification on the complex, whereas guanine and adenine preserve their structural integrity. The complex undergoes nitrate ligand exchange and co-condenses with the silica oligomers, but the nitrosyl groups remain stable, which is an unusual behavior in Ru nitrosyl complexes. In turn, the doping molecules affect the sol-gel reactions and eventually the silica structure as it forms: the complex yields a microporous structure, and the purine bases are responsible for the creation of macropores due to hydrogen bonding with the silanol groups of the matrix. In a confined environment, the interactions are much stronger for the coencapsulated pair guanine complex. While adenine only establishes hydrogen bonds or van der Waals interactions with the complex, guanine bonds covalently to Ru by one N atom of the imidazole ring, which becomes strongly perturbed, resulting in a deformation of the complex geometry. PMID:19499946

  8. Gout and hyperuricemia in Japan: perspectives for international research on purines and pyrimidines in man.

    PubMed

    Hosoya, Tatsuo; Ohno, Iwao; Ichida, Kimiyoshi; Peters, Godefridus J

    2011-12-01

    One of the best-known disorders in purine metabolism is accumulation of uric acid leading to gout. Gout is a lifestyle disease, which was nicely illustrated in the joint symposium of the Japanese Society of Gout and Nucleic Acid Metabolism and of the Purine and Pyrimidine Society held in February 2011 in Tokyo, Japan. The westernization of the Japanese diet led to an increase in hyperuricemia in Japanese, which subsequently boosted research in this field, as illustrated in this symposium. As a consequence, Japanese nucleotide research also expanded, leading to the development of not only new drugs for treatment of gout, but also for other diseases such as cancer, viral infections, and cardiovascular diseases. The research on inborn errors led to the identification of various genetic polymorphisms affecting drug metabolism, revealing differences between Asians and non-Asians. Such genetic differences may also affect the enzymatic properties of an enzyme or a transporter, necessitating specific inhibitors. This knowledge will help to introduce personalization of treatment. In this symposium, the interaction between various specialties formed an excellent basis for translational research between these specialties but also from the bench to the clinic. PMID:22132949

  9. Xanthine metabolism in Bacillus subtilis: characterization of the xpt-pbuX operon and evidence for purine- and nitrogen-controlled expression of genes involved in xanthine salvage and catabolism.

    PubMed Central

    Christiansen, L C; Schou, S; Nygaard, P; Saxild, H H

    1997-01-01

    The xpt and pbuX genes from Bacillus subtilis were cloned, and their nucleotide sequences were determined. The xpt gene encodes a specific xanthine phosphoribosyltransferase, and the pbuX gene encodes a xanthine-specific purine permease. The genes have overlapping coding regions, and Northern (RNA) blot analysis indicated an operon organization. The translation of the second gene, pbuX, was strongly dependent on the translation of the first gene, xpt. Expression of the operon was repressed by purines, and the effector molecules appear to be hypoxanthine and guanine. When hypoxanthine and guanine were added together, a 160-fold repression was observed. The regulation of expression was at the level of transcription, and we propose that a transcription termination-antitermination control mechanism similar to the one suggested for the regulation of the purine biosynthesis operon exists. The expression of the xpt-pbuX operon was reduced when hypoxanthine served as the sole nitrogen source. Under these conditions, the level of the hypoxanthine- and xanthine-degrading enzyme, xanthine dehydrogenase, was induced more than 80-fold. The xanthine dehydrogenase level was completely derepressed in a glnA (glutamine synthetase) genetic background. Although the regulation of the expression of the xpt-pbuX operon was found to be affected by the nitrogen source, it was normal in a glnA mutant strain. This result suggests the existence of different signalling pathways for repression of the transcription of the xpt-pbuX operon and the induction of xanthine dehydrogenase. PMID:9098051

  10. Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation*

    PubMed Central

    Zhang, Huidong; Beckman, Jeff W.; Guengerich, F. Peter

    2009-01-01

    The human DNA polymerase κ homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces “−1” frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N2-etheno-2′-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of −1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711–36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the “+1” base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of −1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for −1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed “dNTP-stabilized incorporation.” The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base. PMID:19837980

  11. Frameshift deletion by Sulfolobus solfataricus P2 DNA polymerase Dpo4 T239W is selective for purines and involves normal conformational change followed by slow phosphodiester bond formation.

    PubMed

    Zhang, Huidong; Beckman, Jeff W; Guengerich, F Peter

    2009-12-11

    The human DNA polymerase kappa homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces "-1" frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N(2)-etheno-2'-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of -1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711-36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the "+1" base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of -1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for -1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed "dNTP-stabilized incorporation." The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base. PMID:19837980

  12. Increasing Occurrences and Functional Roles for High Energy Purine-Pyrimidine Base-Pairs in Nucleic Acids

    PubMed Central

    Kimsey, Isaac; Al-Hashimi, Hashim M.

    2014-01-01

    There are a growing number of studies reporting the observation of purine-pyrimidine base-pairs that are seldom observed in unmodified nucleic acids because they entail the loss of energetically favorable interactions or require energetically costly base ionization or tautomerization. These high energy purine-pyrimidine base-pairs include G•C+ and A•T Hoogsteen base-pairs, which entail ~180° rotation of the purine base in a Watson-Crick base-pair, protonation of cytosine N3, and constriction of the C1′–C1′ distance by ~2.5 Å. Other high energy pure-pyrimidine base-pairs include G•T, G•U, and A•C mispairs that adopt Watson-Crick like geometry through either base ionization or tautomerization. Although difficult to detect and characterize using biophysical methods, high energy purine-pyrimidine base-pairs appear to be more common than once thought. They further expand the structural and functional diversity of canonical and noncanonical nucleic acid base-pairs. PMID:24721455

  13. Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

    PubMed

    Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

    2016-04-12

    Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity. PMID:26985580

  14. Purification and Properties of a Protein Which Binds Cytokinin-active 6-Substituted Purines 1

    PubMed Central

    Erion, Jack L.; Fox, J. Eugene

    1981-01-01

    A protein which binds 6-substituted purines of the cytokinin type with relatively high affinity has been extensively purified from wheat germ. Conventional chromatographic techniques, as well as an affinity matrix to which a cytokinin was covalently coupled, were used in the purification. The wheat germ cytokinin-binding protein (CBF-1) has four unlike subunits and an apparent molecular weight of 183,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CBF-1 is saturated at one cytokinin molecule per tetramer with a Kd for 6-benzylaminopurine of 5 × 10−7 molar. The protein exists both on the native wheat germ ribosome (1 molecule CBF-1 per 80S ribosome) and free in the cytosol with approximately three copies of the latter for each of the former. Data from affinity chromatography studies and cross-linking experiments strongly suggest that a specific binding site for CBF-1 occurs on the wheat germ ribosome. Images PMID:16661618

  15. Diversity-oriented synthesis of acyclic nucleosides via ring-opening of vinyl cyclopropanes with purines.

    PubMed

    Niu, Hong-Ying; Du, Cong; Xie, Ming-Sheng; Wang, Yong; Zhang, Qian; Qu, Gui-Rong; Guo, Hai-Ming

    2015-02-25

    The diversity-oriented synthesis of acyclic nucleosides has been achieved via ring-opening of vinyl cyclopropanes with purines. With Pd2(dba)3·CHCl3 as a catalyst, the 1,5-ring-opening reaction proceeded well and afforded N9 adducts as the major form, in which the C=C bonds in the side chain were exclusively E-form. In the presence of AlCl3, the 1,3-ring-opening reaction occurred smoothly, giving N9 adducts as the dominate products. Meanwhile, when MgI2 was used as the catalyst, the 1,3-ring-opening reaction also worked well to form N7 adducts. PMID:25572827

  16. The ligand binding mechanism to purine nucleoside phosphorylase elucidated via molecular dynamics and machine learning

    PubMed Central

    Decherchi, Sergio; Berteotti, Anna; Bottegoni, Giovanni; Rocchia, Walter; Cavalli, Andrea

    2015-01-01

    The study of biomolecular interactions between a drug and its biological target is of paramount importance for the design of novel bioactive compounds. In this paper, we report on the use of molecular dynamics (MD) simulations and machine learning to study the binding mechanism of a transition state analogue (DADMe–immucillin-H) to the purine nucleoside phosphorylase (PNP) enzyme. Microsecond-long MD simulations allow us to observe several binding events, following different dynamical routes and reaching diverse binding configurations. These simulations are used to estimate kinetic and thermodynamic quantities, such as kon and binding free energy, obtaining a good agreement with available experimental data. In addition, we advance a hypothesis for the slow-onset inhibition mechanism of DADMe–immucillin-H against PNP. Combining extensive MD simulations with machine learning algorithms could therefore be a fruitful approach for capturing key aspects of drug–target recognition and binding. PMID:25625196

  17. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed. PMID:24630982

  18. Escherichia coli pfs transcription: regulation and proposed roles in autoinducer-2 synthesis and purine excretion.

    PubMed

    Kim, Youngbae; Lew, Chih M; Gralla, Jay D

    2006-11-01

    Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions. PMID:16950920

  19. Are purines mediators of the anticonvulsant/neuroprotective effects of ketogenic diets?

    PubMed Central

    Masino, Susan A.; Geiger, Jonathan D.

    2015-01-01

    Abnormal neuronal signaling caused by metabolic changes characterizes several neurological disorders, and in some instances metabolic interventions provide therapeutic benefits. Indeed, altering metabolism either by fasting or by maintaining a low-carbohydrate (ketogenic) diet might reduce epileptic seizures and offer neuroprotection in part because the diet increases mitochondrial biogenesis and brain energy levels. Here we focus on a novel hypothesis that a ketogenic diet-induced change in energy metabolism increases levels of ATP and adenosine, purines that are critically involved in neuron–glia interactions, neuromodulation and synaptic plasticity. Enhancing brain bioenergetics (ATP) and increasing levels of adenosine, an endogenous anticonvulsant and neuroprotective molecule, might help with understanding and treating a variety of neurological disorders. PMID:18471903

  20. 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

  1. Structural basis for selective inhibition of purine nucleoside phosphorylase from Schistosoma mansoni: kinetic and structural studies.

    PubMed

    Castilho, Marcelo S; Postigo, Matheus P; Pereira, Humberto M; Oliva, Glaucius; Andricopulo, Adriano D

    2010-02-15

    Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity. PMID:20129792

  2. N-Benzyl-9-isopropyl-9H-purin-6-amine

    PubMed Central

    Gergela, David; Rouchal, Michal; Bartoš, Peter; Vícha, Robert

    2013-01-01

    The asymmetric unit of the title compound, C15H17N5, consists of two mol­ecules in which the dihedral angles between the best planes of the purine ring system (r.m.s. deviations = 0.0060 and 0.0190 Å) and the benzene ring are 89.21 (3) and 82.14 (4)°. The mol­ecules within the asymmetric unit are linked into dimers by pairs of N—H⋯N hydrogen bonds. Weak C—H⋯π contacts and π–π inter­actions [centroid–centroid = 3.3071 (1) Å] further connect the mol­ecules into a three-dimensional network. PMID:23795114

  3. 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. PMID:26506131

  4. Solution structures of purine base analogues 9-deazaguanine and 9-deazahypoxanthine.

    PubMed

    Karnawat, Vishakha; Puranik, Mrinalini

    2016-03-01

    Deaza analogues of nucleobases are potential drugs against infectious diseases caused by parasites. A caveat is that apart from binding their target parasite enzymes, they also bind and inhibit enzymes of the host. In order to design derivatives of deaza analogues which specifically bind target enzymes, knowledge of their molecular structure, protonation state, and predominant tautomers at physiological conditions is essential. We have employed resonance Raman spectroscopy at an excitation wavelength of 260 nm, to decipher solution structure of 9-deazaguanine (9DAG) and 9-deazahypoxanthine (9DAH). These are analogues of guanine and hypoxanthine, respectively, and have been exploited to study static complexes of nucleobase binding enzymes. Such enzymes are known to perturb pKa of their ligands, and thus, we also determined solution structures of these analogues at two, acidic and alkaline, pH. Structure of each possible protonation state and tautomer was computed using density functional theoretical calculations. Species at various pHs were identified based on isotopic shifts in experimental wavenumbers and by comparing these shifts with corresponding computed isotopic shifts. Our results show that at physiological pH, N1 of pyrimidine ring in 9DAG and 9DAH bears a proton. At lower pH, N3 is place of protonation, and at higher pH, deprotonation occurs at N1 position. The proton at N7 of purine ring remains intact even at pH 12.5. We have further compared these results with naturally occurring nucleotides. Our results identify key vibrational modes which can report on hydrogen bonding interactions, protonation and deprotonation in purine rings upon binding to the active site of enzymes. PMID:25894214

  5. In vitro inflammatory/anti-inflammatory effects of nitrate esters of purines.

    PubMed

    Maugé, Loranne; Fotopoulou, Theano; Delemasure, Stéphanie; Dutartre, Patrick; Koufaki, Maria; Connat, Jean-Louis

    2014-05-01

    Six purine analogues bearing a nitrate ester group (potential NO donor) were tested on human THP-1 macrophages to investigate their effects on the inflammatory response. Only three analogues increased the basal level of IL-1β. Two analogues exacerbated the inflammatory response induced by ATP but not that induced by H2O2. Only 6-[4-(6-nitroxyacetyl)piperazin-1-yl]-9H-purine (compound MK128) abolished ATP or H2O2-induced IL-1β production in the culture medium. Similar results were reproduced on macrophages differentiated from buffy coats and stimulated with LPS. MK128 was the only analogue to release NO and leading to nitrite formation in the culture medium. The EC50 for inhibition of induced IL-1β production by the cells was estimated to be 10-12µg/ml (about 36µM) and corresponded to the production of around 30µM nitrites in the culture medium. This anti-inflammatory effect of MK128 was mimicked by trinitrin used in 10 fold higher concentrations. Preincubation of cells with NO trapper cPTIO partially abolished the beneficial effect of MK128 while MK137, a ONO2 deprived analogue of MK128, was not able to inhibit induced IL-1β production and proved to be inflammatory. Moreover, purinergic channel inhibitors (oATP and U73122) inhibited the MK137 inflammatory effect. Finally, MK128 reduced the quantity of p20 caspase-1 produced in the culture medium. We suggest that MK128 inhibits IL-1β production via NO production and subsequent inflammasome component nitrosylation. On the opposite MK137, deprived from ONO2 group, could act as agonist of purinergic receptors and could thus activate inflammasome. PMID:24613657

  6. 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. PMID:16899691

  7. Human phosphoribosylformylglycineamide amidotransferase (FGARAT): regional mapping, complete coding sequence, isolation of a functional genomic clone, and DNA sequence analysis.

    PubMed

    Patterson, D; Bleskan, J; Gardiner, K; Bowersox, J

    1999-11-01

    Purines play essential roles in many cellular functions, including DNA replication, transcription, intra- and extra-cellular signaling, energy metabolism, and as coenzymes for many biochemical reactions. The de-novo synthesis of purines requires 10 enzymatic steps for the production of inosine monophosphate (IMP). Defects in purine metabolism are associated with human diseases. Further, many anticancer agents function as inhibitors of the de-novo biosynthetic pathway. Genes or cDNAs for most of the enzymes comprising this pathway have been isolated from humans or other mammals. One notable exception is the phosphoribosylformylglycineamide amidotransferase (FGARAT) gene, which encodes the fourth step of this pathway. This gene has been cloned from numerous microorganisms and from Drosophila melanogaster and C. elegans. We report here the identification of a human cDNA containing the coding region of the FGARAT mRNA and the isolation of a P1 clone that contains an intact human FGARAT gene. The P1 clone corrects the purine auxotrophy and protein deficiency of Chinese hamster ovary (CHO) cell mutants (AdeB) deficient in both the activity and the protein for FGARAT. The P1 clone was used to regionally map the FGARAT gene to chromosome region 17p13, a location consistent with our prior assignment of this gene to chromosome 17. A comparison of the DNA sequence of the human FGARAT and FGARAT DNA sequence from 17 other organisms is reported. The isolation of this gene means that DNA clones for all the 10 steps of IMP synthesis have been isolated from humans or other mammals. PMID:10548741

  8. Giant prolactinoma presenting as a skull base tumor with erosion of the cervical vertebrae: pronounced responsiveness to dopamine agonist treatment.

    PubMed

    Bjerg, Mia L; Rosendal, Frederikke; Nielsen, Edith; Ulhøi, Benedikte Palm; Jørgensen, Jens O L

    2014-08-27

    Giant prolactinomas are rare and usually associated with symptoms attributable to hypopituitarism and compression of juxtasellar structures such as the cranial nerves of the cavernous sinus and the optic chiasm. Occasionally, they masquerade as skull base tumors with atypical symptoms. We describe a patient who presented with a low-energy trauma in the neck region that led to the initial diagnosis of a large skull base tumor eroding the cervical vertebrae. After stabilizing surgery, the patient responded to dopamine agonist therapy with normalization of serum prolactin levels and pronounced reduction in tumor volume. PMID:25162753

  9. Postgenomic Analysis of Streptococcus thermophilus Cocultivated in Milk with Lactobacillus delbrueckii subsp. bulgaricus: Involvement of Nitrogen, Purine, and Iron Metabolism▿ †

    PubMed Central

    Herve-Jimenez, Luciana; Guillouard, Isabelle; Guedon, Eric; Boudebbouze, Samira; Hols, Pascal; Monnet, Véronique; Maguin, Emmanuelle; Rul, Françoise

    2009-01-01

    Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yoghurt manufacture, where it is associated with Lactobacillus delbrueckii subsp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG 18311 in response to the presence of L. delbrueckii subsp. bulgaricus ATCC 11842 during growth in milk at two growth stages. Seventy-seven different genes or proteins (4.1% of total coding sequences), implicated mainly in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in coculture. One of the most unpredicted results was a significant decrease of most of the transcripts and enzymes involved in purine biosynthesis. Interestingly, the expression of nearly all genes potentially encoding iron transporters of S. thermophilus decreased, whereas that of iron-chelating dpr as well as that of the fur (perR) regulator genes increased, suggesting a reduction in the intracellular iron concentration, probably in response to H2O2 production by L. bulgaricus. The present study reveals undocumented nutritional exchanges and regulatory relationships between the two yoghurt bacteria, which provide new molecular clues for the understanding of their associative behavior. PMID:19114510

  10. The purine-rich trinucleotide repeat sequences d(CAG)15 and d(GAC)15 form hairpins.

    PubMed

    Yu, A; Dill, J; Mitas, M

    1995-10-25

    The structures of single-stranded (ss) oligonucleotides containing (CAG)15 [ss(CAG)15] or (GAC)15 [ss(GAC)15] were examined. At 10 degrees C, the electrophoretic mobilites of the two DNAs were similar to ss(CTG)15, a DNA that forms a hairpin containing base paired and/or stacked thymines. At 37 degrees C in 50 mM NaCl, single-strand-specific P1 nuclease cleaved the G33-G36 phosphodiesters of ss(GAC)15, and the G32-A34, G35-C36 phosphodiesters of ss(CAG)15 (where the loop apex of both DNAs = A34). Electrophoretic mobility melting profiles indicated that the melting temperature (Tm) of ss(CAG)15 in low (approximately 1 mM Na+) ionic strength was 38 degrees C. In contrast, the Tm of ss(GAC)15 was 49 degrees C, a value similar to the Tm of ss(CTG)15. These results provide evidence that ss(GAC)15 and ss(CAG)15 form similar, but distinguishable hairpin structures. PMID:7479064

  11. N-(2-Meth­oxy­benz­yl)-9-(oxolan-2-yl)-9H-purin-6-amine

    PubMed Central

    Trávníček, Zdeněk; Popa, Igor; Dvořák, Zdeněk; Štarha, Pavel

    2013-01-01

    The title compound, C17H19N5O2, features an almost planar purine skeleton (r.m.s. deviation = 0.009 Å) substituted by a tetra­hydro­furan ring, which adopts an envelope conformation. The purine and benzene rings subtend a dihedral angle of 66.70 (3)°. In the crystal, pairs of N—H⋯N hydrogen bonds connect adjacent mol­ecules into inversion dimers. C—H⋯N, C—H⋯O, C—H⋯π and π–π inter­actions [pyrimidine ring centroid–centroid distance = 3.3909 (1) Å] connect the dimers into a three-dimensional architecture. PMID:23634119

  12. N-(2-Meth-oxy-benz-yl)-9-(oxolan-2-yl)-9H-purin-6-amine.

    PubMed

    Trávníček, Zdeněk; Popa, Igor; Dvořák, Zdeněk; Starha, Pavel

    2013-04-01

    The title compound, C17H19N5O2, features an almost planar purine skeleton (r.m.s. deviation = 0.009 Å) substituted by a tetra-hydro-furan ring, which adopts an envelope conformation. The purine and benzene rings subtend a dihedral angle of 66.70 (3)°. In the crystal, pairs of N-H⋯N hydrogen bonds connect adjacent mol-ecules into inversion dimers. C-H⋯N, C-H⋯O, C-H⋯π and π-π inter-actions [pyrimidine ring centroid-centroid distance = 3.3909 (1) Å] connect the dimers into a three-dimensional architecture. PMID:23634119

  13. Asymmetric Hydrogenation of α-Purine Nucleobase-Substituted Acrylates with Rhodium Diphosphine Complexes: Access to Tenofovir Analogues.

    PubMed

    Sun, Huan-Li; Chen, Fei; Xie, Ming-Sheng; Guo, Hai-Ming; Qu, Gui-Rong; He, Yan-Mei; Fan, Qing-Hua

    2016-05-01

    The first asymmetric hydrogenation of α-purine nucleobase-substituted α,β-unsaturated esters, catalyzed by a chiral rhodium (R)-Synphos catalyst, has been developed. A wide range of mono- and disubstituted acrylates were successfully hydrogenated under very mild conditions in high yields with good to excellent enantioselectivities (up to 99% ee). This method provides a convenient approach to the synthesis of a new kind of optically pure acyclic nucleoside and Tenofovir analogues. PMID:27112983

  14. 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.

  15. CuBr catalyzed C-N cross coupling reaction of purines and diaryliodonium salts to 9-arylpurines.

    PubMed

    Niu, Hong-Ying; Xia, Chao; Qu, Gui-Rong; Zhang, Qian; Jiang, Yi; Mao, Run-Ze; Li, De-Yang; Guo, Hai-Ming

    2011-07-21

    CuBr was found to be an efficient catalyst for the C-N cross coupling reaction of purine and diaryliodonium salts. 9-Arylpurines were synthesized in excellent yields with short reaction times (2.5 h). The method represents an alternative to the synthesis of 9-arylpurines via Cu(II) catalyzed C-N coupling reaction with arylboronic acids as arylating agents. PMID:21660365

  16. 2-Chloro-6-[(2,4-dimeth-oxy-benz-yl)amino]-9-isopropyl-9H-purine.

    PubMed

    Novotná, Radka; Trávníček, Zdeněk

    2013-03-01

    In the title compound, C17H20ClN5O2, the benzene ring and the purine ring system make a dihedral angle of 78.56 (4)°. In the crystal, mol-ecules are linked by pairs of N-H⋯N hydrogen bonds, forming inversion dimers. C-H⋯O and C-H⋯Cl contacts further link the mol-ecules, forming a three-dimensional network. PMID:23476575

  17. Purine metabolism enzyme pattern, cytochemical characteristics and clinicopathologic features of CD10-positive childhood T-cell leukemia.

    PubMed

    Babusíková, O; Cáp, J; Hrivnáková, A; Klobusická, M; Mesárosová, A; Koníková, E

    1991-01-01

    Purine metabolism enzyme pattern, cytochemical markers and clinicopathologic features of common acute lymphoblastic leukemia antigen (cALLA; CD10)-positive, CD10-negative T acute lymphoblastic leukemia (ALL), and cALLA-positive non-T, non-B ALL (common ALL; C ALL) of children were compared. The results of immunophenotyping of blast cells in 61 children with ALL who were treated and followed during the last 7 years at the Second Pediatric Clinic in Bratislava are presented. The aim of our study was to determine the correlation of CD10 marker expression with purine enzyme activities and clinical course in ALL of children. Immunologic phenotype performed by a panel of monoclonal antibodies in indirect immunofluorescence assay revealed 3 main ALL groups: Common ALL (C ALL), T ALL and CD10+ T ALL (C + T ALL). An additional exact cytochemical marker analysis was performed in these three ALL immunologic subtypes. Two enzymes of purine metabolism, i.e. adenosine deaminase (ADA) and purine nucleosidephosphorylase (PNP) were investigated in blast cells by paper radiochromatography. Life-table analysis revealed significant prognostic differences with regard to event-free survival and overall survival in followed groups of ALL patients. Our results showed a rather high frequency of mixed (C + T) ALL phenotype. The characteristic T ALL enzyme pattern (high ADA, low PNP) was present not only in T, but also in CD10+ T ALL blast cells. The T cell marker showed to be dominant in the determination of clinical course and prognostic significance in children with ALL; children with T and CD10+ T ALL phenotype, in contrast to C ALL phenotype, experienced more frequent relapses and a shorter event-free survival. PMID:1837333

  18. Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma.

    PubMed

    Giuliani, Patricia; Zuccarini, Mariachiara; Buccella, Silvana; Rossini, Margherita; D'Alimonte, Iolanda; Ciccarelli, Renata; Marzo, Matteo; Marzo, Antonio; Di Iorio, Patrizia; Caciagli, Francesco

    2016-01-15

    Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions. PMID:26720700

  19. Effects of different lipid sources on intake, digestibility and purine derivatives in hair lambs.

    PubMed

    Pereira, E S; Pereira, M W F; Arruda, P C L; Cabral, L S; Oliveira, R L; Mizubuti, I Y; Pinto, A P; Campos, A C N; Gadelha, C R F; Carneiro, M S S

    2016-08-01

    An experiment was conducted to evaluate the effects of different lipid sources on the nutrient intake, digestibility and purine derivative excretion of lambs. Thirty-five 60-day-old, male, non-castrated Santa Ines lambs with an initial average body weight (BW) of 13.00 ± 1.80 kg were used in a randomized complete block design with seven blocks and five treatments. The experimental treatments consisted of a control diet without supplemental lipids and four test diets with different lipid supplements, selected according to the degree of ruminal protection from hydrogenation: supplementation, being supplementation with whole cottonseed (WC), supplementation with cashew nut meal (CNM), supplementation with both cottonseed and cashew nut meal (WC-CNM) and supplementation with calcium salts of long-chain fatty acids (Ca-LCFA). The lambs were slaughtered after reaching 28 kg average BW for each treatment. The ether extract intake (EEI) was higher (p < 0.01) for the lipid supplemented compared to control diet lambs. Supplementation with WC decreased the digestibility of dry matter (DM), organic matter (OM), neutral detergent fibre (NDF) and total carbohydrate (TC) (p < 0.01), whereas supplementation with CNM, WC-CNM and Ca-LCFA reduced non-fibrous carbohydrate (NFC) digestibility (p < 0.01). The ether extract (EE) digestibility coefficient was higher with CNM, followed by Ca-LCFA and WC, when compared to WC-CNM and control diets. Nitrogen balance (NB) was not influenced (p > 0.05) by the different lipid sources. A lower purine derivative (PD) excretion and thus lower microbial protein supply (MPS) was observed for animals supplemented with Ca-LCFA (p < 0.01) compared to the WC-CNM and control diets. In conclusion, WC, CNM and WC-CNM supplementation did not have negative effects on MPS, although negative effects have been observed on nutrient digestibility. PMID:26854276

  20. Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis[W

    PubMed Central

    Dahncke, Kathleen; Witte, Claus-Peter

    2013-01-01

    Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2’-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed. PMID:24130159

  1. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

    PubMed Central

    2012-01-01

    Background Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Results Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation. Conclusions The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization. PMID:23092390

  2. Regio- and Enantioselective N-Allylations of Imidazole, Benzimidazole, and Purine Heterocycles Catalyzed by Single-Component Metallacyclic Iridium Complexes

    PubMed Central

    Stanley, Levi M.

    2010-01-01

    Highly regio- and enantioselective iridium-catalyzed N-allylations of benzimidazoles, imidazoles, and purines have been developed. N-Allylated benzimidazoles and imidazoles were isolated in high yields (up to 97%) with high branched-to-linear selectivity (up to 99:1) and enantioselectivity (up to 98% ee) from the reactions of benzimidazole and imidazole nucleophiles with unsymmetrical allylic carbonates in the presence of single component, ethylene-bound, metallacyclic iridium catalysts. N-Allylated purines were also obtained in high yields (up to 91%) with high N9:N7 selectivity (up to 96:4), high branched-to-linear selectivity (98:2), and high enantioselectivity (up to 98% ee) under similar conditions. The reactions encompass a range of benzimidazole, imidazole, and purine nucleophiles, as well as a variety of unsymmetrical aryl, heteroaryl, and aliphatic allylic carbonates. Competition experiments between common amine nucleophiles and the heterocyclic nitrogen nucleophiles studied in this work illustrate the effect of nucleophile pKa on the rate of iridium-catalyzed N-allylation reactions. Kinetic studies on the allylation of benzimidazole catalyzed by metallacyclic iridium-phosphoramidite complexes, in combination with studies on the deactivation of these catalysts in the presence of heterocyclic nucleophiles, provide insight into the effects of the structure of the phosphoramidite ligands on the stability of the metallacyclic catalysts. The data obtained from these studies has led to the development of N-allylations of benzimidazoles and imidazoles in the absence of an exogenous base. PMID:19480431

  3. Dependence of gamma-aminobutyric acid modulation of cholinergic transmission on nitric oxide and purines in cat terminal ileum.

    PubMed

    Pencheva, N

    1997-11-27

    The possible involvement of purines and/or nitric oxide (NO) in the gamma-aminobutyric acid (GABA)A receptor-mediated effects on the spontaneous activity of isolated preparations from longitudinal and circular muscles of cat terminal ileum was investigated. GABA had biphasic effects, which were neurogenic and muscarinic. ATP and adenosine dose dependently inhibited the activity of the muscles. A contractile response evoked by the nucleotide only was also observed. The effects of the purines were equipotent and resistant to Nomega-nitro-L-arginine (L-NNA), tetrodotoxin and to desensitization by alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP), except for the contractile effect of ATP, which was abolished by alpha,beta-meATP. Pretreatment of the preparations with ATP or adenosine produced: (i) desensitization to the effects of the respective purinoceptor agonist only; and (ii) suppression of the GABA-induced responses of longitudinal and circular muscles. Hemoglobin and L-NNA greatly reduced or completely blocked the GABA(A)-induced relaxation and decreased the GABA(A)-induced contraction. Our results indicate that purines and NO, to a different extent, mediate the relaxant phase of the GABA effects in both layers. Interactions between muscarinic cholinoceptors and GABA-nitrergic pathway and a concomitant activation of postjunctional P1 and P2y purinoceptors are suggested to explain the prejunctional biphasic effects of GABA. PMID:9473135

  4. 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

  5. Effect of root application of 6-benzylamine purine on auxin transport in Malus domestica

    SciTech Connect

    Stutte, G.

    1987-04-01

    The effect of 6-benzylamine purine (BA) on movement of napthalene acetic acid (NAA) applied to the shoot was investigated. Three-month-old York apple seedlings in sand culture were placed under mercury halide lamps (285 ..mu..mol m/sup -2/ s/sup -1/) at 22/sup 0/C for 24 hours; then 13.8, 27.5 or 55 ..mu..Mol of BA was applied as a soil drench. (/sup 14/C)-NAA was injected into the shoot at petiole base of 4th mature leaf. After 24 hours plants were harvested and movement of label acropetally to new shoot growth and basipetally to old shoot growth or roots determined. Seventeen (S.E. 3.3) percent of applied label was recovered away from application site. In controls, 67% of /sup 14/C that moved was recovered from new shoot growth, 9% in roots and 24% in old shoot tissue. The 13.8 ..mu..Mol treatment resulted in 37% of label being recovered in root tissue and 48% in new growth. The 27.5 and 55 ..mu..Mol applications increased the amount recovered in old shoot tissue but did not increase labelling of root tissues.

  6. Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency.

    PubMed Central

    Agarwal, R P; Crabtree, G W; Parks, R E; Nelson, J A; Keightley, R; Parkman, R; Rosen, F S; Stern, R C; Polmar, S H

    1976-01-01

    Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients... PMID:947948

  7. 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. PMID:26295691

  8. 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.

  9. An insight into purine, tyrosine and tryptophan derived marine antineoplastic alkaloids.

    PubMed

    Palkar, Mahesh B; Rane, Rajesh A; Thapliyal, Neeta; Shaikh, Mahamadhanif S; Alwan, Wesam S; Jain, Kavita S; Karunanidhi, Sivanandhan; Patel, Harun M; Hampannavar, Girish A; Karpoormath, Rajshekhar

    2015-01-01

    There is an ever-increasing need for the development of new drugs with safe and improved profile for the treatment of cancer. From time immemorial, nature has been considered as an abundant source of medicinal compounds having therapeutic properties. An enormous chemical diversity is present in thousands and millions of species of microorganisms, marine organisms, plants and animals that can act as potential therapeutic agents against various types of human cancer. Literature survey revealed that many alkaloids isolated from marine cyanobacteria, fungi, algae, sponges and tunicates displayed a wide range of anticancer properties like antiproliferative, antiangiogenic, induction of apoptosis, promoting cytotoxicity by inhibition of topoisomerase activities and tubulin polymerization. In this context, bastadins derived from tyrosine-based alkaloids have been reported as one the important class of anticancer agents. In particular bastadin 6 (24), seems to be a promising natural lead compound for the development of marine natural product-based anticancer therapeutic agents. This review mainly highlights the pharmacologically active scaffolds like purine, tyrosine and tryptophan containing marine alkaloids that exhibit biological activity, including anti-angiogenesis, cytotoxicity and anticancer activity. PMID:25553433

  10. A defect in the p53 response pathway induced by de novo purine synthesis inhibition.

    PubMed

    Bronder, Julie L; Moran, Richard G

    2003-12-01

    p53 is believed to sense cellular ribonucleotide depletion in the absence of DNA strand breaks and to respond by imposition of a p21-dependent G1 cell cycle arrest. We now report that the p53-dependent G1 checkpoint is blocked in human carcinoma cell lines after inhibition of de novo purine synthesis by folate analogs inhibitory to glycinamide ribonucleotide formyltransferase (GART). p53 accumulated in HCT116, MCF7, or A549 carcinoma cells upon GART inhibition, but, surprisingly, transcription of several p53 targets, including p21cip1/waf1, was impaired. The mechanism of this defect was examined. The p53 accumulating in these cells was nuclear but was not phosphorylated at serines 6, 15, and 20, nor was it acetylated at lysines 373 or 382. The DDATHF-stabilized p53 bound to the p21 promoter in vitro and in vivo but did not activate histone acetylation over the p53 binding sites in the p21 promoter that is an integral part of the transcriptional response mediated by the DNA damage pathway. We concluded that the robust initial response of the p53 pathway to GART inhibitors is not transcriptionally propagated to target genes due to a defect in p53 post-translational modifications and a failure to open chromatin structure despite promoter binding of this unmodified p53. PMID:14517211

  11. Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.

    PubMed

    de Moraes, Marcela Cristina; Ducati, Rodrigo Gay; Donato, Augusto José; Basso, Luiz Augusto; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra

    2012-04-01

    The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The K(M) value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 ± 29.2 μM and 133 ± 14.9 μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC(50)=40.6 ± 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. PMID:22099222

  12. A novel bis(pinacolato)diboron-mediated N-O bond deoxygenative route to C6 benzotriazolyl purine nucleoside derivatives.

    PubMed

    Basava, Vikram; Yang, Lijia; Pradhan, Padmanava; Lakshman, Mahesh K

    2016-08-01

    Reaction of amide bonds in t-butyldimethylsilyl-protected inosine, 2'-deoxyinosine, guanosine, 2'-deoxyguanosine, and 2-phenylinosine with commercially available peptide-coupling agents (benzotriazol-1H-yloxy)tris(dimethylaminophosphonium) hexafluorophosphate (BOP), (6-chloro-benzotriazol-1H-yloxy)trispyrrolidinophosphonium hexafluorophosphate (PyClocK), and (7-azabenzotriazol-1H-yloxy)trispyrrolidinophosphonium hexafluorophospate (PyAOP) gave the corresponding O(6)-(benzotriazol-1-yl) nucleoside analogues containing a C-O-N bond. Upon exposure to bis(pinacolato)diboron and base, the O(6)-(benzotriazol-1-yl) and O(6)-(6-chlorobenzotriazol-1-yl) purine nucleoside derivatives obtained from BOP and PyClocK, respectively, underwent N-O bond reduction and C-N bond formation, leading to the corresponding C6 benzotriazolyl purine nucleoside analogues. In contrast, the 7-azabenzotriazolyloxy purine nucleoside derivatives did not undergo efficient deoxygenation, but gave unsymmetrical nucleoside dimers instead. This is consistent with a prior report on the slow reduction of 1-hydroxy-1H-4-aza and 1-hydroxy-1H-7-azabenzotriazoles. Because of the limited number of commercial benzotriazole-based peptide coupling agents, and to show the applicability of the method when such coupling agents are unavailable, 1-hydroxy-1H-5,6-dichlorobenzotriazole was synthesized. Using this compound, silyl-protected inosine and 2'-deoxyinosine were converted to the O(6)-(5,6-dichlorobenzotriazol-1-yl) derivatives via in situ amide activation with PyBroP. The O(6)-(5,6-dichlorobenzotriazol-1-yl) purine nucleosides so obtained also underwent smooth reduction to afford the corresponding C6 5,6-dichlorobenzotriazolyl purine nucleoside derivatives. A total of 13 examples were studied with successful reactions occurring in 11 cases (the azabenzotriazole derivatives, mentioned above, being the only unreactive entities). To understand whether these reactions are intra or intermolecular processes, a

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Root growth reacts rapidly and more pronounced than shoot growth towards increasing light intensity in tobacco seedlings.

    PubMed

    Walter, Achim; Nagel, Kerstin A

    2006-09-01

    Light intensity is crucial for plant growth and often fluctuates on a small time scale due to altering climate conditions or sunflecks. Recently, we performed a study that looked into the growth effect of a sudden elevation of light intensity on Nicotiana tabacum seedlings.1 It was shown that an increase in light intensity leads to a pronounced increase of root-shoot-ratio as root growth reacts strongly and rapidly to an increase of light intensity. In transition experiments from low (60 micromol m(-2) s(-1)) to high (300 micromol m(-2) s(-1)) light intensity, root growth increased by a factor of four within four days, reaching the steady-state level measured in plants that were cultivated in high-light conditions. During the first three hours after light increase, strong fluctuations of the velocity of the root tip were observed that were putatively caused by a superposition of hydraulic and photosynthetic acclimation to the altered conditions. Experiments with externally applied sucrose and with transgenic plants having reduced capacity for sucrose synthesis indicated clearly that increasing light intensity rapidly enhanced root growth by elevating sucrose export from shoot to root. PMID:19704663

  18. 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. PMID:26257746

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

    PubMed

    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

  20. Circulating intestinal fibroblast growth factor 19 has a pronounced diurnal variation and modulates hepatic bile acid synthesis in man.

    PubMed

    Lundåsen, T; Gälman, C; Angelin, B; Rudling, M

    2006-12-01

    Bile acids (BAs) traversing the enterohepatic circulation exert several important metabolic effects. Their hepatic synthesis, controlled by the enzyme cholesterol 7alpha-hydroxylase (CYP7A1), has a unique diurnal variation in man. Here we provide evidence that the transintestinal flux of BAs regulates serum levels of intestinal fibroblast growth factor 19 (FGF19) that in turn modulate BA production in human liver. Basal FGF19 levels varied by 10-fold in normal subjects, and were reduced following treatment with a BA-binding resin and increased upon feeding the BA chenodeoxycholic acid. Serum FGF19 levels exhibited a pronounced diurnal rhythm with peaks occurring 90-120 min after the postprandial rise in serum BAs. The FGF19 peaks in turn preceded the declining phase of BA synthesis. The diurnal rhythm of serum FGF19 was abolished upon fasting. We conclude that, in humans, circulating FGF19 has a diurnal rhythm controlled by the transintestinal BA flux, and that FGF19 modulates hepatic BA synthesis. Through its systemic effects, circulating FGF19 may also mediate other known BA-dependent effects on lipid and carbohydrate metabolism. PMID:17116003

  1. 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

  2. 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.

  3. Neighboring Group Participation in the Transition State of Human Purine Nucleoside Phosphorylase

    SciTech Connect

    Murkin,A.; Birck, M.; Rinaldo-Matthis, A.; Shi, W.; Taylor, E.; Almo, S.; Schramm, V.

    2007-01-01

    The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition-state analogues show His{sup 257} within hydrogen bonding distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (K{sub m}/K{sub i}) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His{sup 257} serves an important role in the early stages of transition-state formation. Whereas mutation of His{sup 257} resulted in little variation in the PNP{center_dot}DADMe-ImmH{center_dot}SO{sub 4} structures, His257Phe{center_dot}ImmH{center_dot}PO{sub 4} showed distortion at the 5'-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies of the remote 5'-{sup 3}H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5'-{sup 3}H intrinsic KIE to -3, -14, and 7%, respectively, while the BIEs contributed 2, 2, and -2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state.

  4. 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

  5. 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.

  6. Plasma Membrane-Located Purine Nucleotide Transport Proteins Are Key Components for Host Exploitation by Microsporidian Intracellular Parasites

    PubMed Central

    Heinz, Eva; Hacker, Christian; Dean, Paul; Mifsud, John; Goldberg, Alina V.; Williams, Tom A.; Nakjang, Sirintra; Gregory, Alison; Hirt, Robert P.; Lucocq, John M.; Kunji, Edmund R. S.; Embley, T. Martin

    2014-01-01

    Microsporidia are obligate intracellular parasites of most animal groups including humans, but despite their significant economic and medical importance there are major gaps in our understanding of how they exploit infected host cells. We have investigated the evolution, cellular locations and substrate specificities of a family of nucleotide transport (NTT) proteins from Trachipleistophora hominis, a microsporidian isolated from an HIV/AIDS patient. Transport proteins are critical to microsporidian success because they compensate for the dramatic loss of metabolic pathways that is a hallmark of the group. Our data demonstrate that the use of plasma membrane-located nucleotide transport proteins (NTT) is a key strategy adopted by microsporidians to exploit host cells. Acquisition of an ancestral transporter gene at the base of the microsporidian radiation was followed by lineage-specific events of gene duplication, which in the case of T. hominis has generated four paralogous NTT transporters. All four T. hominis NTT proteins are located predominantly to the plasma membrane of replicating intracellular cells where they can mediate transport at the host-parasite interface. In contrast to published data for Encephalitozoon cuniculi, we found no evidence for the location for any of the T. hominis NTT transporters to its minimal mitochondria (mitosomes), consistent with lineage-specific differences in transporter and mitosome evolution. All of the T. hominis NTTs transported radiolabelled purine nucleotides (ATP, ADP, GTP and GDP) when expressed in Escherichia coli, but did not transport radiolabelled pyrimidine nucleotides. Genome analysis suggests that imported purine nucleotides could be used by T. hominis to make all of the critical purine-based building-blocks for DNA and RNA biosynthesis during parasite intracellular replication, as well as providing essential energy for parasite cellular metabolism and protein synthesis. PMID:25474405

  7. 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.

  8. 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

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

    PubMed

    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

  10. 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.

  11. New synthetic routes to protected purine 2'-O-methylriboside-3'-O-phosphoramidites using a novel alkylation procedure.

    PubMed Central

    Sproat, B S; Beijer, B; Iribarren, A

    1990-01-01

    A highly selective alkylation procedure has been developed enabling new synthetic routes to be established for protected purine 2'-O-methylriboside-3'-O-phosphoramidites; building blocks for the assembly of 2'-O-methyloligoribonucleotides. The new procedure avoids the use of the highly toxic and potentially explosive reagent diazomethane and is far superior to the use of silver oxide/methyl iodide. Moreover, the use of highly versatile key intermediates will enable the synthesis of a wide variety of base modified analogues as well as other 2'-O-alkylriboside derivatives. PMID:2308835

  12. 4-(1-Adamantylmeth­yl)-N-(2-chloro-9-isopropyl-9H-purin-6-yl)aniline

    PubMed Central

    Rouchal, Michal; Nečas, Marek; Vícha, Robert

    2009-01-01

    The asymmetric unit of the title compound, C25H30ClN5, consists of two mol­ecules with slightly different geometrical parameters. The dihedral angles between the purine and benzene rings are 39.54 (5) and 23.69 (5)° in the two mol­ecules. The adamantane cages consist of three fused cyclo­hexane rings in classical chair conformations, with C—C—C angles in the range 108 (2)–111 (2)°. In the crystal, mol­ecules are linked into dimers via two N—H⋯N hydrogen bonds. PMID:21582935

  13. Discovery of Disubstituted Imidazo[4,5-b]pyridines and Purines as Potent TrkA Inhibitors

    PubMed Central

    2012-01-01

    Trk receptor tyrosine kinases have been implicated in cancer and pain. A crystal structure of TrkA with AZ-23 (1a) was obtained, and scaffold hopping resulted in two 5/6-bicyclic series comprising either imidazo[4,5-b]pyridines or purines. Further optimization of these two fusion series led to compounds with subnanomolar potencies against TrkA kinase in cellular assays. Antitumor effects in a TrkA-driven mouse allograft model were demonstrated with compounds 2d and 3a. PMID:24900538

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

    PubMed

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

    2016-08-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

    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.

  16. 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

  17. New (RS)-benzoxazepin-purines with antitumour activity: The chiral switch from (RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine.

    PubMed

    López-Cara, Luisa C; Conejo-García, Ana; Marchal, Juan A; Macchione, Giuseppe; Cruz-López, Olga; Boulaiz, Houria; García, María A; Rodríguez-Serrano, Fernando; Ramírez, Alberto; Cativiela, Carlos; Jiménez, Ana I; García-Ruiz, Juan M; Choquesillo-Lazarte, Duane; Aránega, Antonia; Campos, Joaquín M

    2011-01-01

    Completing an 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 has been prepared under microwave-assisted conditions. Their antiproliferative activities on MCF-7 and MDA-MB-231 cancerous cell lines are presented, being the majority of the IC(50) values below 1μM. The most active compound (RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine (14) presents an IC(50) of 0.166μM against the human cancerous cell line MDA-MB-231. Compound 14 was the most selective against the human breast adenocarcinoma MCF-7 and MDA-MB-231 cancer cell lines (Therapeutic Indexes, TIs=5.1 and 11.0, respectively) in relation to the normal one MCF-10A. (RS)-14 was resolved into its enantiomers. Both enantiomers are equally potent, but more potent than the corresponding racemic mixture. (R)-14 induces apoptosis against MCF-7 up to 52.50% of cell population after 48h, being more potent than the clinical-used drug paclitaxel (43%). (RS)-14 induces no acute toxicity in mice after two weeks of treatment. PMID:21126804

  18. Pronounced short-term temporal dynamics of methane fluxes during the snowmelt period at a boreal peatland in NW Russia

    NASA Astrophysics Data System (ADS)

    Gazovic, M.; Kutzbach, L.; Schreiber, P.; Wille, C.; Wilmking, M.

    2009-04-01

    Peatlands are one of the major natural sources of methane (CH4), but the quantification of efflux is uncertain especially during winter, fall and the highly dynamic spring thaw period. Here we report unexpected diurnal variations in methane fluxes as measured using the eddy correlation technique lasting several days during the snow thawing period at a boreal peatland in NW Russia. The beginning of snowmelt in 2008 was characterized by moderate CH4 fluxes of ~0.5-1.8 mg m-2 h-1. However, we found unexpected pattern in CH4 fluxes from 21.04-3.05.2008 when fluxes showed strong diurnal variability apparently controlled by changes in the surface temperature. Hourly CH4 fluxes were ~0.5 mg m-2 h-1 during night and reached as much as ~5 mg m-2 h-1 during midday. An empirical model based on linear reduced major axis regression was used to gap-fill the time series with surface temperature as a predictor. There was a lag of one hour between the diurnal maxima of surface temperature and CH4 flux, respectively. The fluxes started to decrease earlier than the surface temperature reached its maximum. The most likely explanation is that thawing and refreezing of an ice layer at the wet peatland micro-sites due to oscillating surface temperatures above 0 ˚ C during the days and below 0 ˚ C during the nights, which was observed during this period of the year, led to a pronounced diurnal variability of the resistance for gas diffusion from the soil and open water bodies to the atmosphere. Apparently, physical factors influencing the gas transport processes have a stronger effect on CH4 efflux than microbiological ones during the spring whereas the control of CH4 efflux is dominated by biological processes during the vegetation period. The evidence of strong temporal variation on an hourly time scale is important when estimates of cold season fluxes are made to quantify seasonal or annual CH4 emissions, especially when static chambers are applied under climatic conditions

  19. 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.

  20. A cycloartane glycoside derived from Actaea racemosa L. modulates GABAA receptors and induces pronounced sedation in mice.

    PubMed

    Strommer, Barbara; Khom, Sophia; Kastenberger, Iris; Cicek, Serhat Sezai; Stuppner, Hermann; Schwarzer, Christoph; Hering, Steffen

    2014-11-01

    23-O-Acetylshengmanol 3-O-β-D-xylopyranoside (Ac-SM) isolated from Actaea racemosa L.-an herbal remedy for the treatment of mild menopausal disorders-has been recently identified as a novel efficacious modulator of GABAA receptors composed of α1-, β2-, and γ2S-subunits. In the present study, we analyzed a potential subunit-selective modulation of GABA-induced chloride currents (IGABA) at GABA concentrations eliciting 3-8% of the maximal GABA response (EC3-8) through nine GABAA receptor isoforms expressed in Xenopus laevis oocytes by Ac-SM with two-microelectrode voltage clamp and behavioral effects 30 minutes after intraperitoneal application in a mouse model. Efficacy of IGABA enhancement by Ac-SM displayed a mild α-subunit dependence with α2β2γ2S (maximal IGABA potentiation [Emax] = 1454 ± 97%) and α5β2γ2S (Emax = 1408 ± 87%) receptors being most efficaciously modulated, followed by slightly weaker IGABA enhancement through α1β2γ2S (Emax = 1187 ± 166%), α3β2γ2S (Emax = 1174 ± 218%), and α6β2γ2S (Emax = 1171 ± 274%) receptors and less pronounced effects on receptors composed of α4β2γ2S (Emax = 752 ± 53%) subunits, whereas potency was not affected by the subunit composition (EC50 values ranging from α1β2γ2S = 35.4 ± 12.3 µM to α5β2γ2S = 50.9 ± 11.8 µM). Replacing β2- with β1- or β3-subunits as well as omitting the γ2S-subunit affected neither efficacy nor potency of IGABA enhancement by Ac-SM. Ac-SM shifted the GABA concentration-response curve toward higher GABA sensitivity (about 3-fold) and significantly increased the maximal GABA response by 44 ± 13%, indicating a pharmacological profile distinct from a pure allosteric GABAA receptor modulator. In mice, Ac-SM significantly reduced anxiety-related behavior in the elevated plus maze test at a dose of 0.6 mg/kg, total ambulation in the open field test at doses ≥6 mg/kg, stress-induced hyperthermia at doses ≥0.6 mg/kg, and significantly elevated seizure threshold at

  1. 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. PMID:27188426

  2. Desilylative activation of TMSCN in chemoselective Strecker-Ugi type reaction: functional fused imidazoles as building blocks as an entry route to annulated purines.

    PubMed

    Guchhait, Sankar K; Chaudhary, Vikas

    2014-09-14

    A pathway of desilylative activation of TMSCN as a functional isonitrile equivalent, and DABCO-THF as an appropriate system for activation in a chemoselective Strecker-Ugi type reaction, has rendered ethyl glyoxalate and various heterocyclic-2-amidines as feasible substrates, and afforded the successful synthesis of 3-amino-2-carboxyethyl substituted fused imidazoles as useful building blocks. This class of functional scaffold has provided, via construction of the fused pyrimidinone motif, the synthesis of biologically important C8-N9 annulated purines, adenines and their oxo/thio analogs. This new approach is convenient and flexible for the preparation of versatile purine-condensed heterocycles. PMID:25032666

  3. Primordia Vita. Deconvolution from Modern Sequences.

    NASA Astrophysics Data System (ADS)

    Trifonov, Edward N.; Gabdank, Idan; Barash, Danny; Sobolevsky, Yehoshua

    2006-12-01

    Evolution of the triplet code is reconstructed on the basis of consensus temporal order of appearance of amino acids. Several important predictions are confirmed by computational sequence analyses. The earliest amino acids, alanine and glycine, have been encoded by GCC and GGC codons, as today. They were succeeded, respectively, by A- and G-series of amino acids, encoded by pyrimidine-central and purine-central codons. The length of the earliest proteins is estimated to be 6 7 residues. The earliest mRNAs were short G+C-rich molecules. These short sequences could have formed hairpins. This is confirmed by analysis of modern prokaryotic mRNA sequences. Predominant size of detected ancient hairpins also corresponds to 6 7 amino acids, as above. Vestiges of last common ancestor can be found in extant proteins in form of entirely conserved short sequences of size six to nine residues present in all or almost all sequenced prokaryotic proteomes (omnipresent motifs). The functions of the topmost conserved octamers are not involved in the basic elementary syntheses. This suggests an initial abiotic supply of amino acids, bases and sugars.

  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. Disturbance of Antioxidant Enzymes and Purine Metabolism in the Ejaculate of Men Living in Disadvantaged Areas of Kyzylorda Region

    PubMed Central

    Kislitskaya, Valentihna N.; Kenzhin, Zhandos D.; Kultanov, Berikbay Zh.; Dosmagambetova, Raushan S.; Turmuhambetova, Anar A.

    2015-01-01

    AIM: Objective of the study was to evaluate the state of the main indicators of antioxidant status and enzymes of purine metabolism in the germ cells of men living in the zone of ecological catastrophe Aral Sea region. METHODS: The criterion for inclusion is the stay of an adult in the Aral Sea area is not less than 5 years, employment in occupations with no more than 2 hazard class. Determination of the activity of adenosine deaminase (ADA) was conducted in semen by the method of Nemechek et al., 1993. Determination of the activity of catalase (CAT) was performed according by the method of Korolyuk et al., 1988. RESULTS: Results of the study indicate a change in the activity of catalase and adenosine deaminase, due to increased levels of oxidative stress and the development of the pathological process. CONCLUSIONS: According to the results of study, it was put the influence of negative factors of the Aral Sea region in men’s sperm of reproductive age gives to disability free-radical processes, that proves changing of ferments of ant oxidative protection Catalase and adenosine deaminase (ADA). This disturbance in men’s sperm of reproductive age leading to increased level of oxidative stress and impaired activity of antioxidant enzymes and purine metabolism, responsible for the abnormal transmembrane and intracellular processes, reflecting the degree of imbalance of enzymes.

  6. 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

  7. Identification of potential inhibitors for AIRS from de novo purine biosynthesis pathway through molecular modeling studies - a computational approach.

    PubMed

    Rao, R Guru Raj; Biswal, Jayashree; Dhamodharan, Prabhu; Kanagarajan, Surekha; Jeyaraman, Jeyakanthan

    2016-10-01

    In cancer, de novo pathway plays an important role in cell proliferation by supplying huge demand of purine nucleotides. Aminoimidazole ribonucleotide synthetase (AIRS) catalyzes the fifth step of de novo purine biosynthesis facilitating in the conversion of formylglycinamidine ribonucleotide to aminoimidazole ribonucleotide. Hence, inhibiting AIRS is crucial due to its involvement in the regulation of uncontrollable cancer cell proliferation. In this study, the three-dimensional structure of AIRS from P. horikoshii OT3 was constructed based on the crystal structure from E. coli and the modeled protein is verified for stability using molecular dynamics for a time frame of 100 ns. Virtual screening and induced fit docking were performed to identify the best antagonists based on their binding mode and affinity. Through mutational studies, the residues necessary for catalytic activity of AIRS were identified and among which the following residues Lys35, Asp103, Glu137, and Thr138 are important in determination of AIRS function. The mutational studies help to understand the structural and energetic characteristics of the specified residues. In addition to Molecular Dynamics, ADME properties, binding free-energy, and density functional theory calculations of the compounds were carried out to find the best lead molecule. Based on these analyses, the compound from the NCI database, NCI_121957 was adjudged as the best molecule and could be suggested as the suitable inhibitor of AIRS. In future studies, experimental validation of these ligands as AIRS inhibitors will be carried out. PMID:26524231

  8. 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. PMID:25195883

  9. Synthesis and evaluation of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purines and 2-amino-9-(3-alkoxycarbonyloxymethyl-4-alkoxycarbonyloxybut-1- yl)purines as potential prodrugs of penciclovir.

    PubMed

    Kim, D K; Lee, N; Ryu, D H; Kim, Y W; Kim, J S; Chang, K; Im, G J; Choi, W S; Cho, Y B; Kim, K H; Colledge, D; Locarnini, S

    1999-08-01

    A series of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purin es (1-8) and 2-amino-9-(3-alkoxycarbonyl-oxymethyl-4-alkoxycarbonyloxybut -1-yl)purines (9-12) were synthesized as potential prodrugs of penciclovir. Treatment of 6-deoxypenciclovir with trimethyl orthoacetate or triethyl orthopropionate (1.2 equiv) in DMF in the presence of p-TsOH.H2O (0.1 equiv) followed by quenching with excess H2O gave the corresponding mono-O-acetyl or mono-O-propionyl compound, 17 or 18, in excellent yields of 95 and 92%, respectively. Reactions of 17 or 18 with an appropriate alkyl (Me, Et, n-Pr, and i-Pr) 4-nitrophenyl carbonate (1.2 equiv) in pyridine in the presence of a catalytic amount of DMAP (0.1 equiv) at 80 degrees C afforded the monoacyl, monocarbonate derivatives of 6-deoxypenciclovir, 1-8, in 86 94% yields. Similar reactions of 6-deoxypenciclovir with 2.1 equiv of alkyl 4-nitrophenyl carbonate produced the dicarbonate derivatives 9 12 in 81-83% yields. Of the prodrugs tested in rats, 2-amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine (4) achieved the highest mean urinary recovery of penciclovir (36%), followed in order by compounds 2 (35%), 6 (35%), 7 (34%), 10 (34%), 8 (32%), 3 (32%), and famciclovir (31%). The mean urinary recovery of penciclovir and concentrations of penciclovir in the blood from 4 in mice were also slightly higher than those from famciclovir. The in vivo antiviral efficacy of 4 in HSV-1-infected normal BALB/c mice was higher than those of famciclovir and valaciclovir in terms of mortality (100, 80, and 40%) and mean survival time ( > 21, 13+/-5.0 (SEM), and 13+/-1.6 days). Compound 4 demonstrated an effective anti-hepadnaviral response with intrahepatic viral load being reduced by 90%, the viral supercoiled DNA levels reduced by 70% and Pre-S expression inhibited by 30% against duck hepatitis B virus (DHBV) in vivo, and did not cause any significant hepatotoxicity after 4 weeks of treatment. PMID:10482463

  10. Locations and contexts of sequences that hybridize to poly(dG-dT).(dC-dA) in mammalian ribosomal DNAs and two X-linked genes.

    PubMed Central

    Braaten, D C; Thomas, J R; Little, R D; Dickson, K R; Goldberg, I; Schlessinger, D; Ciccodicola, A; D'Urso, M

    1988-01-01

    Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences. Images PMID:3267216

  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. 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. PMID:26237090

  13. Temperature-dependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation.

    PubMed

    Cherkasov, Anton A; Overton, Robert A; Sokolov, Eugene P; Sokolova, Inna M

    2007-01-01

    Temperature and heavy metals such as cadmium (Cd) are important environmental stressors that can strongly affect mitochondrial function of marine poikilotherms. In this study, we investigated the combined effects of temperature (20 degrees C and 30 degrees C) and Cd stress on production of reactive oxygen species (ROS) and oxidative stress in a marine poikilotherm Crassostrea virginica (the eastern oyster) using mitochondrial aconitase as a sensitive biomarker of oxidative damage. We also assessed potential involvement of mitochondrial uncoupling proteins (UCPs) in antioxidant protection in oyster mitochondria using purine nucleotides (GDP, ATP and ADP) as specific inhibitors, and free fatty acids as stimulators, of UCPs. Our results show that exposure to Cd results in elevated ROS production and oxidative damage as indicated by aconitase inactivation which is particularly pronounced at elevated temperature. Unexpectedly, oyster mitochondrial aconitase was inhibited by physiologically relevant levels of ATP (IC(50)=1.93 and 3.04 mmol l(-1) at 20 degrees C and 30 degrees C, respectively), suggesting that allosteric regulation of aconitase by this nucleotide may be involved in regulation of the tricarboxylic acid flux in oysters. Aconitase was less sensitive to ATP inhibition at 30 degrees C than at 20 degrees C, consistent with the elevated metabolic flux at higher temperatures. ADP and GDP also inhibited mitochondrial aconitase but at the levels well above the physiological concentrations of these nucleotides (6-11 mmol l(-1)). Our study shows expression of at least three UCP isoforms in C. virginica gill tissues but provides no indication that UCPs protect mitochondrial aconitase from oxidative inactivation in oysters. Overall, the results of this study indicate that temperature stress exaggerates toxicity of Cd leading to elevated oxidative stress in mitochondria, which may have important implications for survival of poikilotherms in polluted environments during

  14. 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…

  15. Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring.

    PubMed

    Bouhss, A; Sakamoto, H; Palibroda, N; Chiriac, M; Sarfati, R; Smith, J M; Craescu, C T; Bârzu, O

    1995-02-10

    GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N. PMID:7778777

  16. 6-(3,5-Dimeth-oxy-benzyl-amino)-9-(oxan-2-yl)-9H-purine.

    PubMed

    Starha, Pavel; Popa, Igor; Dvořák, Zdeněk; Trávníček, Zdeněk

    2013-04-01

    The mol-ecule of the title compound, C19H23N5O3, contains six-membered pyrimidine and five-membered imidazole rings merged into the essentially planar purine skeleton (r.m.s. deviation = 0.01 Å). In the crystal, pairs of N-H⋯N hydrogen bonds link mol-ecules into inversion dimers. The dimers are linked via C-H⋯O hydrogen bonds, forming double-stranded chains propagating along [001]. These chains are linked via C-H⋯π and parallel slipped π-π inter-actions [centroid-centroid distance = 3.467 (1) Å; slippage 0.519 Å], forming a three-dimensional network. PMID:23634074

  17. 6-(3,5-Dimeth­oxy­benzyl­amino)-9-(oxan-2-yl)-9H-purine

    PubMed Central

    Štarha, Pavel; Popa, Igor; Dvořák, Zdeněk; Trávníček, Zdeněk

    2013-01-01

    The mol­ecule of the title compound, C19H23N5O3, contains six-membered pyrimidine and five-membered imidazole rings merged into the essentially planar purine skeleton (r.m.s. deviation = 0.01 Å). In the crystal, pairs of N—H⋯N hydrogen bonds link mol­ecules into inversion dimers. The dimers are linked via C—H⋯O hydrogen bonds, forming double-stranded chains propagating along [001]. These chains are linked via C—H⋯π and parallel slipped π–π inter­actions [centroid–centroid distance = 3.467 (1) Å; slippage 0.519 Å], forming a three-dimensional network. PMID:23634074

  18. 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

  19. Protonation of azines and purines as a model for the electrophilic aromatic substitution - rationalization by triadic formula.

    PubMed

    Vianello, Robert

    2011-09-01

    First gas-phase carbon proton affinities of eleven azines and purines (pyrrole, pyrazole, imidazole, pyridine, pyridazine, pyrimidine, pyrazine, bicyclic purine, pyridine-N-oxide, 2-aminopyrimidine and uracil) were calculated by a composite G3B3 methodology and used to probe their susceptibility to undergo electrophilic aromatic substitution (EAS), taking benzene as a reference molecule. The results revealed excellent agreement with the available experimental data and were interpreted using the triadic approach. We found out that pyrroles, which are more reactive towards EAS reaction than benzene, are stronger carbon bases than the latter compound, whereas pyridines exhibit lower carbon basicity, being at the same time less reactive toward substitution by electrophiles than benzene. In all of the investigated molecules the frontier orbital describing the corresponding p-electron density at the carbon atom to be protonated is HOMO as calculated by the HF/G3large//B3LYP/6-31G(d) level of theory. Our results are in a disagreement with the work by D'Auria (M. D'Auria, Tetrahedron Lett. 2005, 46, 6333-6336; Lett. Org. Chem. 2005, 2, 659-661), who at B3LYP/6-311+G(d, p) level found out that in some of systems investigated here the HOMO orbital is not of -symmetry, which was used to rationalize the lower reactivity of these systems towards EAS. It turned out that energies of HOMO orbitals alone correlate very poorly with carbon proton affinities, unlike the difference in proton affinities between the most basic carbon atom and thermodynamically the most favourable site of protonation, which performs much better. Triadic analysis demonstrated the importance of considering a complete picture of the protonation process and all three terms appearing in the triadic scheme individually when discussing trends in basicity/nucleophilicity of closely related molecules. PMID:24062111

  20. A unique surface membrane anchored purine-salvage enzyme is conserved among a group of primitive eukaryotic human pathogens.

    PubMed

    Debrabant, A; Bastien, P; Dwyer, D M

    2001-04-01

    Previously, we isolated and characterized the gene encoding the 3'-Nucleotidase/Nuclease (Ld3'NT/NU) from the human pathogen, Leishmania donovani. This unique cell surface enzyme has been shown to be involved in the salvage of host-derived purines, which are essential for the survival of this important protozoan parasite. In this report, we assessed whether the 3'-Nucleotidase/Nuclease was conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that a Ld3'NT/NU gene homolog was present in each of the visceral and cutaneous Leishmania species tested (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of colorimetric assays using 3'-adenosine monophosphate as substrate demonstrated that each of these organisms also expressed significant levels of 3'-nucleotidase enzyme activity. In addition, we showed that a Ld3'NT/NU gene homolog was expressed in each of these Leishmania species as a > 40 kDa 3'-nucleotidase enzyme activity. A Ld3'NT/NU gene homolog was also identified in two Crithidia species (C. fasciculata and C. luciliae) and Leptomonas seymouri but was only marginally detectable in Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens. Cumulatively, results of this study showed that an Ld3'NT/NU homolog was conserved amongst pathogenic Leishmania sp. which suggests that this enzyme must play an critical role in purine salvage for all members of this group of human pathogens. PMID:11451370

  1. Prevention of lethal graft vs Host disease following bone marrow transplantation (pretreatment of the inoculum with purine metabolic enzyme inhibitors)

    SciTech Connect

    Kennedy, D.W.

    1983-01-01

    A correlation between lymphocyte function and enzymes of the purine metabolic pathway has been shown. Abnormal levels of three of these enzymes - adenosine deaminase (ADA), 5'-nucleotidase (5'-NT), and purine nucleoside phosphorylase (PNP) - have been associated with defective lymphoid functions. Selective inhibition of one or more of these enzymes may result in elimination of specific lymphocyte populations from a bone marrow (BM) graft and thus prevent graft-vs-host disease (GVHD). To test this hypothesis, BM and spleen cells were pretreated with inhibitors of ADA or PNP before transplant to histoincompatible recipients. Germfree (GF) mice approximately 11 weeks of age were lethally X-irradiated with 1000 Rads. At 24 hours post irradiation the mice received 5 x 10/sup 6/ BM cells and 5 x 10/sup 6/ BM cells and 5 x 10/sup 6/ spleen cells i.v. from syngeneic donors or allogeneic donors. Prior to injection the mice were divided into 5 groups: (1) the inoculum was treated with a final concentration of 10 ..mu..M deoxycoformycin (dCF), and ADA inhibitor, and 100 ..mu..M deoxyadenosine (dAdo); (2) the inoculum was treated with a final concentration of 100 ..mu..M 8-aminoguanosine (8AGuo), an inhibitor of PNP, and 100 ..mu..M 2'-deoxyguanosine (2'dGuo); (3) allogenic controls; (4) syngeneic controls; and (5) radiation controls. In vitro experiments utilizing human peripheral blood and bone marrow demonstrated a severe immunosuppressive effect by the combination of 2'dCF and dAdo which was quick acting and not easily washed away. Treatment with 8A Guo + 2'dGuo also was immunosuppressive, but not as effective as the dCF and dAdo treatment. Both groups of drugs deserve further investigation for possible clinical application to BM transplantation.

  2. Repetitive Sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Repetitive sequences, or repeats, account for a substantial portion of the eukaryotic genomes. These sequences include very different types of DNA with respect to mode of origin, function, structure, and genomic distribution. Two large families of repetitive sequences can be readily recognized, ta...

  3. 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

  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

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-11-01

    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. 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

  6. Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source.

    PubMed

    Summers, Ryan M; Louie, Tai Man; Yu, Chi Li; Subramanian, Mani

    2011-02-01

    N-Demethylation of many xenobiotics and naturally occurring purine alkaloids such as caffeine and theobromine is primarily catalysed in higher organisms, ranging from fungi to mammals, by the well-studied membrane-associated cytochrome P450s. In contrast, there is no well-characterized enzyme for N-demethylation of purine alkaloids from bacteria, despite several reports on their utilization as sole source of carbon and nitrogen. Here, we provide what we believe to be the first detailed characterization of a purified N-demethylase from Pseudomonas putida CBB5. The soluble N-demethylase holoenzyme is composed of two components, a reductase component with cytochrome c reductase activity (Ccr) and a two-subunit N-demethylase component (Ndm). Ndm, with a native molecular mass of 240 kDa, is composed of NdmA (40 kDa) and NdmB (35 kDa). Ccr transfers reducing equivalents from NAD(P)H to Ndm, which catalyses an oxygen-dependent N-demethylation of methylxanthines to xanthine, formaldehyde and water. Paraxanthine and 7-methylxanthine were determined to be the best substrates, with apparent K(m) and k(cat) values of 50.4±6.8 μM and 16.2±0.6 min(-1), and 63.8±7.5 μM and 94.8±3.0 min(-1), respectively. Ndm also displayed activity towards caffeine, theobromine, theophylline and 3-methylxanthine, all of which are growth substrates for this organism. Ndm was deduced to be a Rieske [2Fe-2S]-domain-containing non-haem iron oxygenase based on (i) its distinct absorption spectrum and (ii) significant identity of the N-terminal sequences of NdmA and NdmB with the gene product of an uncharacterized caffeine demethylase in P. putida IF-3 and a hypothetical protein in Janthinobacterium sp. Marseille, both predicted to be Rieske non-haem iron oxygenases. PMID:20966097

  7. Draft Genome Sequence of the Bacterium Pseudomonas putida CBB5, Which Can Utilize Caffeine as a Sole Carbon and Nitrogen Source

    PubMed Central

    Quandt, Erik M.; Summers, Ryan M.; Subramanian, Mani V.

    2015-01-01

    Pseudomonas putida CBB5 was isolated from soil by enriching for growth on caffeine (1,3,7-trimethylxanthine). The draft genome of this strain is 6.9 Mb, with 5,941 predicted coding sequences. It includes the previously studied Alx gene cluster encoding alkylxanthine N-demethylase enzymes and other genes that enable the degradation of purine alkaloids. PMID:26067973

  8. The Cipher Code of Simple Sequence Repeats in "Vampire Pathogens".

    PubMed

    Zou, Geng; Bello-Orti, Bernardo; Aragon, Virginia; Tucker, Alexander W; Luo, Rui; Ren, Pinxing; Bi, Dingren; Zhou, Rui; Jin, Hui

    2015-01-01

    Blood inside mammals is a forbidden area for the majority of prokaryotic microbes; however, red blood cells tropism microbes, like "vampire pathogens" (VP), succeed in matching scarce nutrients and surviving strong immunity reactions. Here, we found VP of Mycoplasma, Rhizobiales, and Rickettsiales showed significantly higher counts of (AG)n dimeric simple sequence repeats (Di-SSRs) in the genomes, coding and non-coding regions than non Vampire Pathogens (N_VP). Regression analysis indicated a significant correlation between GC content and the span of (AG)n-Di-SSR variation. Gene Ontology (GO) terms with abundance of (AG)3-Di-SSRs shared by the VP strains were associated with purine nucleotide metabolism (FDR < 0.01), indicating an adaptation to the limited availability of purine and nucleotide precursors in blood. Di-amino acids coded by (AG)n-Di-SSRs included all three six-fold code amino acids (Arg, Leu and Ser) and significantly higher counts of Di-amino acids coded by (AG)3, (GA)3, and (TC)3 in VP than N_VP. Furthermore, significant differences (P < 0.001) on the numbers of triplexes formed from (AG)n-Di-SSRs between VP and N_VP in Mycoplasma suggested the potential role of (AG)n-Di-SSRs in gene regulation. PMID:26215592

  9. Crystal structure of disodium 2-amino-6-oxo-6,7-di­hydro-1H-purine-1,7-diide hepta­hydrate

    PubMed Central

    Gur, Dvir; Shimon, Linda J. W.

    2015-01-01

    In the title compound, disodium 2-amino-6-oxo-6,7-di­hydro-1H-purine-1,7-diide hepta­hydrate, 2Na+·C5H3N5O2−·7H2O, the structure is composed of alternating (100) layers of guanine mol­ecules and hydrated Na+ ions. Within the guanine layer, the mol­ecules 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 inter­actions between the Na+ cations and the guanine anions. Guanine mol­ecules are linked to neighboring water mol­ecules by O—H⋯N and O—H⋯O hydrogen bonds into a network structure. PMID:25844188

  10. 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).

  11. A possible prebiotic synthesis of purine, adenine, cytosine, and 4(3H)-pyrimidinone from formamide: implications for the origin of life.

    PubMed

    Saladino, R; Crestini, C; Costanzo, G; Negri, R; Di Mauro, E

    2001-05-01

    The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO(3) and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution. PMID:11377183

  12. trans-Dichloridobis{2-chloro-6-[(3-fluoro-benz-yl)amino]-9-isopropyl-9H-purine-κN (7)}platinum(II).

    PubMed

    Trávníček, Zdeněk; Starha, Pavel

    2013-06-01

    In the title compound, trans-[PtCl2(C15H15ClFN5)2], the Pt(II) atom, located on an inversion centre, is coordinated by the purine N atoms of the 2-chloro-6-[(3-fluoro-benz-yl)amino]-9-isopropyl-9H-purine ligands and two Cl atoms in a slightly distorted trans-square-planar coordination geometry [N-Pt-Cl angles = 89.91 (5) and 90.09 (5)°]. Weak intra-molecular N-H⋯Cl contacts occur. In the crystal, C-H⋯Cl and C-H⋯F contacts, as well as weak π-π stacking inter-actions [centroid-centroid distances = 3.5000 (11) and 3.6495 (12) Å] connect the mol-ecules into a three-dimensional architecture. PMID:23794993

  13. trans-Dichloridobis{2-chloro-6-[(3-fluoro­benz­yl)amino]-9-isopropyl-9H-purine-κN 7}platinum(II)

    PubMed Central

    Trávníček, Zdeněk; Štarha, Pavel

    2013-01-01

    In the title compound, trans-[PtCl2(C15H15ClFN5)2], the PtII atom, located on an inversion centre, is coordinated by the purine N atoms of the 2-chloro-6-[(3-fluoro­benz­yl)amino]-9-isopropyl-9H-purine ligands and two Cl atoms in a slightly distorted trans-square-planar coordination geometry [N—Pt—Cl angles = 89.91 (5) and 90.09 (5)°]. Weak intra­molecular N—H⋯Cl contacts occur. In the crystal, C—H⋯Cl and C—H⋯F contacts, as well as weak π–π stacking inter­actions [centroid–centroid distances = 3.5000 (11) and 3.6495 (12) Å] connect the mol­ecules into a three-dimensional architecture. PMID:23794993

  14. 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. PMID:25750754

  15. 2-Chloro-6-[(2,4-dimeth­oxy­benz­yl)amino]-9-isopropyl-9H-purine

    PubMed Central

    Novotná, Radka; Trávníček, Zdeněk

    2013-01-01

    In the title compound, C17H20ClN5O2, the benzene ring and the purine ring system make a dihedral angle of 78.56 (4)°. In the crystal, mol­ecules are linked by pairs of N—H⋯N hydrogen bonds, forming inversion dimers. C—H⋯O and C—H⋯Cl contacts further link the mol­ecules, forming a three-dimensional network. PMID:23476575

  16. 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

  17. Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

    PubMed

    Farthing, Don; Gehr, Lynne; Karnes, H Thomas; Sica, Domenic; Gehr, Todd; Larus, Terri; Farthing, Christine; Xi, Lei

    2007-01-01

    Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo

  18. Synthesis of carbon-11-labeled imidazopyridine- and purine-thioacetamide derivatives as new potential PET tracers for imaging of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1).

    PubMed

    Gao, Mingzhang; Wang, Min; Zheng, Qi-Huang

    2016-03-01

    The target tracer carbon-11-labeled imidazopyridine- and purine-thioacetamide derivatives, N-(3-[(11)C]methoxy-4-methoxyphenyl)-2-((5-methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)acetamide (3-[(11)C]4a) and N-(4-[(11)C]methoxy-3-methoxyphenyl)-2-((5-methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)acetamide (4-[(11)C]4a); 2-((6-amino-9H-purin-8-yl)thio)-N-(3-[(11)C]methoxy-4-methoxyphenyl)acetamide (3-[(11)C]8a) and 2-((6-amino-9H-purin-8-yl)thio)-N-(4-[(11)C]methoxy-3-methoxyphenyl)acetamide (4-[(11)C]8a), were prepared by O-[(11)C]methylation of their corresponding precursors with [(11)C]CH3OTf under basic condition (2N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50-60% radiochemical yields based on [(11)C]CO2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185-555GBq/μmol. PMID:26856922

  19. 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

    2009-01-01

    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. PMID:19001417

  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. 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

  2. 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.

  3. 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

    2016-01-01

    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

  4. Borrelia burgdorferi bb0426 encodes a 2′-deoxyribosyltransferase that plays a central role in purine salvage

    PubMed Central

    Lawrence, Kevin A; Jewett, Mollie W; Rosa, Patricia A; Gherardini, Frank C

    2009-01-01

    Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5′-diphosphates to deoxynucleosides-5′-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2′-deoxyribosyltransferase (DRTase, encoded by bb0426), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues. PMID:19460093

  5. N-phosphonocarbonylpyrrolidine derivatives of guanine: a new class of bi-substrate inhibitors of human purine nucleoside phosphorylase.

    PubMed

    Rejman, Dominik; Panova, Natalya; Klener, Pavel; Maswabi, Bokang; Pohl, Radek; Rosenberg, Ivan

    2012-02-23

    A complete series of pyrrolidine nucleotides, (3R)- and (3S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acids and (3S,4R)-, (3R,4S)-, (3S,4S)-, and (3R,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphosphonic acids, were synthesized and evaluated as potential inhibitors of purine nucleoside phosphorylase (PNP) isolated from peripheral blood mononuclear cells (PBMCs) and cell lines of myeloid and lymphoid origin. Two compounds, (S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acid (2a) and (3S,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphosphonic acid (6a), were recognized as nanomolar competitive inhibitors of PNP isolated from cell lines with K(i) values within the ranges of 16-100 and 10-24 nM, respectively. The low (MESG)K(i) and (Pi)K(i) values of both compounds for PNP isolated from PBMCs suggest that these compounds could be bisubstrate inhibitors that occupy both the phosphate and nucleoside binding sites of the enzyme. PMID:22264015

  6. 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

  7. p38α Activates Purine Metabolism to Initiate Hematopoietic Stem/Progenitor Cell Cycling in Response to Stress.

    PubMed

    Karigane, Daiki; Kobayashi, Hiroshi; Morikawa, Takayuki; Ootomo, Yukako; Sakai, Mashito; Nagamatsu, Go; Kubota, Yoshiaki; Goda, Nobuhito; Matsumoto, Michihiro; Nishimura, Emi K; Soga, Tomoyoshi; Otsu, Kinya; Suematsu, Makoto; Okamoto, Shinichiro; Suda, Toshio; Takubo, Keiyo

    2016-08-01

    Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery. PMID:27345838

  8. Purine (N)-Methanocarba Nucleoside Derivatives Lacking an Exocyclic Amine as Selective A3 Adenosine Receptor Agonists.

    PubMed

    Tosh, Dilip K; Ciancetta, Antonella; Warnick, Eugene; O'Connor, Robert; Chen, Zhoumou; Gizewski, Elizabeth; Crane, Steven; Gao, Zhan-Guo; Auchampach, John A; Salvemini, Daniela; Jacobson, Kenneth A

    2016-04-14

    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

  9. Antifolates targeting purine synthesis allow entry of tumor cells into S phase regardless of p53 function.

    PubMed

    Bronder, Julie L; Moran, Richard G

    2002-09-15

    The class of folate antimetabolites typified by (6R)-dideazatetrahydrofolate (lometrexol, DDATHF) are specific inhibitors of de novo purine synthesis because of potent inhibition of glycinamide ribonucleotide formyltransferase (GART) but do not induce detectable levels of DNA strand breaks. As such, they are a test case of the concept that ribonucleotide depletion can be sensed by p53, resulting in a G(1) cell cycle block. The GART inhibitors have been proposed previously to be cytotoxic in tumor cells lacking p53 function but only cytostatic in p53 wild-type tumor cells. We have investigated this concept. Cell cycle progression into and through S phase was slowed by DDATHF, but both p53 +/+ and -/- human colon carcinoma cells entered and completed one S phase in the presence of drug. This inability of p53 to initiate a G(1) arrest after DDATHF treatment was mirrored by an independence of the cytotoxicity of DDATHF on p53 function. We conclude that carcinoma cells are killed equally well by DDATHF and related compounds whether or not the p53 pathway is intact and that the utility of GART inhibitors would not be limited to p53-negative tumors. PMID:12234990

  10. Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

    PubMed Central

    Suarez, Javier; Schramm, Vern L.

    2015-01-01

    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. PMID:26305965

  11. Investigation of riboflavin sensitized degradation of purine and pyrimidine derivatives of DNA and RNA under UVA and UVB.

    PubMed

    Joshi, Prakash C; Keane, Thomas C

    2010-10-01

    DNA and RNA undergo photodegradation in UVC (200-290nm) due to direct absorption by the purine and pyrimidine bases. Limited effects are observed under UVB (290-320nm) or UVA (320-400nm). We have observed that an endogenous photosensitizer, riboflavin (RF), upon exposure to UVB or UVA can extensively damage the DNA and RNA bases. Guanine, uracil, thymine, adenine and cytosine were degraded by 100%, 82%, 60.4%, 46.3% and 10.3% under UVA (12J) and by 100%, 54.1%, 38.9%, 42.2% and <1.0% under UVB (6J), respectively. Guanosine and deoxyguanosine were degraded by 98±1.0% and 80±1.0% under UVA (4J) and UVB (12J), respectively. With an exception of GMP (53-82%), dGMP (51-88%) and to some extent TMP (3-4%) the remaining nucleosides and nucleotides were resistant to RF-induced photodecomposition. The photodegradation of G derivatives by RF was 2-fold higher than a well known photodynamic agent rose bengal. A comparison of the intensities of UVA and UVB sources used in this study with natural sunlight suggests that exposure with the latter along with an endogenous photosensitizer can have similar effects on DNA and RNA depending upon the duration of exposure. PMID:20816939

  12. 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.

  13. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    PubMed

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  14. Uptake of CdSe and CdSe/ZnS quantum dots into bacteria via purine-dependent mechanisms.

    PubMed

    Kloepfer, J A; Mielke, R E; Nadeau, J L

    2005-05-01

    Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type Bacillus subtilis incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In Escherichia coli, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light. PMID:15870345

  15. Oligomerization of the UapA Purine Transporter Is Critical for ER-Exit, Plasma Membrane Localization and Turnover.

    PubMed

    Martzoukou, Olga; Karachaliou, Mayia; Yalelis, Vassilis; Leung, James; Byrne, Bernadette; Amillis, Sotiris; Diallinas, George

    2015-08-14

    Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER (endoplasmic reticulum) through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated bimolecular fluorescence complementation assay, we provide evidence that a UapA oligomerization is associated with ER-exit and turnover, as ER-retained mutants due to either modification of a Tyr-based N-terminal motif or partial misfolding physically associate but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells. PMID:26049015

  16. Sperm of Galeorhinus galeus (Elasmobranchii, Triakidae) Traverse an Excurrent Duct System Characterized by Pronounced Regionalization: A Scanning Electron and Light Microscopy Study.

    PubMed

    Mcclusky, Leon Mendel

    2015-11-01

    The transport and subsequent maturation of spermatozoa in the vertebrate excurrent duct require the creation of a series of biochemically defined luminal milieus along the length of the duct. Such specialization is accomplished, among others, by changes in the epididymal histoarchitecture. Here we show that the intratesticular and extratesticular genital ducts of mating Galeorhinus galeus exhibit pronounced regionalization both in terms of epithelial histology and lumen diameter size. Findings also reveal distinct differences in the manner in which the spermatozoa were found in each segment of the duct. Novel scanning electron microscopy evidence is presented showing that the wide lumen ductuli epididymides, which ultimately convey the spermatozoa to the proximal epididymis, show functional specialization as well. The wall of the former consisted of cuboidal ciliated and nonciliated cells whose spatial arrangement in the duct wall resulted in a luminal surface showing lengthy rows of cilia-free areas, with each row bordered on both sides by a single row of cilia. The proximal epididymis comprised several subregions whose epithelial histology varied widely. The distal epididymis and ampulla of the epididymis possessed many fingerlike projections and transverse septa, respectively. As the main storage site for spermatozoa, the ampulla completed the bundling of spermatozoa into spermatozeugmata. These were circular sperm masses in which the heads of the spermatozoa were aligned side by side and embedded in a seminal matrix, while their tails extended outward. These findings of pronounced regionalization differ greatly from the rather uniform epididymal histology seen in some rays. PMID:26248611

  17. Hydrolysis of oligoribonucleotides: influence of sequence and length.

    PubMed Central

    Kierzek, R

    1992-01-01

    The chemical stability of phosphodiester bonds of some oligoribonucleotides in the presence of a cofactor like polyvinylpyrolidine (PVP) is sequence dependent. It was found that pyrimidine-A (YA) and pyrimidine-C (YC) are especially susceptible to hydrolysis. The hydrolyzability of this same phosphodiester bond is dependent on its position in the oligomer. The presence of 3' and 5'-adjacent nucleotides enhances hydrolysis of the UA phosphodiester bond. The acceleration of the hydrolysis of UA by a 5'-adjacent nucleotide is not base dependent. However, a 3'-adjacent purine increases hydrolysis of a UA phosphodiester bond more than a 3'-pyrimidine. The presence of the exoamino group on the 3'-side base (on 6 and 4 position for adenosine and cytidine, respectively) of YA or YZ phosphodiester bond is required for hydrolysis. Images PMID:1408823

  18. Pronounced Effects of HERG-Blockers E-4031 and Erythromycin on APD, Spatial APD Dispersion and Triangulation in Transgenic Long-QT Type 1 Rabbits

    PubMed Central

    Franke, Gerlind; Perez Feliz, Stefanie; Hartmann, Maximilian; Koren, Gideon; Zehender, Manfred; Bode, Christoph; Brunner, Michael; Odening, Katja E.

    2014-01-01

    Background Prolongation of action potential duration (APD), increased spatial APD dispersion, and triangulation are major factors promoting drug-induced ventricular arrhythmia. Preclinical identification of HERG/IKr-blocking drugs and their pro-arrhythmic potential, however, remains a challenge. We hypothesize that transgenic long-QT type 1 (LQT1) rabbits lacking repolarizing IKs current may help to sensitively detect HERG/IKr-blocking properties of drugs. Methods Hearts of adult female transgenic LQT1 and wild type littermate control (LMC) rabbits were Langendorff-perfused with increasing concentrations of HERG/IKr-blockers E-4031 (0.001–0.1 µM, n = 9/7) or erythromycin (1–300 µM, n = 9/7) and APD, APD dispersion, and triangulation were analyzed. Results At baseline, APD was longer in LQT1 than in LMC rabbits in LV apex and RV mid. Erythromycin and E-4031 prolonged APD in LQT1 and LMC rabbits in all positions. However, erythromycin-induced percentaged APD prolongation related to baseline (%APD) was more pronounced in LQT1 at LV base-lateral and RV mid positions (100 µM, LQT1, +40.6±9.7% vs. LMC, +24.1±10.0%, p<0.05) and E-4031-induced %APD prolongation was more pronounced in LQT1 at LV base-lateral (0.01 µM, LQT1, +29.6±10.6% vs. LMC, +19.1±3.8%, p<0.05) and LV base-septal positions. Moreover, erythromycin significantly increased spatial APD dispersion only in LQT1 and increased triangulation only in LQT1 in LV base-septal and RV mid positions. Similarly, E-4031 increased triangulation only in LQT1 in LV apex and base-septal positions. Conclusions E-4031 and erythromycin prolonged APD and increased triangulation more pronouncedly in LQT1 than in LMC rabbits. Moreover, erythromycin increased APD dispersion only in LQT1, indicating that transgenic LQT1 rabbits could serve as sensitive model to detect HERG/IKr-blocking properties of drugs. PMID:25244401

  19. Distortional binding of transition state analogs to human purine nucleoside phosphorylase probed by magic angle spinning solid-state NMR.

    PubMed

    Vetticatt, Mathew J; Itin, Boris; Evans, Gary B; Schramm, Vern L

    2013-10-01

    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

  20. 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. PMID:23902343

  1. Role of Purine Nucleoside Phosphorylase in Interactions between 2′,3′-Dideoxyinosine and Allopurinol, Ganciclovir, or Tenofovir

    PubMed Central

    Ray, Adrian S.; Olson, Loren; Fridland, Arnold

    2004-01-01

    The level of systemic exposure to 2′,3′-dideoxyinosine (ddI) is increased 40 to 300% when it is coadministered with allopurinol (Allo), ganciclovir (GCV), or tenofovir. However, the mechanism for these drug interactions remains undefined. A metabolic route for ddI clearance is its breakdown by purine nucleoside phosphorylase (PNP). Consistent with previous reports, enzymatic inhibition assays showed that acyclic nucleotide analogs can inhibit the phosphorolysis of inosine. It was further established that the mono- and diphosphate forms of tenofovir were inhibitors of PNP-dependent degradation of ddI (Kis, 38 nM and 1.3 μM, respectively). Allo and its metabolites were found to be relatively weak inhibitors of PNP (Kis, >100 μM). Coadministration of tenofovir, GCV, or Allo decreased the amounts of intracellular ddI breakdown products in CEM cells, while they increased the ddI concentrations (twofold increase with each drug at approximately 20 μM). While inhibition of the physiological function of PNP is unlikely due to the ubiquitous presence of high levels of enzymatic activity, phosphorylated metabolites of GCV and tenofovir may cause the increased level of exposure to ddI by direct inhibition of its phosphorolysis by PNP. The discrepancy between the cellular activity of Allo and the weak enzyme inhibition by Allo and its metabolites may be explained by an indirect mechanism of PNP inhibition. This mechanism may be facilitated by the unfavorable equilibrium of PNP and the buildup of one of its products (hypoxanthine) through the inhibition of xanthine oxidase by Allo. These findings support the inhibition of PNP-dependent ddI degradation as the molecular mechanism of these drug interactions. PMID:15047506

  2. 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

  3. Guanine-based purines modulate the effect of L-NAME on learning and memory in rats.

    PubMed

    Giuliani, P; Buccella, P; Ballerini, P; Ciccarelli, R; D'alimonte, I; Cicchitti, S; Petragnani, N; Natale, S; Iacovella, G; Caciagli, F; Di Iorio, P

    2012-11-01

    Guanosine has been reported to exert neuroprotective effects. We recently reported that, following intraperitoneal (i.p.) injection to rats, it resulted to be widely distributed. Its metabolic product guanine also rapidly increased in all the tissues, including brain, after i.p. injection of guanosine and consistently we found a significant enzymatic activity of a soluble purine nucleoside phosphorylase in the plasma of the treated animals. In this study the effect of per os administration of guanosine or guanine to rats submitted to passive avoidance task has been evaluated. Guanosine (4 and 8 mg/kg) administered pretraining impaired retention in the passive avoidance task and was unable to prevent the amnesic effect caused by 100 mg/kg N-omega-nitro-l-arginine methyl ester (L-NAME), an inhibitor of the nitric oxide synthase (NOS) known to reduce the capability of treated animals to acquire or retain informations in several learning tasks. On the contrary, guanine (4 and 8 mg/kg), which per se did not modify the latency to step-trough in the passive avoidance task, when administered pretraining 15 min before L-NAME prevented, in a dose dependent manner, the amnesic effect of the NOS inihibitor. Moreover the nucleobase was able to rescue the memory trace also when administered after training. Neither guanosine nor guanine had effects on locomotor activity. These results indicate that guanine can exert important biological activities which may be different from those mediated by its precursor guanosine, thus this evenience should be taken into account when the biological effects of guanosine are evaluated. PMID:23138719

  4. Guanine-based purines modulate the effect of L-NAME on learning and memory in rats.

    PubMed

    Giuliani, P; Buccella, S; Ballerini, P; Ciccarelli, R; D'Alimonte, I; Cicchitti, S; Petragnani, N; Natale, S; Iacovella, G; Caciagli, F; Di Iorio, P

    2012-12-01

    Guanosine has been reported to exert neuroprotective effects. We recently reported that, following intraperitoneal (i.p.) injection to rats, it resulted to be widely distributed. Its metabolic product guanine also rapidly increased in all the tissues, including brain, after i.p. injection of guanosine and consistently we found a significant enzymatic activity of a soluble purine nucleoside phosphorylase in the plasma of the treated animals. In this study the effect of per os administration of guanosine or guanine to rats submitted to passive avoidance task has been evaluated. Guanosine (4 and 8 mg/kg) administered pretraining impaired retention in the passive avoidance task and was unable to prevent the amnesic effect caused by 100 mg/kg N-omega-nitro-l-arginine methyl ester (L-NAME), an inhibitor of the nitric oxide synthase (NOS) known to reduce the capability of treated animals to acquire or retain informations in several learning tasks. On the contrary, guanine (4 and 8 mg/kg), which per se did not modify the latency to step-trough in the passive avoidance task, when administered pretraining 15 min before L-NAME prevented, in a dose dependent manner, the amnesic effect of the NOS inhibitor. Moreover the nucleobase was able to rescue the memory trace also when administered after training. Neither guanosine nor guanine had effects on locomotor activity. These results indicate that guanine can exert important biological activities which may be different from those mediated by its precursor guanosine, thus this evenience should be taken into account when the biological effects of guanosine are evaluated. PMID:23241935

  5. 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

  6. 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

  7. A large database DNA sequence handling program with generalized searching specifications.

    PubMed

    Stockwell, P A

    1982-01-11

    The program described allows for the creation and manipulation of files of DNA sequence data up to very great lengths. The program uses its own paging system to load segments of the sequence into a small internal buffer so that the program does not have excessive memory requirements. The program offers a menu of functions to the user, and has been written to be forgiving of user errors. A code for the generalised specification of bases as a series of groups (i.e. A or T, Purine, etc.) has been devised and can be used in search specifications or in sequence files. Versions of the program have been developed to run with special efficiency under DIGITAL's RT11 operating system or to run under systems with a suitable implementation of FORTRAN VI. PMID:7063398

  8. A 26-base-pair repetitive sequence specific for Neisseria gonorrhoeae and Neisseria meningitidis genomic DNA.

    PubMed Central

    Correia, F F; Inouye, S; Inouye, M

    1986-01-01

    Two-dimensional heteroduplex mapping of Neisseria gonorrhoeae genomic DNA revealed a number of spots, indicating the existence of repetitive sequences. When one of the spots was extracted and used as a probe for Southern blot analysis, two HindIII bands (11.0 and 3.6 kilobases [kb]) of the genomic digest hybridized with approximately equal intensity. The 3.6-kb fragment was cloned and found to contain two different types of repeated sequence. One type was approximately 1.1 kb in length and was found at least twice in the entire genome. The other consisted of a 26-base-pair family GT(C/A)C(Py)G(Pu)TTTTTGTTAAT(Py)C(Pu)CTATA (Py, pyrimidine; Pu, purine) that was repeated at least 20 times in the entire genome. This repetitive sequence was found also in Neisseria meningitidis but not in various other gram-negative bacteria. Images PMID:3091577

  9. [Sequencing babies?].

    PubMed

    Jordan, Bertrand

    2015-10-01

    An extension of newborn screening to genome sequencing is now feasible but raises a number of scientific, organisational and ethical issues. This is being explored in discussions and in several funded trials, in order to maximize benefits and avoid some identified risks. As some companies are already offering such a service, this is quite an urgent matter. PMID:26481033

  10. 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

  11. 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.

  12. 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.

  13. Purine biosynthesis, riboflavin production, and trophic-phase span are controlled by a Myb-related transcription factor in the fungus Ashbya gossypii.

    PubMed

    Mateos, Laura; Jiménez, Alberto; Revuelta, José L; Santos, María A

    2006-07-01

    Ashbya gossypii is a natural riboflavin overproducer used in the industrial production of the vitamin. We have isolated an insertional mutant exhibiting higher levels of riboflavin production than the wild type. DNA analysis of the targeted locus in the mutant strain revealed that a syntenic homolog of the Saccharomyces cerevisiae BAS1 gene, a member of the Myb family of transcription factors, was inactivated. Directed gene disruption of AgBAS1 confirmed the phenotype observed for the insertional mutant, and the Deltabas1 mutant also showed auxotrophy for adenine and several growth defects, such as a delay in the germination of the spores and an abnormally prolonged trophic phase. Additionally, we demonstrate that the DNA-binding domain of AgBas1p is able to bind to the Bas1-binding motifs in the AgADE4 promoter; we also show a clear nuclear localization of a green fluorescent protein-Bas1 fusion protein. Real-time quantitative PCR analyses comparing the wild type and the Deltabas1 mutant revealed that AgBAS1 was responsible for the adenine-mediated regulation of the purine and glycine pathways, since the transcription of the ADE4 and SHM2 genes was virtually abolished in the Deltabas1 mutant. Furthermore, the transcription of ADE4 and SHM2 in the Deltabas1 mutant did not diminish during the transition from the trophic to the productive phase did not diminish, in contrast to what occurred in the wild-type strain. A C-terminal deletion in the AgBAS1 gene, comprising a hypothetical regulatory domain, caused constitutive activation of the purine and glycine pathways, enhanced riboflavin overproduction, and prolonged the trophic phase. Taking these results together, we propose that in A. gossypii, AgBAS1 is an important transcription factor that is involved in the regulation of different physiological processes, such as purine and glycine biosynthesis, riboflavin overproduction, and growth. PMID:16820505

  14. 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. PMID:25314375

  15. Demonstration in vitro of inhibition in normal rat tissues yet stimulation in Jensen sarcoma cells of 5-fluorouracil anabolism by purine nucleosides

    SciTech Connect

    Beltz, R.E.; Haddad-Zackrison, L.

    1986-05-01

    It has been shown previously that the ability of tumor cells to anabolize 5-fluorouracil (FUra) to nucleotides can often be enhanced by exposing the cells to various purine nucleosides. Increases in FUra cytotoxicity have been observed to accompany this enhancement. In the present study the effects of purine nucleosides on FUra anabolism in rat tumor cells and in normal rat tissues sensitive to FUra were compared. Pieces of small intestine (SI), bone marrow suspensions (BM) and Jensen tumor cells were incubated in culture medium at 37/sup 0/ for 1 hr in the presence (or absence) of a selected purine nucleoside, then (2-/sup 14/C)FUra was added and the incubation was continued for another hr. Incorporation of radioactivity into the trichloroacetic acid-insoluble fraction in each case was determined as a measure of FUra anabolism. Inosine, adenosine and N/sup 6/-methyl-adenosine, 1 mM, stimulated FUra incorporation into the acid-insoluble fraction 2-3 fold in the tumor cells but inhibited this incorporation 59-70% in SI and 31-70% in BM. Attempts to further suppress FUra anabolism in the normal tissues resulted in a maximal inhibition of 92% in SI, using 1 mM alloxanthine, and a maximal inhibition of 84% in BM, employing combined 1 mM alloxanthine and 1 mM 5-aminoimidazole-4-carbox-amide ribonucleoside. These data suggest ways of selectively altering FUra anabolism in normal tissue and in tumor tissue of the tumor-bearing rat to improve the therapeutic index of FUra.

  16. Allantoin, a stress-related purine metabolite, can activate jasmonate signaling in a MYC2-regulated and abscisic acid-dependent manner.

    PubMed

    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

    2016-04-01

    Allantoin is a metabolic intermediate of purine catabolism that often accumulates in stressed plants. Recently, we used Arabidopsis knockout mutants (aln) ofALLANTOINASEto 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 analnmutant (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, thealn-1mutant displayed increased JA levels and enhanced responses to mechanical wounding and exogenous JA. Moreover,alnmutants demonstrated modestly increased susceptibility toPseudomonas syringaeandPectobacterium 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, includingMYC2, in wild-type plants, supporting the idea that allantoin might be responsible for the observed JA-related phenotypes ofalnmutants. However, mutants deficient in bioactive JA (jar1-1), insensitive to JA (myc2-3), or deficient in ABA (aba2-1andbglu18) suppressed the effect of exogenous allantoin. The suppression was further confirmed inaln-1 jar1-1andaln-1 bglu18double 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

  17. DNA sequence-dependent ionic currents in ultra-small solid-state nanopores.

    PubMed

    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. PMID:27103233

  18. 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

  19. 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

  20. Purine 3':5'-cyclic nucleotides with the nucleobase in a syn orientation: cAMP, cGMP and cIMP.

    PubMed

    Řlepokura, Katarzyna Anna

    2016-06-01

    Purine 3':5'-cyclic nucleotides are very well known for their role as the secondary messengers in hormone action and cellular signal transduction. Nonetheless, their solid-state conformational details still require investigation. Five crystals containing purine 3':5'-cyclic nucleotides have been obtained and structurally characterized, namely adenosine 3':5'-cyclic phosphate dihydrate, C10H12N5O6P·2H2O or cAMP·2H2O, (I), adenosine 3':5'-cyclic phosphate 0.3-hydrate, C10H12N5O6P·0.3H2O or cAMP·0.3H2O, (II), guanosine 3':5'-cyclic phosphate pentahydrate, C10H12N5O7P·5H2O or cGMP·5H2O, (III), sodium guanosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H11N5O7P(-)·4H2O or Na(cGMP)·4H2O, (IV), and sodium inosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H10N4O7P(-)·4H2O or Na(cIMP)·4H2O, (V). Most of the cyclic nucleotide zwitterions/anions [two from four cAMP present in total in (I) and (II), cGMP in (III), cGMP(-) in (IV) and cIMP(-) in (V)] are syn conformers about the N-glycosidic bond, and this nucleobase arrangement is accompanied by Crib-H...Npur hydrogen bonds (rib = ribose and pur = purine). The base orientation is tuned by the ribose pucker. An analysis of data obtained from the Cambridge Structural Database made in the context of syn-anti conformational preferences has revealed that among the syn conformers of various purine nucleotides, cyclic nucleotides and dinucleotides predominate significantly. The interactions stabilizing the syn conformation have been indicated. The inter-nucleotide contacts in (I)-(V) have been systematized in terms of the chemical groups involved. All five structures display three-dimensional hydrogen-bonded networks. PMID:27256694

  1. Discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as inhibitors of the human poly(A)-selective ribonuclease Caf1

    PubMed Central

    Jadhav, Gopal P.; Kaur, Ishwinder; Maryati, Maryati; Airhihen, Blessing; Fischer, Peter M.; Winkler, G. Sebastiaan

    2015-01-01

    Eukaryotic mRNA contains a 3′ poly(A) tail, which plays important roles in the regulation of mRNA stability and translation. Well-characterized enzymes involved in the shortening of the poly(A) tail include the multi-subunit Ccr4-Not deadenylase, which contains the Caf1 (Pop2) and Ccr4 catalytic components, and poly(A)-specific ribonuclease (PARN). Two Mg2+ ions present in the active sites of these ribonucleases are required for RNA cleavage. Here, we report the discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as (sub)micromolar inhibitors of Caf1. PMID:26299350

  2. 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. PMID:26844769

  3. 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

  4. 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

  5. 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

  6. 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. PMID:26895222

  7. Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA

    PubMed Central

    2016-01-01

    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. PMID:26895222

  8. Purine Nucleoside Analog - Sulfinosine Modulates Diverse Mechanisms of Cancer Progression in Multi-Drug Resistant Cancer Cell Lines

    PubMed Central

    Dačević, Mirjana; Isaković, Aleksandra; Podolski-Renić, Ana; Isaković, Andelka M.; Stanković, Tijana; Milošević, Zorica; Rakić, Ljubisav; Ruždijić, Sabera; Pešić, Milica

    2013-01-01

    Achieving an effective treatment of cancer is difficult, particularly when resistance to conventional chemotherapy is developed. P-glycoprotein (P-gp) activity governs multi-drug resistance (MDR) development in different cancer cell types. Identification of anti-cancer agents with the potential to kill cancer cells and at the same time inhibit MDR is important to intensify the search for novel therapeutic approaches. We examined the effects of sulfinosine (SF), a quite unexplored purine nucleoside analog, in MDR (P-gp over-expressing) non-small cell lung carcinoma (NSCLC) and glioblastoma cell lines (NCI-H460/R and U87-TxR, respectively). SF showed the same efficacy against MDR cancer cell lines and their sensitive counterparts. However, it was non-toxic for normal human keratinocytes (HaCaT). SF induced caspase-dependent apoptotic cell death and autophagy in MDR cancer cells. After SF application, reactive oxygen species (ROS) were generated and glutathione (GSH) concentration was decreased. The expression of key enzyme for GSH synthesis, gamma Glutamyl-cysteine-synthetase (γGCS) was decreased as well as the expression of gst-π mRNA. Consequently, SF significantly decreased the expression of hif-1α, mdr1 and vegf mRNAs even in hypoxic conditions. SF caused the inhibition of P-gp (coded by mdr1) expression and activity. The accumulation of standard chemotherapeutic agent – doxorubicin (DOX) was induced by SF in concentration- and time-dependent manner. The best effect of SF was obtained after 72 h when it attained the effect of known P-gp inhibitors (Dex-verapamil and tariquidar). Accordingly, SF sensitized the resistant cancer cells to DOX in subsequent treatment. Furthermore, SF decreased the experssion of vascular endothelial growth factor (VEGF) on mRNA and protein level and modulated its secretion. In conclusion, the effects on P-gp (implicated in pharmacokinetics and MDR), GSH (implicated in detoxification) and VEGF (implicated in tumor-angiogenesis and

  9. Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis.

    PubMed

    Cheng, Xue; Auger, Andréanne; Altaf, Mohammed; Drouin, Simon; Paquet, Eric; Utley, Rhea T; Robert, François; Côté, Jacques

    2015-06-01

    Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257-2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966-15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for

  10. Eaf1 Links the NuA4 Histone Acetyltransferase Complex to Htz1 Incorporation and Regulation of Purine Biosynthesis

    PubMed Central

    Cheng, Xue; Auger, Andréanne; Altaf, Mohammed; Drouin, Simon; Paquet, Eric; Utley, Rhea T.; Robert, François

    2015-01-01

    Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257–2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966–15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for

  11. Metagenomic Analysis of the Stool Microbiome in Patients Receiving Allogeneic Stem Cell Transplantation: Loss of Diversity Is Associated with Use of Systemic Antibiotics and More Pronounced in Gastrointestinal Graft-versus-Host Disease

    PubMed Central

    Holler, Ernst; Butzhammer, Peter; Schmid, Karin; Hundsrucker, Christian; Koestler, Josef; Peter, Katrin; Zhu, Wentao; Sporrer, Daniela; Hehlgans, Thomas; Kreutz, Marina; Holler, Barbara; Wolff, Daniel; Edinger, Matthias; Andreesen, Reinhard; Levine, John E.; Ferrara, James L.; Gessner, Andre; Spang, Rainer; Oefner, Peter J.

    2016-01-01

    Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 µmol/L to 11.8 ± 2.8 µmol/L in all post-transplant samples and to 3.5 ± 3 µmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT. PMID:24492144

  12. Ontogeny of pronounced female-biased sexual size dimorphism in the Malaysian cat gecko (Aeluroscalabotes felinus: Squamata: Eublepharidae): a test of the role of testosterone in growth regulation.

    PubMed

    Kubička, Lukáš; Golinski, Alison; John-Alder, Henry; Kratochvíl, Lukáš

    2013-07-01

    Species differences in the effect of male gonadal androgens on male growth are considered a possible mechanism allowing shifts in magnitude and even direction of sexual size dimorphism (SSD) in squamate reptiles. According to the bipotential growth regulation hypothesis, the androgen testosterone (T) enhances male growth in species with male-biased SSD and conversely inhibits male growth in males of female-larger species. In the present study, we describe the ontogeny of the pronounced female-biased SSD and report the effect of T on growth via hormonal manipulations in males and females of the Malaysian cat gecko (Aeluroscalabotes felinus). In accord with the predictions of the bipotential growth regulation hypothesis, growth was inhibited by replacement of T in castrated males. Additionally, exogenous T inhibited growth of females to male-typical levels. Nevertheless, male castration alone did not significantly affect growth, contrary to the prediction of the bipotential growth regulation hypothesis, which contradicts the generality of this hypothesis. Application of exogenous T to females can interfere with normal ovarian function. Therefore, although not directly tested in this study, we suggest that ovarian effects on the ontogeny of SSD in A. felinus are consistent with our results. The development of SSD is a function of differential growth between the sexes, and potential sex-specific growth regulation in both males and females should be taken into account as possible proximate mechanisms responsible for SSD. PMID:23545460

  13. Quantum spin Hall insulators in functionalized arsenene (AsX, X = F, OH and CH3) monolayers with pronounced light absorption.

    PubMed

    Zhao, Jun; Li, Yanle; Ma, Jing

    2016-05-14

    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. PMID:27101795

  14. 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

    De Sanctis, Pierfilippo; Foxe, John J; Czobor, Pal; Wylie, Glenn R; Kamiel, Stephanie M; Huening, Jessica; Nair-Collins, Mike; Krakowski, Menahem I

    2013-08-01

    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

  15. Are mammographic changes in the tumor bed more pronounced after intraoperative radiotherapy for breast cancer? Subgroup analysis from a randomized trial (TARGIT-A).

    PubMed

    Engel, Dorothee; Schnitzer, Andreas; Brade, Joachim; Blank, Elena; Wenz, Frederik; Suetterlin, Marc; Schoenberg, Stefan; Wasser, Klaus

    2013-01-01

    Intraoperative radiotherapy (IORT) with low-energy x-rays is increasingly used in breast-conserving therapy (BCT). Previous non-randomized studies have observed mammographic changes in the tumor bed to be more pronounced after IORT. The purpose of this study was to reassess the postoperative changes in a randomized single-center subgroup of patients from a multicenter trial (TARGIT-A). In this subgroup (n = 48) 27 patients received BCT with IORT, 21 patients had BCT with standard whole-breast radiotherapy serving as controls. Overall 258 postoperative mammograms (median follow-up 4.3 years, range 3-8) were retrospectively evaluated by two radiologists in consensus focusing on changes in the tumor bed. Fat necroses showed to be significantly more frequent (56% versus 24%) and larger (8.7 versus 1.6 sq cm, median) after IORT than those in controls. Scar calcifications were also significantly more frequent after IORT (63% versus 19%). The high incidence of large fat necroses in our study confirms previous study findings. However, the overall higher incidence of calcifications in the tumor bed after IORT represents a new finding, requiring further attention. PMID:23173717

  16. Surface modification for patterned cell growth on substrates with pronounced topographies using sacrificial photoresist and parylene-C peel-off

    NASA Astrophysics Data System (ADS)

    Larramendy, Florian; Yoshida, Shotaro; Jalabert, Laurent; Takeuchi, Shoji; Paul, Oliver

    2016-09-01

    A range of methods including soft lithography are available for patterning protein layers for cell adhesion on quasi-planar substrates. Suitably structured, these layers favor the geometrically constrained, controlled growth of cells and the development of cellular extensions on them. For this purpose, the ability to control the shape and dimension of cell-adhesive areas with high precision is crucial. For more advanced studies of cell interactions, the surface modification or functionalization of substrates with complex topographies is desirable. This paper describes a simple technique allowing to produce surface modification patterns using delicate molecules such as laminin on substrates exhibiting pronounced topographies with recessed and protruding microstructures. The technique is based on the combination of sacrificial photoresist structures with a connected parylene-C layer. This layer locally adheres to the substrate wherever the substrate needs to be protected against the surface modification. After surface modification, the parylene-C layer is peeled off. Patterns comprising arbitrary networks of modified and unmodified substrate areas can thus be realized. We demonstrate the technique with the guided growth of neuron-like PC12 cells on networks of laminin lines on substrates structured with micropillars and microwells.

  17. 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)

  18. Pronounced -Log T Divergence in Specific Heat of Nonmetallic CeOBiS2: A Mother Phase of BiS2-Based Superconductor

    NASA Astrophysics Data System (ADS)

    Higashinaka, Ryuji; Asano, Takuya; Nakashima, Takuya; Fushiya, Kengo; Mizuguchi, Yoshikazu; Miura, Osuke; Matsuda, Tatsuma D.; Aoki, Yuji

    2015-02-01

    The low-temperature properties of CeOBiS2 single crystals are studied by electrical resistivity, magnetization, and specific heat measurements. Ce 4f-electrons are found to be in a well-localized state split by crystalline-electric-field (CEF) effects. The CEF ground state is a pure Jz = ±1/2 doublet, and excited doublets are located far above. At low temperatures in zero field, we observe pronounced -log T divergence in the specific heat, revealing the presence of quantum critical fluctuations of 4f magnetic moments near a quantum critical point (QCP). Considering that CeOBiS2 is a nonmetal, this phenomenon cannot be attributed to the competition between Kondo and the Ruderman-Kittel-Kasuya-Yosida (RKKY) interactions as in numerous f-electron-based strongly correlated metals, indicating an unconventional mechanism. We suggest that CeOBiS2 is the first material found to be located at a QCP among geometrically frustrated nonmetallic magnets.

  19. 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.

  20. Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase.

    PubMed

    Parker, W B; Allan, P W; Waud, W R; Hong, J S; Sorscher, E J

    2011-06-01

    The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT. PMID:21394111

  1. 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

  2. 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.

  3. In Vivo Evidence for Alcohol-Induced Neurochemical Changes in Rat Brain Without Protracted Withdrawal, Pronounced Thiamine Deficiency, or Severe Liver Damage

    PubMed Central

    Zahr, Natalie M; Mayer, Dirk; Vinco, Shara; Orduna, Juan; Luong, Richard; Sullivan, Edith V; Pfefferbaum, Adolf

    2009-01-01

    Magnetic resonance spectroscopy (MRS) studies in human alcoholics report decreases in N-acetylaspartate (NAA) and choline-containing (Cho) compounds. Whether alterations in brain metabolite levels are attributable to alcohol per se or to physiological effects of protracted withdrawal or impaired nutritional or liver status remains unclear. Longitudinal effects of alcohol on brain metabolites measured in basal ganglia with single-voxel MRS were investigated in sibling pairs of wild-type Wistar rats, with one rat per pair exposed to escalating doses of vaporized alcohol, the other to vapor chamber air. MRS was conducted before alcohol exposure and twice during exposure. After 16 weeks of alcohol exposure, rats achieved average blood alcohol levels (BALs) of ~ 293 mg per 100 ml and had higher Cho and a trend for higher glutamine + glutamate (Glx) than controls. After 24 weeks of alcohol exposure, BALs rose to ~ 445 mg per 100 ml, and alcohol-exposed rats had higher Cho, Glx, and glutamate than controls. Thiamine and thiamine monophosphate levels were significantly lower in the alcohol than the control group but did not reach levels low enough to be considered clinically relevant. Histologically, livers of alcohol-exposed rats exhibited greater steatosis and lower glycogenosis than controls, but were not cirrhotic. This study demonstrates a specific pattern of neurobiochemical changes suggesting excessive membrane turnover or inflammation, indicated by high Cho, and alterations to glutamate homeostasis in the rat brain in response to extended vaporized alcohol exposure. Thus, we provide novel in vivo evidence for alcohol exposure as causing changes in brain chemistry in the absence of protracted withdrawal, pronounced thiamine deficiency, or severe liver damage. PMID:18704091

  4. Reconstructing of a Sequence Using Similar Sequences

    Energy Science and Technology Software Center (ESTSC)

    1995-11-28

    SIMSEQ reconstructs sequences from oligos. Similar known sequences are used as a reference. At present, simulated data are being used to develop the algorithm. SIMSEQ generates an initial random sequence, then generates a second sequence that is 60 to 90 percent similar to the first. Next, the second sequence is chopped into its appropriate oligos. All possible sequences are reconstructed to determine the most similar. Those with the highest similarity are printed as output.

  5. 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

    2016-01-01

    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. PMID:27023513

  6. Action of a mammalian AP-endonuclease on DNAs of defined sequences.

    PubMed Central

    Haukanes, B I; Helland, D E; Kleppe, K

    1989-01-01

    An apurinic/apyrimidinic (AP) specific endonuclease from mouse plasmacytoma cells (line MPC-11), was observed to cleave apurinic sites in oligonucleotides 9, 11, 12, 39 and 40 nucleotides in length. However, the enzyme failed to cleave AP-sites in two oligonucleotides 7 nucleotides in length. The maximum rates of digestion observed on these short single-stranded DNA (ssDNA) fragments were approximately 1/30 of the rates observed on double-stranded DNA (dsDNA). In studies using the Maxam-Gilbert DNA sequencing analysis, apurinic sites in purine-rich regions were preferentially cleaved in dsDNA but not in ssDNA, indicating that the enzyme has a sequence preference on dsDNA. These results suggest that some sites on DNA might be more efficiently repaired than others. Images PMID:2466239

  7. Glutathione-dependent reduction of arsenate in human erythrocytes--a process independent of purine nucleoside phosphorylase.

    PubMed

    Németi, Balázs; Gregus, Zoltán

    2004-12-01

    Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is toxicologically important, yet its mechanism is unknown. To clarify this, AsV reduction was investigated in human red blood cells (RBC), as they possess a simple metabolism. RBC were incubated with AsV in gluconate buffer, and the formed AsIII was quantified by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The observations are compatible with the following conclusions. (1) Human RBC reduce AsV intracellularly, because 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, inhibitor of the chloride-bicarbonate exchanger, which also mediates phosphate and AsV uptake), as well as chloride and phosphate, countered AsIII formation. (2) Purine nucleoside phosphorylase (PNP), whose AsV reductase activity has been directly demonstrated, cannot be a physiologically relevant AsV reductase, because its inhibitor (BCX-1777) failed to decrease the basal erythrocytic AsV reduction, although it prevented the increase in AsIII formation caused by artificial activation of PNP with inosine and dithiothreitol. (3) The basal (PNP-independent) AsV reduction requires glutathione (GSH), because the GSH depletor diethylmaleate strongly diminished AsIII formation. (4) The erythrocytic AsV reduction apparently depends on NAD(P) supply, because oxidants of NAD(P)H (i.e., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, 4-dimethylaminophenol) enhanced AsIII formation from AsV. The oxidant-stimulated AsV reduction is PNP-independent, because BCX-1777 failed to affect it, but is GSH-dependent, because diethylmaleate impaired it. (5) Pyruvate-induced glucose depletion, which causes NAD enrichment in the erythrocytes at the expense of NADH, enhanced AsV reduction. This suggests that the erythrocytic AsV reduction requires both NAD supply and operation of the lower part of the glycolytic pathway starting from glyceraldehyde-3

  8. Products of the direct reaction of the diazonium ion of a metabolite of the carcinogen N-nitrosomorpholine with purines of nucleosides and DNA.

    PubMed

    Zink, Charles N; Soissons, Nicolas; Fishbein, James C

    2010-07-19

    A number of putative purine nucleoside and nucleobase adducts of the diazonium ion derived from 3-hydroxy-N-nitrosomorpholine have been synthesized as dimethylacetals. These are converted, in most cases nearly quantitatively, to the aldehydes, or in two cases to their derivatives, on treatment with mild acid to yield standards for a quantitative investigation of alkylation of purine nucleosides and DNA by the above metabolite of the powerful carcinogen N-nitrosomorpholine. The stability of the resulting nucleobase ethoxyacetaldehyde (EA) adducts has been characterized under a number of conditions with respect to their propensity to decompose. The stabilities, compared to that of the previously characterized adduct of the model benzimidazole, are generally unexceptional. Deposition of adducts on purine nucleosides and DNA were quantified in reactions in which 3-hydroperoxy-N-nitrosomorpholine was reduced to the hydroxy metabolite by a water-soluble phosphine at 21 +/- 2 degrees C. The adduct profile is highly similar to that observed from simpler alpha-hydroxy metabolites of acyclic dialkylnitrosamines, with the three most abundant ethoxyacetaldehyde (EA) adducts in reactions of duplex DNA being N7-EA-Gua approximately O(6)-EA-Gua > N3-EA-Ade. The initial rate kinetics of formation of hydroxyethyl (HE) lesions from the initially formed EA lesions have been determined in the case of the major products in the cases of both the nucleoside and DNA adducts. The rates of formation of HE adducts are accelerated in DNA, relative to the nucleosides in the cases of the N7-EA-Ade, N7-EA-Gua, and O(6)-EA-Gua adducts by factors of 7, 14, and 54, respectively. The initial rates of depurination of the N3-EA-Ade, N7-EA-Gua, and N7-EA-Gua adducts have also been quantified, and they are unexceptional in comparison with what has been previously reported for simple alkyl adducts. The adduct profiles reported here stand in significant contrast to what has been reported previously for

  9. P2Y1 and cysteinyl leukotriene receptors mediate purine and cysteinyl leukotriene co-release in primary cultures of rat microglia.

    PubMed

    Ballerini, P; Di Iorio, P; Ciccarelli, R; Caciagli, F; Poli, A; Beraudi, A; Buccella, S; D'Alimonte, I; D'Auro, M; Nargi, E; Patricelli, P; Visini, D; Traversa, U

    2005-01-01

    Inflammation is widely recognized as contributing to the pathology of acute and chronic neurodegenerative conditions. Microglial cells are pathologic sensors in the brain and activated microglia have been viewed as detrimental. Leukotriene, including cysteinyl leukotrienes (CysLTs) are suggested to be involved in brain inflammation and neurological diseases and ATP, by its receptors is a candidate for microglia activation. A23187 (10 microM) stimulated microglia to co-release CysLTs and [3H] adenine based purines ([3H] ABPs), mainly ATP. The biosynthetic production of CysLTs was abolished by 10 microM MK-886, an inhibitor of 5-lipoxygenase-activating protein activity. RT-PCR analysis showed that microglia expressed both CysLT1 / CysLT2 receptors, P2Y1ATP receptors and several members of the ATP binding cassette (ABC) transporters including MRP1, MRP4 and Pgp. The increase in [Ca2+]i elicited by LTD4 (0.1 microM) and 2MeSATP (100 microM), agonists for CysLT- and P2Y1-receptors, was abolished by the respective antagonists, BAYu9773 (0.5 microM) and suramin (50 microM). The stimulation of both receptor subtypes, induced a concomitant increase in the release of both [3H] ABPs and CysLTs that was blocked by the antagonists and significantly reduced by a cocktail of ABC transporter inhibitors, BAPTA/AM (intracellular Ca2+ chelator) and staurosporine (0.1 microM, PKC blocker). P2Y antagonist was unable to antagonise the effects of LTD4 and BAYu9773 did not reduce the effects of 2MeSATP. These data suggest that: i) the efflux of purines and cysteinyl-leukotrienes is specifically and independently controlled by the two receptor types, ii) calcium, PKC and the ABC transporter system can reasonably be considered common mechanisms underlying the release of ABPs and CysLTs from microglia. The blockade of P2Y1 or CysLT1/CysLT2 receptors by specific antagonists that abolished the raise in [Ca2+]i and drastically reduced the concomitant efflux of both compounds, as well as the

  10. Polyarthritis in primary Sjögren's syndrome represents a distinct subset with less pronounced B cell proliferation a Dutch cohort with long-term follow-up.

    PubMed

    Ter Borg, E J; Kelder, J C

    2016-03-01

    infrequently (32 %). All patients were treated with a classic (c) disease-modifying antirheumatic drug (DMARD), but in two cases, treatment was necessary with a tumour necrosis factor (TNF) inhibitor. PA+ pSS patients are more frequently anti-CCP positive and have a less pronounced B cell proliferation than PA- patients. PSS patients with PA seem to have a relatively mild articular expression with a favourable course. PMID:26791875

  11. The Cipher Code of Simple Sequence Repeats in “Vampire Pathogens”

    PubMed Central

    Zou, Geng; Bello-Orti, Bernardo; Aragon, Virginia; Tucker, Alexander W.; Luo, Rui; Ren, Pinxing; Bi, Dingren; Zhou, Rui; Jin, Hui

    2015-01-01

    Blood inside mammals is a forbidden area for the majority of prokaryotic microbes; however, red blood cells tropism microbes, like “vampire pathogens” (VP), succeed in matching scarce nutrients and surviving strong immunity reactions. Here, we found VP of Mycoplasma, Rhizobiales, and Rickettsiales showed significantly higher counts of (AG)n dimeric simple sequence repeats (Di-SSRs) in the genomes, coding and non-coding regions than non Vampire Pathogens (N_VP). Regression analysis indicated a significant correlation between GC content and the span of (AG)n-Di-SSR variation. Gene Ontology (GO) terms with abundance of (AG)3-Di-SSRs shared by the VP strains were associated with purine nucleotide metabolism (FDR < 0.01), indicating an adaptation to the limited availability of purine and nucleotide precursors in blood. Di-amino acids coded by (AG)n-Di-SSRs included all three six-fold code amino acids (Arg, Leu and Ser) and significantly higher counts of Di-amino acids coded by (AG)3, (GA)3, and (TC)3 in VP than N_VP. Furthermore, significant differences (P < 0.001) on the numbers of triplexes formed from (AG)n-Di-SSRs between VP and N_VP in Mycoplasma suggested the potential role of (AG)n-Di-SSRs in gene regulation. PMID:26215592

  12. Disruption of de novo purine biosynthesis in Pseudomonas fluorescens Pf0-1 leads to reduced biofilm formation and a reduction in cell size of surface-attached but not planktonic cells

    PubMed Central

    Newell, Peter D.

    2016-01-01

    Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25 ∼ 30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium. PMID:26788425

  13. Cytosines, but not purines, determine recombination activating gene (RAG)-induced breaks on heteroduplex DNA structures: implications for genomic instability.

    PubMed

    Naik, Abani Kanta; Lieber, Michael R; Raghavan, Sathees C

    2010-03-01

    The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C((d))C((S))C((S))" (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability. PMID:20051517

  14. Development of Purine-Derived 18F-Labeled Pro-drug Tracers for Imaging of MRP1 Activity with PET

    PubMed Central

    2014-01-01

    Multidrug resistance-associated protein 1 (MRP1) is a drug efflux transporter that has been implicated in the pathology of several neurological diseases and is associated with development of multidrug resistance. To enable measurement of MRP1 function in the living brain, a series of 6-halopurines decorated with fluorinated side chains have been synthesized and evaluated as putative pro-drug tracers. The tracers were designed to undergo conjugation with glutathione within the brain and hence form the corresponding MRP1 substrate tracers in situ. 6-Bromo-7-(2-[18F]fluoroethyl)purine showed good brain uptake and rapid metabolic conversion. Dynamic PET imaging demonstrated a marked difference in brain clearance rates between wild-type and mrp1 knockout mice, suggesting that the tracer can allow noninvasive assessment of MRP1 activity in vivo. PMID:24456310

  15. Factors related to the growth of psittacosis virus (strain 6BC) II. Purines, pyrimidines, and other components related to nucleic acid.

    PubMed

    MORGAN, H R

    1952-03-01

    In various amounts and mixtures, adenine, guanine, xanthine, hypoxanthine, thymine, thymidine, cytidylic acid, and an enzymatic digest of desoxyribonucleic acid all failed to influence the inhibition by sulfadiazine of the growth of psittacosis virus (6BC) in embryonated eggs. A number of purine analogues, including benzimidazole, 2,6-diaminopurine, and 8-azaguanine, inhibited the growth of psittacosis virus (6BC) in tissue cultures at concentrations which had no obvious toxic effects on the host tissues. The virus inhibitory action of 2,6-diaminopurine was reversed by addition of adenine and that of 8-azaguanine by guanine. The growth of psittacosis virus (6BC) was inhibited by the pteridine compounds 2-ammo-4-hydroxy-6-formylpteridine and xanthopterin, while other related substances had little or no inhibitory activity. Xanthine reversed the inhibitory effects of 2-amino-4-hydroxy-6-formylpteridine. There was no correlation between the inhibitory activity of the pteridines on xanthine oxidase and multiplication of the virus. PMID:14927793

  16. Investigation of free amino acid, total phenolics, antioxidant activity and purine alkaloids to assess the health properties of non-Camellia tea.

    PubMed

    Bi, Wu; He, Chunnian; Ma, Yunyun; Shen, Jie; Zhang, Linghua Harris; Peng, Yong; Xiao, Peigen

    2016-03-01

    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

  17. Purine Analog-Like Properties of Bendamustine Underlie Rapid Activation of DNA Damage Response and Synergistic Effects with Pyrimidine Analogues in Lymphoid Malignancies

    PubMed Central

    Hiraoka, Nobuya; Kikuchi, Jiro; Yamauchi, Takahiro; Koyama, Daisuke; Wada, Taeko; Uesawa, Mitsuyo; Akutsu, Miyuki; Mori, Shigehisa; Nakamura, Yuichi; Ueda, Takanori; Kano, Yasuhiko; Furukawa, Yusuke

    2014-01-01

    Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies. PMID:24626203

  18. 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 Central

    Koissi, Niangoran; Fishbein, James C.

    2013-01-01

    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-oxa-pentanal fragment on O6-Gua, and condensation with N6-Ade, presumably in the vicinity. Decreasing temperature of the reactions and decreasing pH modestly increased the yields of trapped crosslink. 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 times, the amount of cross-link was observed to increase, nearly linearly, by about four-fold over one week. These data indicate that there are a minimum of 2 populations of cross-links, one that forms rapidly, in minutes, and another that grows in with time, over days. Reduced nicotinamide co-factors 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. PMID:23587048

  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. Coronary flow regulation in patients with ischemic heart disease: release of purines and prostacyclin and the effect of inhibitors of prostaglandin formation.

    PubMed

    Edlund, A; Berglund, B; van Dorne, D; Kaijser, L; Nowak, J; Patrono, C; Sollevi, A; Wennmalm, A

    1985-06-01

    The present investigation was undertaken to study cardiac release of adenosine and prostacyclin (prostaglandin [PG] I2) in patients with ischemic heart disease (IHD), and to assess coronary vascular resistance before and after inhibition of synthesis in such patients. In 48 patients with IHD, arterial and coronary sinus blood samples were taken at rest, during atrial pacing to angina, and after pacing. Levels of purines were determined by high-performance liquid chromatography and the PGI2 metabolite 6-keto-PGF1 alpha was measured with radioimmunoassay. Coronary sinus blood flow was determined with retrograde continuous thermodilution before and after oral administration of indomethacin, aspirin, naproxen, or ibuprofen. Atrial pacing induced myocardial ischemia, as evidenced by typical chest pain and arrested lactate extraction. Adenosine was extracted at rest, but during ischemia there was a significant release of its metabolite hypoxanthine, indicating increased myocardial breakdown of high-energy adenine nucleotides. Arterial and coronary sinus concentrations of 6-keto-PGF1 alpha were low and no significant differences between them were found. After administration of the PG-synthesis inhibitor indomethacin, coronary vascular resistance was elevated, as was the cardiac oxygen extraction. The three other PG-synthesis inhibitors (aspirin, naproxen, and ibuprofen) did not, however, induce any change in coronary vascular resistance or in the cardiac extraction of oxygen. On the basis of these data we suggest that in patients with IHD cardiac ischemia results in increased myocardial production and release of purines, cardiac ischemia does not elicit any detectable increase in coronary production of prostacyclin, and the increased coronary resistance induced by indomethacin does not reflect the involvement of locally formed PG in the maintenance of coronary flow, but is rather a direct effect of the drug. PMID:3888437

  1. 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.

  2. Plasma First Resuscitation Reduces Lactate Acidosis, Enhances Redox Homeostasis, Amino Acid and Purine Catabolism in a Rat Model of Profound Hemorrhagic Shock.

    PubMed

    D'Alessandro, Angelo; Moore, Hunter B; Moore, Ernest E; Wither, Matthew J; Nemkov, Travis; Morton, Alexander P; Gonzalez, Eduardo; Chapman, Michael P; Fragoso, Miguel; Slaughter, Anne; Sauaia, Angela; Silliman, Christopher C; Hansen, Kirk C; Banerjee, Anirban

    2016-08-01

    The use of aggressive crystalloid resuscitation to treat hypoxemia, hypovolemia, and nutrient deprivation promoted by massive blood loss may lead to the development of the blood vicious cycle of acidosis, hypothermia, and coagulopathy and, utterly, death. Metabolic acidosis is one of the many metabolic derangements triggered by severe trauma/hemorrhagic shock, also including enhanced proteolysis, lipid mobilization, as well as traumatic diabetes. Appreciation of the metabolic benefit of plasma first resuscitation is an important concept. Plasma resuscitation has been shown to correct hyperfibrinolysis secondary to severe hemorrhage better than normal saline. Here, we hypothesize that plasma first resuscitation corrects metabolic derangements promoted by severe hemorrhage better than resuscitation with normal saline. Ultra-high-performance liquid chromatography-mass spectrometry-based metabolomics analyses were performed to screen plasma metabolic profiles upon shock and resuscitation with either platelet-free plasma or normal saline in a rat model of severe hemorrhage. Of the 251 metabolites that were monitored, 101 were significantly different in plasma versus normal saline resuscitated rats. Plasma resuscitation corrected lactate acidosis by promoting glutamine/amino acid catabolism and purine salvage reactions. Plasma first resuscitation may benefit critically injured trauma patients by relieving the lactate burden and promoting other non-clinically measured metabolic changes. In the light of our results, we propose that plasma resuscitation may promote fueling of mitochondrial metabolism, through the enhancement of glutaminolysis/amino acid catabolism and purine salvage reactions. The treatment of trauma patients in hemorrhagic shock with plasma first resuscitation is likely not only to improve coagulation, but also to promote substrate-specific metabolic corrections. PMID:26863033

  3. Restricting Glutamine or Glutamine-Dependent Purine and Pyrimidine Syntheses Promotes Human T Cells with High FOXP3 Expression and Regulatory Properties.

    PubMed

    Metzler, Barbara; Gfeller, Patrick; Guinet, Elisabeth

    2016-05-01

    T cell subsets differ in their metabolic requirements, and further insight into such differences might be harnessed to selectively promote regulatory T cells (Tregs) for therapies in autoimmunity and transplantation. We found that Gln restriction during human T cell activation favored CD4 T cells with high expression of the Treg transcription factor FOXP3. This resulted from shrinking numbers and reduced proliferation of activated FOXP3(lo/-)CD4 T cells while FOXP3(hi)CD4 T cell numbers increased. This gain was abolished by blocking Gln synthetase, an enzyme that responds to Gln and purine/pyrimidine deficiencies. The shift toward FOXP3(hi)CD4 T cells under Gln restriction was recapitulated with inhibitors of Gln-dependent pyrimidine and purine syntheses that together closely mimicked declining cell numbers and cell cycles, and by small interfering RNA knockdown of the respective rate-limiting Gln-consuming enzymes CAD and PPAT. FOXP3(hi)-enriched CD25(hi)CD4 T cells from these cultures inhibited proliferation, but they also produced effector cytokines, including IL-17A. The latter was largely confined to CTLA-4(hi)-expressing FOXP3(hi)-enriched CD25(hi)CD4 T cells that suppressed proliferation more weakly than did CTLA-4(lo/-)CD25(hi)FOXP3(hi)-enriched T cells. A causal link between high IL-17A production and impaired suppression of proliferation could not be demonstrated, however. Collectively, these results reveal a Gln synthetase-dependent increase and resilience of FOXP3(hi) cells under Gln restriction, and they demonstrate that impaired Gln-dependent nucleotide synthesis promotes FOXP3(hi) cells with regulator properties. It remains to be investigated to what extent the concomitant retention of IL-17A-producing CD4 T cells may limit the therapeutic potential of Tregs enriched through targeting these pathways in vivo. PMID:27022197

  4. The Sequence and Structure Determine the Function of Mature Human miRNAs

    PubMed Central

    Wawrzyniak, Dariusz; Jeleniewicz, Jaroslaw; Barciszewska, Miroslawa Z.; Barciszewski, Jan

    2016-01-01

    Micro RNAs (miRNAs) (19–25 nucleotides in length) belong to the group of non-coding RNAs are the most abundant group of posttranscriptional regulators in multicellular organisms. They affect a gene expression by binding of fully or partially complementary sequences to the 3’-UTR of target mRNA. Furthermore, miRNAs present a mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated at the post-transcriptional level. However, little is known about the specific pathways through which miRNAs with specific sequence or structural motifs regulate the cellular processes. In this paper we showed the broad and deep characteristics of mature miRNAs according to their sequence and structural motifs. We investigated a distinct group of miRNAs characterized by the presence of specific sequence motifs, such as UGUGU, GU-repeats and purine/pyrimidine contents. Using computational function and pathway analysis of their targeted genes, we were able to observe the relevance of sequence and the type of targeted mRNAs. As the consequence of the sequence analysis we finally provide the comprehensive description of pathways, biological processes and proteins associated with the distinct group of characterized miRNAs. Here, we found that the specific group of miRNAs with UGUGU can activate the targets associated to the interferon induction pathway or pathways prominently observed during carcinogenesis. GU-rich miRNAs are prone to regulate mostly processes in neurogenesis, whereas purine/pyrimidine rich miRNAs could be involved rather in transport and/or degradation of RNAs. Additionally, we have also analyzed the simple sequence repeats (SSRs). Their variation within mature miRNAs might be critical for normal miRNA regular activity. Expansion or contraction of SSRs in mature miRNA might directly affect its mRNA interaction or even change the function of that distinct miRNA. Our results prove that due to the specific sequence features, these

  5. Poincaré recurrences of DNA sequences

    NASA Astrophysics Data System (ADS)

    Frahm, K. M.; Shepelyansky, D. L.

    2012-01-01

    We analyze the statistical properties of Poincaré recurrences of Homo sapiens, mammalian, and other DNA sequences taken from the Ensembl Genome data base with up to 15 billion base pairs. We show that the probability of Poincaré recurrences decays in an algebraic way with the Poincaré exponent β≈4 even if the oscillatory dependence is well pronounced. The correlations between recurrences decay with an exponent ν≈0.6 that leads to an anomalous superdiffusive walk. However, for Homo sapiens sequences, with the largest available statistics, the diffusion coefficient converges to a finite value on distances larger than one million base pairs. We argue that the approach based on Poncaré recurrences determines new proximity features between different species and sheds a new light on their evolution history.

  6. Natural and artificial RNAs occupy the same restricted region of sequence space

    PubMed Central

    Kennedy, Ryan; Lladser, Manuel E.; Wu, Zhiyuan; Zhang, Chen; Yarus, Michael; De Sterck, Hans; Knight, Rob

    2010-01-01

    Different chemical and mutational processes within genomes give rise to sequences with different compositions and perhaps different capacities for evolution. The evolution of functional RNAs may occur on a “neutral network” in which sequences with any given function can easily mutate to sequences with any other. This neutral network hypothesis is more likely if there is a particular region of composition that contains sequences that are functional in general, and if many different functions are possible within this preferred region of composition. We show that sequence preferences in active sites recovered by in vitro selection combine with biophysical folding rules to support the neutral network hypothesis. These simple active-site specifications and folding preferences obtained by artificial selection experiments recapture the previously observed purine bias and specific spread along the GC axis of naturally occurring aptamers and ribozymes isolated from organisms, although other types of RNAs, such as miRNA precursors and spliceosomal RNAs, that act primarily through complementarity to other amino acids do not share these preferences. These universal evolved sequence features are therefore intrinsic in RNA molecules that bind small-molecule targets or catalyze reactions. PMID:20032164

  7. Effects of halogenated WNA derivatives on sequence dependency for expansion of recognition sequences in non-natural-type triplexes.

    PubMed

    Taniguchi, Yosuke; Nakamura, Ayako; Senko, Yusuke; Nagatsugi, Fumi; Sasaki, Shigeki

    2006-03-01

    Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but their target duplexes are limited to homopurine/homopyrimidine sequences because of interruption of the pyrimidines bases in the purine region. This problem has not been fully solved despite a wide variety of studies. Recently, we have developed a bicyclic system as a novel scaffold for nucleoside analogues (WNA, W-shaped nucleoside analogues) and determined two useful compounds, WNA-betaT (2) and WNA-betaC (5), for highly stable and selective triplex formation at a TA and a CG interrupting site, respectively. However, subsequent investigations have shown that the triplex formation using WNA is dependent on the neighboring bases of the TFOs. In this study, we have synthesized new WNA derivatives having halogenated recognition bases or benzene rings and evaluated the effects of the modifications on the triplex stability as well as selectivity. It has been found that the WNA-betaT analogues holding 5-halogenated pyrimidine bases (WNA-beta(Br)U (3) and WNA-beta(F)U (4)) exhibit high CG-selectivity. On the other hand, the WNA-betaT derivatives having the bromo-substituted benzene ring (mBr-WNA-betaT (10) and oBr-WNA-betaT (11)) have shown high selectivity to a TA interrupting site with high stability in the sequences to which the original WNA-betaT do not bind. Thus, sequence-dependency has been overcome by the sequence-dependent use of WNA-betaT, mBr-WNA-betaT, and oBr-WNA-betaT. PMID:16497000

  8. 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.

  9. 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

  10. Whole Genome Sequencing

    MedlinePlus

    ... you want to learn. Search form Search Whole Genome Sequencing You are here Home Testing & Services Testing ... the full story, click here . What is whole genome sequencing? Whole genome sequencing is the mapping out ...

  11. 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.

  12. Science sequence design

    NASA Technical Reports Server (NTRS)

    Koskela, P. E.; Bollman, W. E.; Freeman, J. E.; Helton, M. R.; Reichert, R. J.; Travers, E. S.; Zawacki, S. J.

    1973-01-01

    The activities of the following members of the Navigation Team are recorded: the Science Sequence Design Group, responsible for preparing the final science sequence designs; the Advanced Sequence Planning Group, responsible for sequence planning; and the Science Recommendation Team (SRT) representatives, responsible for conducting the necessary sequence design interfaces with the teams during the mission. The interface task included science support in both advance planning and daily operations. Science sequences designed during the mission are also discussed.

  13. Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis.

    PubMed

    Bzowska, Agnieszka; Koellner, Gertraud; Wielgus-Kutrowska, Beata; Stroh, Albrecht; Raszewski, Grzegorz; Holý, Antonin; Steiner, Thomas; Frank, Joachim

    2004-09-17

    The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining

  14. Crystal structure of 6,7-dihy­droxy-6,7-di­hydro-3H-imidazo[1,2-a]purin-9(5H)-one

    PubMed Central

    Guo, Wei; Li, Cheng-Xun; Lv, Jie; Wang, Jing

    2016-01-01

    The title purine derivative, C7H7N5O3, is an adduct of guanine with glyoxal. In the mol­ecule, the di­hydro­imidazole ring adopts a twisted conformation on the C—C bond, and the two hydroxyl groups lie on opposite sides of the mean plane of the ring. In the crystal, the mol­ecules are linked by N—H⋯O, O—H⋯N and N—H⋯N hydrogen bonds forming a three-dimensional framework. The crystal packing is reinforced by C—H⋯O hydrogen bonds and by offset π–π stacking of the purine ring systems of inversion related mol­ecules [inter­centroid distance = 3.4839 (12) Å]. PMID:27536400

  15. (1-Adamant­yl){4-[(2-chloro-9-isopropyl-9H-purin-6-yl)aminometh­yl]phen­yl}methanone trichloro­methane solvate

    PubMed Central

    Rouchal, Michal; Nečas, Marek; Vícha, Robert

    2009-01-01

    In the title compound, C26H30ClN5O·CHCl3, the purine mol­ecule consists of essentially planar benzene and purine ring systems [maximum deviation 0.010 (4) Å for both ring systems] forming a dihedral angle of 85.52 (9)°. Inter­molecular N—H⋯N hydrogen bonds link adjacent mol­ecules into centrosymmetric dimers. The structure also contains inter­molecular C—H⋯O and C—H⋯N inter­actions. The benzene rings form offset face-to-face π–π stacking inter­actions with an inter­planar distance of 3.541 (4) Å and a centroid-to-centroid distance of 4.022 (4) Å. PMID:21583132

  16. DNA sequence analysis of gamma radiation-induced deletions and insertions at the APRT locus of hamster cells

    SciTech Connect

    Miles, C.; Sargent, G.; Phear, G.; Meuth, M. )

    1990-01-01

    Gamma radiation-induced gene rearrangements at the Chinese hamster ovary cell locus coding for the purine salvage enzyme adenine phosphoribosyl transferase (APRT) consist of both simple deletions and more complex alterations that are presumably the result of multiple strand breaks. To characterize these mutations at the DNA sequence level, fragments altered by deletion and insertion mutations were obtained by cloning in lambda phage vectors or by using the polymerase chain reaction. The radiation-induced deletions characterized here eliminate 3-4 kb and have at least one breakpoint in an AT-rich region or near short direct or inverted repeats. Insertions involve small fragments (102 and 456 bp) of repetitive DNA that appear to be related to B2 (short interspersed repetitive) and long interspersed repeat families. The novel fragments bear little resemblance to each other or to sequences at the integration sites, and their introduction is accompanied by a small target site deletion.

  17. DNA sequences at the ends of the genome of bacteriophage Mu essential for transposition.

    PubMed Central

    Groenen, M A; Timmers, E; van de Putte, P

    1985-01-01

    We have determined the minimal DNA sequences at the ends of the genome of bacteriophage Mu that are required for its transposition. A mini-Mu was constructed on a multicopy plasmid that enabled the manipulation of the DNA sequences at its ends without affecting the genes essential for transposition. The genes A and B, which were cloned outside the ends of the mini-Mu on the same plasmid, were both needed for optimal transposition. In our experimental system the predominant end products of the transposition are cointegrates both in the presence and in the absence of B. Two regions ending approximately 25 and 160 bp from the left end and one ending approximately 50 bp from the right end appear to be essential for optimal transposition. Overlapping with these regions, a 22-base-pair sequence was recognized with the consensus Y-G-T-T-C-A-Y-T-N-N-A-A-R-Y-R-C-G-A-A-A-A, where Y and R represent any pyrimidine and purine, respectively. At the left end these sequences occur as direct repeats; at the right end this sequence is inverted with respect to those at the left end. PMID:2984681

  18. Targeted enzyme prodrug therapy for metastatic prostate cancer – a comparative study of L-methioninase, purine nucleoside phosphorylase, and cytosine deaminase

    PubMed Central

    2014-01-01

    Background Enzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3. Results All three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p < 0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV. Conclusions Strong binding of fusion proteins to the prostate cancer cells

  19. 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

  20. Characterization of the pre-mRNA binding site for yeast ribosomal protein L32: the importance of a purine-rich internal loop.

    PubMed

    Li, H; Dalal, S; Kohler, J; Vilardell, J; White, S A

    1995-07-21

    The structure of the RNA binding target for Saccharomyces cerevisiae ribosomal protein L32 was examined using chemical and enzymatic probes as well as thermodynamic methods. In vivo, the production of yeast RPL32 is regulated by a feedback mechanism whereby RPL32 binds to the 5' end of its transcript and inhibits splicing. The binding site of ribosomal protein L32 on the L32 RNA transcript can be reduced to fewer than 30 nucleotides which compromise a stem-internal loop-stem structural motif. The internal loop is closed by a potential G-U pair, is asymmetric and contains mostly purines. The existence of the two helical regions was confirmed by chemical and enzymatic probing. The reactivity of the loop region suggests a structure intermediate between that of single and double-stranded RNA. Base stacking continues into the loop, but two loop bases are extremely reactive to chemical agents. The interaction between the model RNA and the protein is specific and has a dissociation constant of approximately 10 nM. Several of the loop bases are critical for protein binding, as demonstrated by mutational data and chemical protection and modification interference studies. The internal loop destabilizes the RNA, and allows the RNA to melt in an all-or-none fashion. PMID:7616567

  1. Nucleolipids of Canonical Purine ß‐d‐Ribo‐Nucleosides: Synthesis and Cytostatic/Cytotoxic Activities Toward Human and Rat Glioblastoma Cells

    PubMed Central

    Knies, Christine; Hammerbacher, Katharina; Kinscherf, Ralf

    2015-01-01

    Abstract We report on the synthesis of two series of canonical purine ß‐d‐ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterized by elemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by 1H and 13C NMR, and pH‐dependent UV/Vis spectroscopy. A selection of the novel nucleolipids with different lipophilic moieties were first tested on their cytotoxic effect toward human macrophages. Compounds without a significant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligodendroglioma GOS‐3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. In order to additionally investigate the potential molecular mechanisms involved in the cytotoxic effects of the derivatives on GOS‐3 or BT4Ca cells, we evaluated the induction of apoptosis and observed the particular activity of the nucleolipid ethyl 3‐{4‐hydroxymethyl‐2‐methyl‐6‐[6‐oxo‐1‐(3,7,11‐trimethyl‐dodeca‐2,6,10‐trienyl)‐1,6‐dihydro‐purin‐9‐yl]‐tetrahydro‐furo[3,4‐d][1,3]dioxol‐2‐yl}propionate (8 c) toward both human and rat glioblastoma cell lines in vitro. PMID:27308225

  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. The generalized quaternion sequence

    NASA Astrophysics Data System (ADS)

    Deveci, Ömür

    2016-04-01

    In this work, we define the recurrence sequence by using the relation matrix of the generalized quaternion group and then, we obtain miscellaneous properties of this sequence. Also, we obtain the cyclic groups and the semigroups which are produced by generating matrix of the sequence defined when read modulo m. Furthermore, we study this sequence modulo m, and then we derive the relationship among the order the cyclic groups obtained and the periods of the sequence defined.

  5. Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

    PubMed Central

    Kukreti, S; Sun, J S; Garestier, T; Hélène, C

    1997-01-01

    Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates. PMID:9336456

  6. Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.

    PubMed

    Semenyuk, A; Darian, E; Liu, J; Majumdar, A; Cuenoud, B; Miller, P S; Mackerell, A D; Seidman, M M

    2010-09-14

    The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2'-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence. PMID:20701359

  7. Targeting of an Interrupted Polypurine:Polypyrimidine Sequence in Mammalian Cells by a Triplex-Forming Oligonucleotide Containing a Novel Base Analogue†

    PubMed Central

    Semenyuk, A.; Darian, E.; Liu, J.; Majumdar, A.; Cuenoud, B.; Miller, P. S.; MacKerell, A. D.; Seidman, M. M.

    2010-01-01

    The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2′-OMeguanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence. PMID:20701359

  8. 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

  9. Two parallel pathways in the kinetic sequence of the Dihydrofolate Reductase from Mycobacterium tuberculosis

    PubMed Central

    Czekster, Clarissa M.; Vandemeulebroucke, An; Blanchard, John S.

    2011-01-01

    Dihydrofolate reductase from Mycobacterium tuberculosis catalyzes the NAD(P)H dependent reduction of dihydrofolate, yielding NAD(P)+ and tetrahydrofolate, the primary one carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. Previously, steady-state studies revealed that the chemical step significantly contributes to the steady state turnover number, but that a step after the chemical step was likely limiting the reaction rate. Here, we report the first pre-steady state investigation of the kinetic sequence of the MtDHFR aiming to identify kinetic intermediates, and the identity of the rate limiting steps. This kinetic analysis suggests a kinetic sequence comprising two parallel pathways with a rate determining product release. Although product release is likely occurring in a random fashion, there is a slight preference for the release of THF first, a kinetic sequence never observed for a wild type dihydrofolate reductase of any organism studied to date. Temperature studies were conducted to determine the magnitude of the energetic barrier posed by the chemical step, and the pH dependence of the chemical step was studied, demonstrating an acidic shift from the pKa observed under steady-state. The rate constants obtained here were combined with the activation energy for the chemical step to compare energy profiles for each kinetic sequence. The two parallel pathways are discussed, as well as their implications on the catalytic cycle of this enzyme. PMID:21744813

  10. 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.

  11. 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.

  12. Association Between Upstream Purine Complexes of Human Caveolin-1 Gene and Schizophrenia in Qazvin Province of Iran

    PubMed Central

    Najafipour, Reza; Heidari, Abolfazl; Alizadeh, Safar Ali; Ghafelebashi, Hannaneh; Rashvand, Zahra; Javadi, Amir; Moradi, Mohammad; Afshar, Hosein

    2014-01-01

    Background: Caveolin is a multifunctional and scaffolding membrane protein, which involves cholesterol trafficking to plasma lipid microdomain. It organizes and targets synaptic parts of the neurotransmitter and neurotrophic receptor signaling pathways. Caveolins are encoded by CAV-1, 2 and 3 genes. Disruption of the CAV1 would likely ruin the neuronal signaling, which leads to symptoms of schizophrenia in predisposed individuals. Objectives: The upper area of CAV-1 gene is highly conserved and can have a regulatory role in neurodegenerative diseases. This study was designed to find out the possible association of polymorphisms of this area and schizophrenia. Patients and Methods: In a case-control study, 254 blood samples were obtained from 127 patients with schizophrenia and 127 well matched controls referred to 22 Bahman Hospital of Qazvin University of Medical Sciences (QUMS) in Qazvin province, Iran, using simple random sampling method. After extracting DNA, the upper region of the human CAV1- gene was amplified by PCR in all collected samples. The products were visualized by silver staining in 10% polyacrylamide gel and then sequenced. Results: We detected nine homozygotes in patients and 15 in control subjects. Homozygosity was 7.08% and 11.8% in cases and control, respectively. Nine types homozygote haplotype were detected in upper region of the CAV1 gene in cases and controls. Three haplotypes were common in cases and controls; four haplotypes were seen in controls only and two in cases. Conclusions: Our findings implied a significant correlation between some haplotypes of upper region of CAV1 gene and schizophrenia. Existence of some haplotypes and lack of another in CAV1 upstream can suggest a significant correlation between schizophrenia and some haplotypes. PMID:25763243

  13. Brain activation during anticipation of sound sequences.

    PubMed

    Leaver, Amber M; Van Lare, Jennifer; Zielinski, Brandon; Halpern, Andrea R; Rauschecker, Josef P

    2009-02-25

    Music consists of sound sequences that require integration over time. As we become familiar with music, associations between notes, melodies, and entire symphonic movements become stronger and more complex. These associations can become so tight that, for example, hearing the end of one album track can elicit a robust image of the upcoming track while anticipating it in total silence. Here, we study this predictive "anticipatory imagery" at various stages throughout learning and investigate activity changes in corresponding neural structures using functional magnetic resonance imaging. Anticipatory imagery (in silence) for highly familiar naturalistic music was accompanied by pronounced activity in rostral prefrontal cortex (PFC) and premotor areas. Examining changes in the neural bases of anticipatory imagery during two stages of learning conditional associations between simple melodies, however, demonstrates the importance of fronto-striatal connections, consistent with a role of the basal ganglia in "training" frontal cortex (Pasupathy and Miller, 2005). Another striking change in neural resources during learning was a shift between caudal PFC earlier to rostral PFC later in learning. Our findings regarding musical anticipation and sound sequence learning are highly compatible with studies of motor sequence learning, suggesting common predictive mechanisms in both domains. PMID:19244522

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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)…

  20. T. cacao Transcriptome Sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To compliment the T. cacao genome sequencing initiative and to build a reference set of expressed genes for functional studies, a broad and state-of-the-art approach to transcriptome sequencing is underway. Using newly optimized methods, transcriptome sequencing libraries were prepared from RNA of o...

  1. Enhanced virome sequencing using targeted sequence capture

    PubMed Central

    Wylie, Todd N.; Wylie, Kristine M.; Herter, Brandi N.; Storch, Gregory A.

    2015-01-01

    Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viral populations. It is culture independent, requires no a priori knowledge of the viruses in the sample, and may provide useful genomic information. However, MSS can lack sensitivity and may yield insufficient data for detailed analysis. We have created a targeted sequence capture panel, ViroCap, designed to enrich nucleic acid from DNA and RNA viruses from 34 families that infect vertebrate hosts. A computational approach condensed ∼1 billion bp of viral reference sequence into <200 million bp of unique, representative sequence suitable for targeted sequence capture. We compared the effectiveness of detecting viruses in standard MSS versus MSS following targeted sequence capture. First, we analyzed two sets of samples, one derived from samples submitted to a diagnostic virology laboratory and one derived from samples collected in a study of fever in children. We detected 14 and 18 viruses in the two sets, comprising 19 genera from 10 families, with dramatic enhancement of genome representation following capture enrichment. The median fold-increases in percentage viral reads post-capture were 674 and 296. Median breadth of coverage increased from 2.1% to 83.2% post-capture in the first set and from 2.0% to 75.6% in the second set. Next, we analyzed samples containing a set of diverse anellovirus sequences and demonstrated that ViroCap could be used to detect viral sequences with up to 58% variation from the references used to select capture probes. ViroCap substantially enhances MSS for a comprehensive set of viruses and has utility for research and clinical applications. PMID:26395152

  2. 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.

  3. Oxidation of purine nucleotides by Triplet 3,3',4,4'-benzophenone tetracarboxylic acid in aqueous solution: pH-dependence.

    PubMed

    Saprygina, Natalya N; Morozova, Olga B; Abramova, Tatyana V; Grampp, Günter; Yurkovskaya, Alexandra V

    2014-07-10

    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 × 10(9) M(-1) s(-1) (GMP), and kq = 1.2 × 10(9) M(-1) s(-1) (AMP), whereas in neutral and basic solutions it is significantly lower: kq = 2.6 × 10(8) M(-1) s(-1) (GMP, 4.9 < pH < 9.4), kq = 3.5 × 10(7) M(-1) s(-1) (GMP, pH > 9.4), kq = 1.0 × 10(8) 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 × 10(8) M(-1) s(-1) (4.9 < pH < 6.5) to 1.0 × 10(8) M(-1) s(-1) (pH > 6.5). PMID:24926567

  4. Design, synthesis, and in vitro biological evaluation of novel 6-methyl-7-substituted-7-deaza purine nucleoside analogs as anti-influenza A agents.

    PubMed

    Lin, Cai; Sun, Chenghai; Liu, Xiao; Zhou, Yiqian; Hussain, Muzammal; Wan, Junting; Li, Minke; Li, Xue; Jin, Ruiliang; Tu, Zhengchao; Zhang, Jiancun

    2016-05-01

    Among many subtypes of influenza A viruses, influenza A(H1N1) and A(H3N2) subtypes are currently circulating among humans (WHO report 2014-15). Therapeutically, the emergence of viral resistance to currently available drugs (adamantanes and neuraminidase inhibitors) has heightened alarms for developing novel drugs that could address diverse targets in the viral replication cycle in order to improve treatment outcomes. To this regard, the design and synthesis of nucleoside analog inhibitors as potential anti-influenza A agents is a very active field of research nowadays. In this study, we designed and synthesized a series of hitherto unknown 6-methyl-7-substituted-7-deaza purine nucleoside analogs, and evaluated for their biological activities against influenza A virus strains, H1N1 and H3N2. From the viral inhibition assay, we identified some effective compounds, among which, compounds 5x (IC50 = 5.88 μM and 6.95 μM for H1N1 and H3N2, respectively) and 5z (IC50 = 3.95 μM and 3.61 μM for H1N1 and H3N2, respectively) demonstrated potent anti-influenza A activity. On the basis of selectivity index, we conceive that compound 5x may serve as a chemical probe of interest for further lead optimization studies with a general aim of developing novel and effective anti-influenza A virus agents. PMID:26802557

  5. Sequence variation in ligand binding sites in proteins

    PubMed Central

    Magliery, Thomas J; Regan, Lynne

    2005-01-01

    Background The recent explosion in the availability of complete genome sequences has led to the cataloging of tens of thousands of new proteins and putative proteins. Many of these proteins can be structurally or functionally categorized from sequence conservation alone. In contrast, little attention has been given to the meaning of poorly-conserved sites in families of proteins, which are typically assumed to be of little structural or functional importance. Results Recently, using statistical free energy analysis of tetratricopeptide repeat (TPR) domains, we observed that positions in contact with peptide ligands are more variable than surface positions in general. Here we show that statistical analysis of TPRs, ankyrin repeats, Cys2His2 zinc fingers and PDZ domains accurately identifies specificity-determining positions by their sequence variation. Sequence variation is measured as deviation from a neutral reference state, and we present probabilistic and information theory formalisms that improve upon recently suggested methods such as statistical free energies and sequence entropies. Conclusion Sequence variation has been used to identify functionally-important residues in four selected protein families. With TPRs and ankyrin repeats, protein families that bind highly diverse ligands, the effect is so pronounced that sequence "hypervariation" alone can be used to predict ligand binding sites. PMID:16194281

  6. 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.

  7. Uncorrectable sequences and telecommand

    NASA Technical Reports Server (NTRS)

    Ekroot, Laura; Mceliece, R.; Dolinar, S.; Swanson, L.

    1993-01-01

    The purpose of a tail sequence for command link transmission units is to fail to decode, so that the command decoder will begin searching for the start of the next unit. A tail sequence used by several missions and recommended for this purpose by the Consultative Committee on Space Data Standards is analyzed. A single channel error can cause the sequence to decode. An alternative sequence requiring at least two channel errors before it can possibly decode is presented. (No sequence requiring more than two channel errors before it can possibly decode exists for this code.)

  8. Single-stranded DNA-binding proteins PURalpha and PURbeta bind to a purine-rich negative regulatory element of the alpha-myosin heavy chain gene and control transcriptional and translational regulation of the gene expression. Implications in the repression of alpha-myosin heavy chain during heart failure.

    PubMed

    Gupta, Madhu; Sueblinvong, Viranuj; Raman, Jai; Jeevanandam, Valluvan; Gupta, Mahesh P

    2003-11-01

    The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells. PMID:12933792

  9. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids.

    PubMed

    Kim, Ki-Hyun; Nielsen, Peter E; Glazer, Peter M

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences by Watson-Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  10. Using Triple Helix Forming Peptide Nucleic Acids for Sequence-selective Recognition of Double-stranded RNA

    PubMed Central

    Hnedzko, Dziyana; Cheruiyot, Samwel K.; Rozners, Eriks

    2014-01-01

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple helix forming peptide nucleic acids (PNAs) that bind in the major grove of RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M) that enables strong triple helical binding at physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E) that enable recognition of isolated pyrimidines in the purine rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis and HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included. PMID:25199637

  11. Indexing Similar DNA Sequences

    NASA Astrophysics Data System (ADS)

    Huang, Songbo; Lam, T. W.; Sung, W. K.; Tam, S. L.; Yiu, S. M.

    To study the genetic variations of a species, one basic operation is to search for occurrences of patterns in a large number of very similar genomic sequences. To build an indexing data structure on the concatenation of all sequences may require a lot of memory. In this paper, we propose a new scheme to index highly similar sequences by taking advantage of the similarity among the sequences. To store r sequences with k common segments, our index requires only O(n + NlogN) bits of memory, where n is the total length of the common segments and N is the total length of the distinct regions in all texts. The total length of all sequences is rn + N, and any scheme to store these sequences requires Ω(n + N) bits. Searching for a pattern P of length m takes O(m + m logN + m log(rk)psc(P) + occlogn), where psc(P) is the number of prefixes of P that appear as a suffix of some common segments and occ is the number of occurrences of P in all sequences. In practice, rk ≤ N, and psc(P) is usually a small constant. We have implemented our solution and evaluated our solution using real DNA sequences. The experiments show that the memory requirement of our solution is much less than that required by BWT built on the concatenation of all sequences. When compared to the other existing solution (RLCSA), we use less memory with faster searching time.

  12. Unlocking hidden genomic sequence

    PubMed Central

    Keith, Jonathan M.; Cochran, Duncan A. E.; Lala, Gita H.; Adams, Peter; Bryant, Darryn; Mitchelson, Keith R.

    2004-01-01

    Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs. PMID:14973330

  13. Multiplexed Fragaria Chloroplast Genome Sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method to sequence multiple chloroplast genomes that uses the sequencing depth of ultra high throughput sequencing technologies was recently described. Sequencing complete chloroplast genomes can resolve phylogenetic relationships at low taxonomic levels and identify point mutations and indels tha...