Science.gov

Sample records for 5-methyl uracil

  1. Multiphoton ionization of Uracil

    NASA Astrophysics Data System (ADS)

    Prieto, Eladio; Martinez, Denhi; Guerrero, Alfonso; Alvarez, Ignacio; Cisneros, Carmen

    2016-05-01

    Multiphoton ionization and dissociation of Uracil using a Reflectron time of flight spectrometer was performed along with radiation from the second harmonic of a Nd:YAG laser. Uracil is one of the four nitrogen bases that belong to RNA. The last years special interest has been concentrated on the study of the effects under UV radiation in nucleic acids1 and also in the role that this molecule could have played in the origin and development of life on our planet.2 The MPI mass spectra show that the presence and intensity of the resulting ions strongly depend on the density power. The identification of the ions in the mass spectra is presented. The results are compared with those obtained in other laboratories under different experimental conditions and some of them show partial agreement.3 The present work was supported by CONACYT-Mexico Grant 165410 and DGAPA UNAM Grant IN101215 and IN102613.

  2. Methylation of thymine and uracil with free methyl cations formed due to beta-decay of tritiated methane: possible implication in mutagenesis and carcinogenesis.

    PubMed

    Korsakov, M V; Bystrova, M O; Mironiuk, T A; Sinotova, E N; Ivin, B A; Nefedov, V D; Likhachev, A J

    1989-05-01

    Exposure of solid thymine and uracil at room temperature to free methyl cations, produced due to beta-decay of tritiated methane, resulted in formation of their 1-, O2-, 3-, O4-, and 6-methyl derivatives. In addition, uracil formed a 5-methyl derivative (thymine); tritium-containing thymine and uracil were also detected. Both thymine and uracil formed predominantly unidentified products which resulted presumably from their oligomerization. Incubation at -195 degrees C did not markedly change the pattern of reaction products. Aqueous-ammonia solutions of these pyrimidines formed methylated derivatives and considerable amounts of methanol and tritiated water. The possible implication of these reactions in mutagenic and carcinogenic effects of tritium-substituted hydrocarbons is discussed.

  3. Cytosine-to-uracil deamination by SssI DNA methyltransferase.

    PubMed

    Stier, Ildikó; Kiss, Antal

    2013-01-01

    The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5'-amino-5'-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine ((m5)C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of (m5)C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung (+) host proficient in uracil excision repair.

  4. Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs*

    PubMed Central

    Soysa, Radika; Wilson, Zachary N.; Elferich, Johannes; Forquer, Isaac; Shinde, Ujwal; Riscoe, Michael K.; Yates, Phillip A.; Ullman, Buddy

    2013-01-01

    The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent Km values of 20 and 99 μm for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent Km values 6 and 7 μm for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process. PMID:23986453

  5. Dynamics of Dissociative Electron Attachment to Uracil and Furane

    NASA Astrophysics Data System (ADS)

    Fonseca Dos Santos, Samantha; Douguet, Nicolas; Orel, Ann; Rescigno, Thomas

    2016-05-01

    We present the results of a theoretical study of dissociative electron attachment (DEA) to Uracil and Furan. In both cases we will present calculated angular distributions based on analysis of the entrance amplitudes obtained from the results of complex Kohn scattering calculations. For uracil, we will compare our results with available experimentally measured angular distributions obtained using the COLTRIMS method.

  6. Fragmentation Pathways in the Uracil Radical Cation

    SciTech Connect

    Zhou, Congyi; Matsika, Spiridoula; Kotur, Marija; Weinacht, Thomas C.

    2012-08-24

    We investigate pathways for fragmentation in the uracil radical cation using ab initio electronic structure calculations. We focus on the main fragments produced in pump–probe dissociative ionization experiments. These are fragments with mass to charge ratios (m/z) of 69, 28, 41, and 42. Barriers to dissociation along the ground ionic surface are reported, which provide an estimate of the energetic requirements for the production of the main fragments. Finally, direct and sequential fragmentation mechanisms have been analyzed, and it is concluded that sequential fragmentation after production of fragment with m/z 69 is the dominant mechanism for the production of the smaller fragments.

  7. On the Electronically Excited States of Uracil

    SciTech Connect

    Epifanovsky, Evgeny; Kowalski, Karol; Fan, Peng-Dong; Valiev, Marat; Matsika, Spiridoula; Krylov, Anna

    2008-10-09

    Vertical excitation energies in uracil in the gas phase and in water solution are investigated by the equation-of-motion coupled-cluster and multi-reference configuration interaction methods. Basis set effects are found to be important for converged results. The analysis of electronic wave functions reveals that the lowest singlet states are predominantly of a singly excited character and are therefore well described by single-reference equation-of-motion methods augmented by a perturbative triples correction to account for dynamical correlation. Our best estimates for the vertical excitation energies for the lowest singlet n and are 5.0±0.1 eV and 5.3±0.1 eV, respectively. The solvent effects for these states are estimated to be +0.5 eV and ±0.1 eV, respectively. We attribute the difference between the computed vertical excitations and the maximum of the experimental absorption to strong vibronic interaction between the lowest A00 and A0 states leading to intensity borrowing by the forbidden transition.

  8. Uracil salvage pathway in Lactobacillus plantarum: Transcription and genetic studies.

    PubMed

    Arsène-Ploetze, Florence; Nicoloff, Hervé; Kammerer, Benoît; Martinussen, Jan; Bringel, Françoise

    2006-07-01

    The uracil salvage pathway in Lactobacillus plantarum was demonstrated to be dependent on the upp-pyrP gene cluster. PyrP was the only high-affinity uracil transporter since a pyrP mutant no longer incorporated low concentrations of radioactively labeled uracil and had increased resistance to the toxic uracil analogue 5-fluorouracil. The upp gene encoded a uracil phosphoribosyltransferase (UPRT) enzyme catalyzing the conversion of uracil and 5-phosphoribosyl-alpha-1-pyrophosphate to UMP and pyrophosphate. Analysis of mutants revealed that UPRT is a major cell supplier of UMP synthesized from uracil provided by preformed nucleic acid degradation. In a mutant selection study, seven independent upp mutants were isolated and all were found to excrete low amounts of pyrimidines to the growth medium. Pyrimidine-dependent transcription regulation of the biosynthetic pyrimidine pyrR1-B-C-Aa1-Ab1-D-F-E operon was impaired in the upp mutants. Despite the fact that upp and pyrP are positioned next to each other on the chromosome, they are not cotranscribed. Whereas pyrP is expressed as a monocistronic message, the upp gene is part of the lp_2376-glyA-upp operon. The lp_2376 gene encodes a putative protein that belongs to the conserved protein family of translation modulators such as Sua5, YciO, and YrdC. The glyA gene encodes a putative hydroxymethyltransferase involved in C1 unit charging of tetrahydrofolate, which is required in the biosynthesis of thymidylate, pantothenate, and purines. Unlike upp transcription, pyrP transcription is regulated by exogenous pyrimidine availability, most likely by the same mechanism of transcription attenuation as that of the pyr operon.

  9. Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases: Poxvirus Uracil-DNA Glycosylase

    SciTech Connect

    Schormann, Norbert; Zhukovskaya, Natalia; Bedwell, Gregory; Nuth, Manunya; Gillilan, Richard; Prevelige, Peter E.; Ricciardi, Robert P.; Banerjee, Surajit; Chattopadhyay, Debasish

    2016-11-02

    We report that uracil-DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil-DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non-enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus-specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA-binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. In conclusion, the adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three-dimensional structure of D4. We provide an overview of the current state of the knowledge on the structure-function relationship of D4.

  10. Serum "uracil+uridine" levels in pernicious anaemia.

    PubMed

    Parry, T E; Blackmore, J A

    1976-12-01

    The serum "uracil+uridine" level, expressed as uracil, has been measured in 21 cases of vitamin B12 deficiency, in which the serum folate was normal, and compared with the level in 97 normal subjects. The level in the vitamin B12 deficient group (11.9 mumol/1). was significantly lower than in the controls (15.7 mumol/1., P less than 0.005). Nine of the former were complicated by stystemic illness but the clinical and haematological features in the remaining 12 were consistent with the diagnosis of pernicious anaemia in relapse. The serum uracil level in this group was even lower (10.21 mumol/1., P less than 0.01). This finding is unexpected in view of the generally accepted indirect role of vitamine B12 in the methylation of deoxyuridine monophosphate to deoxythymidine monophosphate. Reasons are given for not accepting these results as reflecting the main biochemical lesion in vitamin B12 deficiency. Although they do not give direct support to an impairment in the methylation of deoxyuridine monophosphate, they do not exclude it as they test only one possible metabolic pathway and moreover they could represent the result of more than one action of vitamin B12 on uracil metabolism. They do show, however, that some aspect of uracil metabolism other than methylation is affected in vitamin B12 deficiency in man.

  11. Inelastic collisions of the uracil molecules with electrons.

    PubMed

    Shafranyosh, I I; Sukhoviya, M I

    2012-11-14

    Ionization and excitation of the uracil molecules by electron impact is investigated. Production of positive ions of uracil molecules (nucleic acid base) was studied using a crossed electron and molecular beam technique. The method developed by the authors enabled the molecular beam intensity to be measured and the electron dependences and the absolute values of the total cross sections of production of both positive ions to be determined. It is shown that the total positive uracil ion production cross section reaches its maximal value of (1.0 ± 0.1) × 10(-15) cm(2) at the 95 eV electron energy. Dissociative ionization cross sections were also determined. The luminescence spectra of isolated uracil molecules in the wavelength range of 200-500 nm under the influence of slow electrons are obtained. In the spectrum, more than 20 spectral bands and lines at 100 eV electron energy are observed. It is shown that the uracil radiation spectrum is formed by the processes of molecules dissociative excitation, dissociative excitation with ionization, excitation of electronic levels of the initial molecule and molecular ion.

  12. Electronic structure of uracil-like nucleobases adsorbed on Si(001): uracil, thymine and 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Molteni, Elena; Onida, Giovanni; Cappellini, Giancarlo

    2016-04-01

    We study the electronic properties of the Si(001):Uracil, Si(001):Thymine, and Si(001):5-Fluorouracil systems, focusing on the Si dimer-bridging configuration with adsorption governed by carbonyl groups. While the overall structural and electronic properties are similar, with small differences due to chemical substitutions, much larger effects on the surface band dispersion and bandgap show up as a function of the molecular orientation with respect to the surface. An off-normal orientation of the molecular planes is favored, showing larger bandgap and lower total energy than the upright position. We also analyze the localization of gap-edge occupied and unoccupied surface states. Supplementary material in the form of one pdf file available from the Journal web page at http://dx.doi.org/10.1140/epjb/e2016-70011-1

  13. The regioselective iodination of quinolines, quinolones, pyridones, pyridines and uracil.

    PubMed

    Dutta, Uttam; Deb, Arghya; Lupton, David W; Maiti, Debabrata

    2015-12-28

    A radical based direct C-H iodination protocol for quinolines, quinolones, pyridones, pyridines, and uracil has been developed. The iodination occurs in a C3 selective manner for quinolines and quinolones. Pyridones and pyridines undergo C3 and C5 iodination, while dimethyl uracil undergoes C5 iodination. Scope of the method was demonstrated through the rapid synthesis of both electron rich as well as electron poor heteroaromatic iodides. The protocol was found to be scalable and general, while a mechanism has been proposed.

  14. Uracil misincorporation into DNA and folic acid supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE: We evaluated the relation between UrMis in different ...

  15. Time-resolved radiation chemistry: Dynamics of electron attachment to uracil following UV excitation of iodide-uracil complexes

    SciTech Connect

    King, Sarah B.; Yandell, Margaret A.; Stephansen, Anne B.; Neumark, Daniel M.

    2014-12-14

    Electron attachment to uracil was investigated by applying time-resolved photoelectron imaging to iodide-uracil (I{sup –}U) complexes. In these studies, an ultraviolet pump pulse initiated charge transfer from the iodide to the uracil, and the resulting dynamics of the uracil temporary negative ion were probed. Five different excitation energies were used, 4.00 eV, 4.07 eV, 4.14 eV, 4.21 eV, and 4.66 eV. At the four lowest excitation energies, which lie near the vertical detachment energy of the I{sup –}U complex (4.11 eV), signatures of both the dipole bound (DB) as well as the valence bound (VB) anion of uracil were observed. In contrast, only the VB anion was observed at 4.66 eV, in agreement with previous experiments in this higher energy range. The early-time dynamics of both states were highly excitation energy dependent. The rise time of the DB anion signal was ∼250 fs at 4.00 eV and 4.07 eV, ∼120 fs at 4.14 eV and cross-correlation limited at 4.21 eV. The VB anion rise time also changed with excitation energy, ranging from 200 to 300 fs for excitation energies 4.00–4.21 eV, to a cross-correlation limited time at 4.66 eV. The results suggest that the DB state acts as a “doorway” state to the VB anion at 4.00–4.21 eV, while direct attachment to the VB anion occurs at 4.66 eV.

  16. Targeting tumors with nonreplicating Toxoplasma gondii uracil auxotroph vaccines

    PubMed Central

    Fox, Barbara A.; Sanders, Kiah L.; Chen, Shan; Bzik, David J.

    2013-01-01

    Toxoplasma gondii is an intracellular parasite that has evolved to actively control its invaded host cells. Toxoplasma triggers then actively regulates host innate IL-12 and interferon-γ responses that elicit T cell control of infection. A live, nonreplicating avirulent uracil auxotroph vaccine strain (cps) of Toxoplasma triggers novel innate immune responses that stimulate amplified CD8+ T cell responses and life-long immunity in vaccinated mice. Here, we review recent reports showing that intratumoral treatment with cps activated immune-mediated regression of established solid tumors in mice. We speculate that a better understanding of host-parasite interaction at the molecular level and applying improved genetic models based on Δku80 Toxoplasma strains will stimulate development of highly effective immunotherapeutic cancer vaccine strategies using engineered uracil auxotrophs. PMID:23928100

  17. Shape resonance spectra of uracil, 5-fluorouracil, and 5-chlorouracil

    SciTech Connect

    Kossoski, F.; Varella, M. T. do N.; Bettega, M. H. F.

    2014-01-14

    We report on the shape resonance spectra of uracil, 5-fluorouracil, and 5-chlorouracil, as obtained from fixed-nuclei elastic scattering calculations performed with the Schwinger multichannel method with pseudopotentials. Our results are in good agreement with the available electron transmission spectroscopy data, and support the existence of three π* resonances in uracil and 5-fluorouracil. As expected, the anion states are more stable in the substituted molecules than in uracil. Since the stabilization is stronger in 5-chlorouracil, the lowest π* resonance in this system becomes a bound anion state. The present results also support the existence of a low-lying σ{sub CCl{sup *}} shape resonance in 5-chlorouracil. Exploratory calculations performed at selected C–Cl bond lengths suggest that the σ{sub CCl{sup *}} resonance could couple to the two lowest π* states, giving rise to a very rich dissociation dynamics. These facts would be compatible with the complex branching of the dissociative electron attachment cross sections, even though we cannot discuss any details of the vibration dynamics based only on the present fixed-nuclei results.

  18. Shape resonance spectra of uracil, 5-fluorouracil, and 5-chlorouracil

    NASA Astrophysics Data System (ADS)

    Kossoski, F.; Bettega, M. H. F.; Varella, M. T. do N.

    2014-01-01

    We report on the shape resonance spectra of uracil, 5-fluorouracil, and 5-chlorouracil, as obtained from fixed-nuclei elastic scattering calculations performed with the Schwinger multichannel method with pseudopotentials. Our results are in good agreement with the available electron transmission spectroscopy data, and support the existence of three π* resonances in uracil and 5-fluorouracil. As expected, the anion states are more stable in the substituted molecules than in uracil. Since the stabilization is stronger in 5-chlorouracil, the lowest π* resonance in this system becomes a bound anion state. The present results also support the existence of a low-lying σ _CCl^* shape resonance in 5-chlorouracil. Exploratory calculations performed at selected C-Cl bond lengths suggest that the σ _CCl^* resonance could couple to the two lowest π* states, giving rise to a very rich dissociation dynamics. These facts would be compatible with the complex branching of the dissociative electron attachment cross sections, even though we cannot discuss any details of the vibration dynamics based only on the present fixed-nuclei results.

  19. Bis(5-methyl­pyrazine-2-carboxyl­ato)­diphenyl­tin(IV)

    PubMed Central

    Gao, Zhongjun

    2008-01-01

    In the mol­ecule of the title compound, [Sn(C6H5)2(C6H5N2O2)2], two O and one N atoms from the two 5-methyl­pyrazine-2-carboxyl­ate ligands and one C atom of a phenyl group form a distorted square-planar arrangement in the equatorial plane around the Sn atom, while the distorted octa­hedral coordination is completed by an N atom of one of the 5-methyl­pyrazine-2-carboxyl­ate ligands and a C atom of the other phenyl group in the axial positions. In the crystal structure, inter­molecular C—H⋯O hydrogen bonds link the mol­ecules into centrosymmetric dimers. PMID:21202741

  20. Uracil as an index of lactic acid bacteria contamination of tomato products.

    PubMed

    Hidalgo, Alyssa; Pompei, Carlo; Galli, Antonietta; Cazzola, Sara

    2005-01-26

    The aim of this research was to evaluate the suitability of uracil as an hygienic quality index of tomato products. Whereas uridine was naturally present throughout tomato fruits' ripening, uracil appeared only after microbial contamination. In tomato pulp inoculated with nine different microbial strains, all five lactic acid bacteria (LAB) studied released relevant quantities of uracil (150-1040 mg/kg of dm), with a correlated partial or total decrease of uridine. Uracil production by yeasts and molds was very low or nonexistent; the starting uridine concentration (approximately 960 mg/kg of dm) remained constant or increased. Uracil thermostability was also verified. Twenty-six samples of tomato paste (30 degrees Brix) were collected from bag-in-drums produced in an industrial processing plant, some with evident swelling symptoms. All of the samples with high microbial count presented uracil. Uracil was also present in samples with microbial contamination under the detection limit and Howard mold count below legislation limits, implying the reprocessing, at least partial, of altered tomato product. The results indicate that uracil presence in tomato products is an index of LAB contamination that has occurred before heat treatment.

  1. HYDROGEN-BONDED DIMERS OF ADENINE AND URACIL DERIVATIVES.

    PubMed

    HAMLIN, R M; LORD, R C; RICH, A

    1965-06-25

    In concentrated solutions of either 9-ethyladenine or 1-cyclohexyluracil in deuterochloroform, absorption bands in the infrared spectrum demonstrate hydrogen bonding of the adenine and uracil derivatives with themselves. In dilute solutions, there is very little hydrogen bonding. However, when dilute solutions of 9-ethyladenine and 1-cyclohexyluracil are mixed, a series of bands appear which show that these molecules are hydrogen-bonding with each other much more strongly than with themselves. A study of the stoichiometry of this association indicates formation of 1:1 hydrogen-bonded pairs in solution.

  2. FT-IR and Raman spectra, ab initio and density functional computations of the vibrational spectra, molecular geometries and atomic charges of uracil and 5-halogenated uracils (5-X-uracils; X=F, Cl, Br, I).

    PubMed

    Singh, J S

    2014-01-03

    Raman (200-4000 cm(-1)) and FT-IR (400-4000 cm(-1)) spectra of uracil and 5-halogenated uracils (5-X-uracils; X=F, Cl, Br, I) have been recorded and analyzed in the range 200-4000 cm(-1). The optimized molecular geometries, atomic polar tensor (APT) charges and vibrational characteristics have been studied theoretically using restricted Hartree-Fock (RHF) and density functional theory (DFT) methods. Ab initio and DFT calculations [using Becke's exchange in conjunction with Lee-Yang-Parr's correlation functional and Becke's three-parameter hybrid method (B3LYP)] were carried out to study the optimized molecular fundamental vibrational frequencies for uracil and 5-halogenated uracils by employing Gaussian-03 program. Gauss View software was used to make the vibrational analysis. Raman and IR spectra have been computed theoretically for the uracil and 5-halogenated molecules. The fundamental vibrational frequencies along with their corresponding intensities in IR and Raman activities and depolarization ratios of the Raman lines have also been calculated using the RHF and DFT methods employing different basis sets. Quantum chemical calculations helped in the reassignments of some fundamental vibrational modes. Most of the B3LYP/6-311++G(**) vibrational frequencies are in excellent agreement with available experimental assignments. The ring breathing and kekule stretching modes are found to lower magnitudes compared to those for uracil which could be due to mass effect of halogen atom in place of the hydrogen atom. The C-X (X=F, Cl, Br, I) stretching frequency is distinctly separated from the CH/NH ring stretching frequencies on the pyrimidine ring. All other bands have also been assigned different fundamentals/overtones/combinations.

  3. Fingerprinting DNA oxidation processes: IR characterization of the 5-methyl-2'-deoxycytidine radical cation.

    PubMed

    Bucher, Dominik B; Pilles, Bert M; Pfaffeneder, Toni; Carell, Thomas; Zinth, Wolfgang

    2014-02-24

    Methylated cytidine plays an important role as an epigenetic signal in gene regulation. Its oxidation products are assumed to be involved in active demethylation processes but also in damaging DNA. Here, we report the photochemical production of the 5-methyl-2'-deoxycytidine radical cation via a two-photon ionization process. The radical cation is detected by time-resolved IR spectroscopy and identified by band assignment using density functional theory calculations. Two final oxidation products are characterized with liquid chromatography coupled to mass spectrometry.

  4. Antimicrobial N-brominated hydantoin and uracil grafted polystyrene beads.

    PubMed

    Farah, Shady; Aviv, Oren; Laout, Natalia; Ratner, Stanislav; Domb, Abraham J

    2015-10-28

    Hydantoin-N-halamine derivatives conjugated on polystyrene beads are promising disinfectants with broad antimicrobial activity affected by the gradual release of oxidizing halogen in water. The objective of this work was to identify and test of hydantoin-like molecules possessing urea moiety, which may provide N-haloamines releasing oxidizing halogens when exposed to water at different rates and release profiles for tailored antimicrobial agents. In this work, several hydantoin (five member ring) and for the first time reported, uracil (six member ring) derivatives have been conjugated to polystyrene beads and tested for their lasting antimicrobial activity. Four molecules of each series were conjugated onto polystyrene beads from the reaction of the N-potassium hydantoin or uracil derivatives onto chloromethylated polystyrene beads. A distinct difference in bromine loading capacity and release profiles was found for the different conjugated derivatives. All tested materials exhibit strong antimicrobial activity against Escherichia coli and bacteriophages MS2 of 7 and ~4 log reduction, respectively. These results highlight the antimicrobial potential of halogenated cyclic molecules containing urea groups as water disinfection agents.

  5. Death of Bacillus subtilis Auxotrophs Due to Deprivation of Thymine, Tryptophan, or Uracil

    PubMed Central

    Pritikin, William B.; Romig, W. R.

    1966-01-01

    Pritikin, William B. (University of California, Los Angeles), and W. R. Romig. Death of Bacillus subtilis auxotrophs due to deprivation of thymine, tryptophan, or uracil, J. Bacteriol. 92:291–296. 1966.—Auxotrophic mutants of Bacillus subtilis 168 that require either tryptophan, uracil, or thymine died rapidly when deprived of any of these compounds. Phage PBS1 was produced by infected B. subtilis 168 (thy try-2) deprived of thymine. Phage PBS1 was not produced by infected B. subtilis 168 (try-2) deprived of tryptophan or infected B. subtilis 168-15 (try-2 ura) deprived of uracil. B. subtilis 168 thy try-2 and 168-15 could be transduced by phage PBS1 after prolonged deprivation of tryptophan or uracil, respectively. When B. subtilis 168-15 was transduced to uracil independence by phage PBS1, the uracil-independent transductants became immune to uracil-less death within 10 min of exposure to phage, and began to multiply within 2 hr after exposure to phage at an incubation temperature of 46 C. PMID:16562109

  6. Death of Bacillus subtilis Auxotrophs Due to Deprivation of Thymine, Tryptophan, or Uracil.

    PubMed

    Pritikin, W B; Romig, W R

    1966-08-01

    Pritikin, William B. (University of California, Los Angeles), and W. R. Romig. Death of Bacillus subtilis auxotrophs due to deprivation of thymine, tryptophan, or uracil, J. Bacteriol. 92:291-296. 1966.-Auxotrophic mutants of Bacillus subtilis 168 that require either tryptophan, uracil, or thymine died rapidly when deprived of any of these compounds. Phage PBS1 was produced by infected B. subtilis 168 (thy try-2) deprived of thymine. Phage PBS1 was not produced by infected B. subtilis 168 (try-2) deprived of tryptophan or infected B. subtilis 168-15 (try-2 ura) deprived of uracil. B. subtilis 168 thy try-2 and 168-15 could be transduced by phage PBS1 after prolonged deprivation of tryptophan or uracil, respectively. When B. subtilis 168-15 was transduced to uracil independence by phage PBS1, the uracil-independent transductants became immune to uracil-less death within 10 min of exposure to phage, and began to multiply within 2 hr after exposure to phage at an incubation temperature of 46 C.

  7. Bis-(2-amino-5-methyl-pyridinium) fumarate-fumaric acid (1/1).

    PubMed

    Hemamalini, Madhukar; Fun, Hoong-Kun

    2010-07-24

    In the crystal structure of the title compound, C(6)H(9)N(2) (+)·0.5C(4)H(2)O(4) (2-)·0.5C(4)H(6)O(4), the fumarate dianion and fumaric acid mol-ecule are located on inversion centres. The 2-amino-5-methyl-pyrimidinium cation inter-acts with the carboxyl-ate group of the fumarate anion through a pair of N-H⋯O hydrogen bonds, forming an R(2) (2)(8) ring motif. These motifs are centrosymmetrically paired via N-H⋯O hydrogen bonds, forming a complementary DDAA array. The carboxyl groups of the fumaric acid mol-ecules and the carboxyl-ate groups of the fumarate anions are hydrogen bonded through O-H⋯O hydrogen bonds, leading to a supra-molecular chain along [101]. The crystal structure is further stabilized by weak C-H⋯O hydrogen bonds.

  8. Production of uracil from methane by a newly isolated Methylomonas sp. SW1.

    PubMed

    Kim, Sangwoo; Lee, Wangjun; Song, Insu; Kwon, Yuhyun; Yun, Seokhun; Park, Soohyun; Cho, Sukhyeong; Oh, Byung-Keun; Oh, Han Bin; Lee, Jinwon

    2016-12-20

    Methane is an abundant, inexpensive one-carbon feedstock and one of the most powerful greenhouse gases. Because it does not compete with food demand, it is considered a promising carbon feedstock for the production of valuable products using methanotrophic bacteria. Here, we isolated a novel methanotrophic bacterium, Methylomonas sp. SW1, from a sewage sample obtained from Wonju City Water Supply Drainage Center, Republic of Korea. The conditions for uracil production by Methylomonas sp. SW1, such as Cu(2+) concentration and temperature were investigated and optimized. As a result, Methylomonas sp. SW1 produced uracil from methane as a sole carbon source with a titer of 2.1mg/L in 84h without genetic engineering under the optimized condition. The results in this study demonstrate the feasibility of using Methylomonas sp. SW1 for the production of uracil from methane. This is the first report of uracil production from gas feedstock by methanotrophic bacteria.

  9. Effect of amino substitution on the excited state dynamics of uracil.

    PubMed

    Bányász, Akos; Gustavsson, Thomas; Keszei, Erno; Improta, Roberto; Markovitsi, Dimitra

    2008-07-01

    The excited state deactivation of two amino-substituted uracils, 5-aminouracil (5AU) and 6-aminouracil (6AU) in aqueous solution was studied by femtosecond fluorescence upconversion. The fluorescence of 6AU decays as fast as that of uracil with a unique time constant of about 100 femtoseconds. The fluorescence of 5AU exhibits a more complex behavior, fundamentally different from what we found in any other uracils: the decays are globally slower (up to several picoseconds) and depend strongly on the wavelength. This difference is attributed to the particular character of the amino group, affecting the out-of-plane motion of the 5-substituent which has been shown to be crucial for the ultrafast internal conversion occurring in uracils. Our observations indicate instead the formation of a transient fluorescent state which in turn is deactivated by a different relaxation process specific to the amino group.

  10. Carbon nanotube-nucleobase hybrids: nanorings from uracil-modified single-walled carbon nanotubes.

    PubMed

    Singh, Prabhpreet; Toma, Francesca Maria; Kumar, Jitendra; Venkatesh, V; Raya, Jesus; Prato, Maurizio; Verma, Sandeep; Bianco, Alberto

    2011-06-06

    Single-walled carbon nanotubes (SWCNTs) have been covalently functionalized with uracil nucleobase. The hybrids have been characterized by using complementary spectroscopic and microscopic techniques including solid-state NMR spectroscopy. The uracil-functionalized SWCNTs are able to self-assemble into regular nanorings with a diameter of 50-70 nm, as observed by AFM and TEM. AFM shows that the rings do not have a consistent height and thickness, which indicates that they may be formed by separate bundles of CNTs. The simplest model for the nanoring formation likely involves two bundles of CNTs interacting with each other via uracil-uracil base-pairing at both CNT ends. These nanorings can be envisaged for the development of advanced electronic circuits.

  11. Barrier-Free Intermolecular Proton Transfer Induced by Excess Electron Attachment to the Complex of Alanine with Uracil

    SciTech Connect

    Dabkowska, Iwona; Rak, Janusz; Gutowski, Maciej S.; Nilles, J.M.; Stokes, Sarah; Bowen, Kit H.

    2004-04-01

    The photoelectron spectrum of the uracil-alanine anionic complex (UA)- has been recorded with 2.540 eV photons. This spectrum reveals a broad feature with a maximum between 1.6-2.1 eV. The vertical electron detachment energy is too large to be attributed to an (UA)- anionic complex in which an intact uracil anion is solvated by alanine, or vice versa. The neutral and anionic complexes of uracil and alanine were studied at the B3LYP and second order Moeller-Plesset level of theory with 6-31++G** basis sets. The neutral complexes form cyclic hydrogen bonds and the three most stable neutral complexes are bound by 0.72, 0.61 and 0.57 eV. The electron hole in complexes of uracil with alaninie is localized on uracil, but the formation of a complex with alanine strongly modulates the vertical ionization energy of uracil. The theoretical results indicate that the excess electron in (UA)- occupies a p* orbital localized on uracil. The excess electron attachment to the complex can induce a barrier-free proton transfer (BFPT) from the carboxylic group of alanine to the O8 atom of uracil. As a result, the four most stable structures of the uracil-alanine anionic complex can be characterized as the neutral radical of hydrogenated uracil solvated by the anion of deprotonated alanine. Our current results for the anionic complex of uracil with alanine are similar to our previous results for the anion of uracil with glycine [Eur. Phys. J. D 20, 431 (2002)], and together they indicate that the BFPT process is not very sensitive to the nature of the amino acid's hydrophobic residual group. The BFPT to the O8 atom of uracil may be relevant to the damage suffered by nucleic acid bases due to exposure to low energy electrons.

  12. Scaffold hopping: exploration of acetanilide-containing uracil analogues as potential NNRTIs.

    PubMed

    Babkov, Denis A; Valuev-Elliston, Vladimir T; Paramonova, Maria P; Ozerov, Alexander A; Ivanov, Alexander V; Chizhov, Alexander O; Khandazhinskaya, Anastasia L; Kochetkov, Sergey N; Balzarini, Jan; Daelemans, Dirk; Pannecouque, Christophe; Seley-Radtke, Katherine L; Novikov, Mikhail S

    2015-03-01

    In order to identify novel nonnucleoside inhibitors of HIV-1 reverse transcriptase two series of amide-containing uracil derivatives were designed as hybrids of two scaffolds of previously reported inhibitors. Subsequent biological evaluation confirmed acetamide uracil derivatives 15a-k as selective micromolar NNRTIs with a first generation-like resistance profile. Molecular modeling of the most active compounds 15c and 15i was employed to provide insight on their inhibitory properties and direct future design efforts.

  13. A QM/MM study of absorption spectra of uracil derivatives in aqueous solution

    NASA Astrophysics Data System (ADS)

    Nakayama, Akira

    2016-12-01

    The absorption spectra of three representative uracil derivatives (uracil, thymine, and 5-fluorouracil) in aqueous solution are investigated by the QM/MM approach, where the CASPT2 method is employed to evaluate the excitation energies. The computed absorption spectra are in good agreement with the experimental results, and in particular, the relative values of the absorption maximum between these derivatives are well reproduced in the simulations.

  14. Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

    PubMed Central

    Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

    2016-01-01

    We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

  15. Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

    PubMed Central

    Róna, Gergely; Scheer, Ildikó; Nagy, Kinga; Pálinkás, Hajnalka L.; Tihanyi, Gergely; Borsos, Máté; Békési, Angéla; Vértessy, Beáta G.

    2016-01-01

    The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell. PMID:26429970

  16. P2 receptors activated by uracil nucleotides--an update.

    PubMed

    Brunschweiger, Andreas; Müller, Christa E

    2006-01-01

    Pyrimidine nucleotides, including UTP, UDP and UDP-glucose, are important signaling molecules which activate G protein-coupled membrane receptors (GPCRs) of the P2Y family. Four distinct pyrimidine nucleotide-sensitive P2Y receptor subtypes have been cloned, P2Y2, P2Y4, P2Y6 and P2Y14. P2Y2 and P2Y4 receptors are activated by UTP (the P2Y2, and the rat but not the human P2Y4 receptor are also activated by ATP), the P2Y6 receptor is activated by UDP, and the P2Y14 receptor by UDP-glucose. Furthermore, non-P2Y GPCRs, the cysteinylleukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinylleukotrienes. While P2Y2, P2Y4, and P2Y6 receptor activation results in stimulation of phospholipase C, the P2Y14 receptor is coupled to inhibition of adenylate cyclase. Derivatives and analogs of the physiological nucleotides UTP, UDP and ATP have been synthesized and evaluated in order to obtain enzymatically stable, subtype-selective agonists. The P2Y2 receptor agonists diuridine tetraphosphate (diquafosol) and the uracil-cytosine dinucleotide denufosol are currently undergoing clinical trials for dry eye disease, retinal detachment disease, upper respiratory tract symptoms, and cystic fibrosis, respectively. The first antagonists for P2Y2 and P2Y6 receptors that appear to be selective versus other P2Y receptor subtypes have recently been described. Selective antagonists for P2Y4 and P2Y14 receptors are still lacking. Uracil nucleotide-sensitive P2Y receptor subtypes may constitute future targets for the treatment of certain cancer types, vascular diseases, inflammatory diseases, and immunomodulatory intervention. They have also been proposed to play a role in neurodegenerative diseases. This article is an updated version of "P2-Pyrimidinergic Receptors and Their Ligands" by C. E. Müller published in Curr. Pharm. Des. 2002, 8, 2353-2369.

  17. Molecular characterization of methanogenic N(5)-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase.

    PubMed

    Upadhyay, Vikrant; Ceh, Katharina; Tumulka, Franz; Abele, Rupert; Hoffmann, Jan; Langer, Julian; Shima, Seigo; Ermler, Ulrich

    2016-09-01

    Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.

  18. Androgen receptor mutations are associated with altered epigenomic programming as evidenced by HOXA5 methylation.

    PubMed

    Bens, S; Ammerpohl, O; Martin-Subero, J I; Appari, M; Richter, J; Hiort, O; Werner, R; Riepe, F G; Siebert, R; Holterhus, P-M

    2011-01-01

    Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level.

  19. Electroanalytical study of proflavine intercalation in 5-methyl or inosine-containing amplicons.

    PubMed

    Alexiadou, Despina K; Ioannou, Andrea K; Kouidou-Andreou, Sofia A; Voulgaropoulos, Anastasios N; Girousi, Stella Th

    2008-10-01

    Amplicons corresponding to the GC-rich p53 exon 5 and its analogues, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, or 60% of guanosine by inosine and GC-poor p53 exon 6 were synthesized and investigated electrochemically, in the presence and absence of proflavine, by differential pulse voltammetry (DPV). Incorporation of base analogues and the thermal stability of the resulting amplicons were tested in the presence of a fluorescent probe (Sybr-Green). Peak current at 1.0 V was lower for methylated than for unmethylated PCR amplicons and was similarly affected by proflavine intercalation. In contrast, considerable peak current differences were observed in the presence of proflavine for unmodified exon 5 v.s. exon 6 or inosine-containing amplicons. Thermal analysis verified the expected shifts in melting temperature (T (m)) due to the base analogue incorporation and GC-content variations. In conclusion, methylated and unmethylated PCR amplicons could be distinguished in model DNA systems using differential pulse voltammetry (DPV) and use of proflavine could serve as an electrochemical probe for identifying different DNA conformations.

  20. Simultaneous determination of 2-furfural and 5-methyl-2-furfural using corona discharge ion mobility spectrometry.

    PubMed

    Jafari, M T; Khayamian, T

    2009-06-01

    A novel technique, corona discharge ion mobility spectrometry (CD-IMS), was developed for the qualitative and quantitative determination of 2-furfural (F) and 5-methyl-2-furfural (MF) in aqueous solutions. The limits of detection (LODs) were 5.3 x 10(-3) microg/mL for F and 6.7 x 10(-3) microg/mL for MF. The linear dynamic ranges of 1.16 x 10(-2) to 1.04 microg/mL and 2.20 x 10(-2) to 1.10 microg/mL were obtained for F and MF, respectively. The relative standard deviation was below 12% for both compounds. In addition to analysis of the individual compound, simultaneous determination of F and MF was also investigated. It was realized that F imposes a matrix effect on the MF signal and vice versa. The standard addition method was used to deal with the matrix effect. The recovery of the compounds in the synthetic samples validates the capability of the method.

  1. Prebiotic synthesis of 5-substituted uracils: a bridge between the RNA world and the DNA-protein world

    NASA Technical Reports Server (NTRS)

    Robertson, M. P.; Miller, S. L.

    1995-01-01

    Under prebiotic conditions, formaldehyde adds to uracil at the C-5 position to produce 5-hydroxymethyluracil with favorable rates and equilibria. Hydroxymethyluracil adds a variety of nucleophiles, such as ammonia, glycine, guanidine, hydrogen sulfide, hydrogen cyanide, imidazole, indole, and phenol, to give 5-substituted uracils with the side chains of most of the 20 amino acids in proteins. These reactions are sufficiently robust that, if uracil had been present on the primitive Earth, then these substituted uracils would also have been present. The ribozymes of the RNA world would have included many of the functional groups found in proteins today, and their catalytic activities may have been considerably greater than presently assumed.

  2. Effect of uracil addition on proteomic profiles and 1,3-β-glucan production in Agrobacterium sp.

    PubMed

    Jin, Li-Hua; Lee, Jung-Heon

    2014-01-01

    Uridine diphosphate (UDP)-glucose is a precursor of 1,3-β-glucan and is synthesized from glucose-1-phosphate and uridine triphosphate (UTP). Uracil was used as a precursor for UTP, which resulted in an increase in both the UDP-glucose level and the rate of 1,3-β-glucan synthesis. 1,3-β-Glucan production metabolism was reactivated after uracil addition during fermentation. 2D-PAGE was used to examine the changes in the expression level of the key metabolic enzymes in the production of 1,3-β-glucan after uracil addition. The results showed that the expression levels of UTP-glucose-1-phosphate uridylytransferase, phosphoglucomutase, and 1,3-β-glucan synthase catalytic subunit, key metabolic enzymes in the curdlan biosynthesis pathway, were increased after uracil addition by 3.5%, 30%, and 35%, respectively. Uracil phosphoribosyltransferase, which converts uracil to uridine monophosphate (UMP), was also upregulated 79% more than that without uracil addition. However, the expression levels of orotidine 5-phosphate decarboxylase, glucose-1-phosphate adenylyltransferase, and glucose-6-phosphate isomerase were decreased after uracil addition. This proteomic information is useful for predicting changes in the pathway of uracil utilization. This work provides proteomic information for the integrative analysis of bioinformatic databases, which can be used to predict and understand the metabolism of glucan synthesis at the cellular level.

  3. An efficient prebiotic synthesis of cytosine and uracil

    NASA Technical Reports Server (NTRS)

    Robertson, M. P.; Miller, S. L.

    1995-01-01

    In contrast to the purines, the routes that have been proposed for the prebiotic synthesis of pyrimidines from simple precursors give only low yields. Cytosine can be synthesized from cyanoacetylene and cyanate; the former precursor is produced from a spark discharge in a CH4/N2 mixture and is an abundant interstellar molecule. But this reaction requires relatively high concentrations of cyanate (> 0.1 M), which are unlikely to occur in aqueous media as cyanate is hydrolysed rapidly to CO2 and NH3. An alternative route that has been explored is the reaction of cyanoacetaldehyde (formed by hydrolysis of cyanoacetylene) with urea. But at low concentrations of urea, this reaction produces no detectable quantities of cytosine. Here we show that in concentrated urea solution--such as might have been found in an evaporating lagoon or in pools on drying beaches on the early Earth--cyanoacetaldehyde reacts to form cytosine in yields of 30-50%, from which uracil can be formed by hydrolysis. These reactions provide a plausible route to the pyrimidine bases required in the RNA world.

  4. Singlet excited-state behavior of uracil and thymine in aqueous solution: a combined experimental and computational study of 11 uracil derivatives.

    PubMed

    Gustavsson, Thomas; Bányász, Akos; Lazzarotto, Elodie; Markovitsi, Dimitra; Scalmani, Giovanni; Frisch, Michael J; Barone, Vincenzo; Improta, Roberto

    2006-01-18

    The excited-state properties of uracil, thymine, and nine other derivatives of uracil have been studied by steady-state and time-resolved spectroscopy. The excited-state lifetimes were measured using femtosecond fluorescence upconversion in the UV. The absorption and emission spectra of five representative compounds have been computed at the TD-DFT level, using the PBE0 exchange-correlation functional for ground- and excited-state geometry optimization and the Polarizable Continuum Model (PCM) to simulate the aqueous solution. The calculated spectra are in good agreement with the experimental ones. Experiments show that the excited-state lifetimes of all the compounds examined are dominated by an ultrafast (<100 fs) component. Only 5-substituted compounds show more complex behavior than uracil, exhibiting longer excited-state lifetimes and biexponential fluorescence decays. The S(0)/S(1) conical intersection, located at CASSCF (8/8) level, is indeed characterized by pyramidalization and out of plane motion of the substituents on the C5 atom. A thorough analysis of the excited-state Potential Energy Surfaces, performed at the PCM/TD-DFT(PBE0) level in aqueous solution, shows that the energy barrier separating the local S(1) minimum from the conical intersection increases going from uracil through thymine to 5-fluorouracil, in agreement with the ordering of the experimental excited-state lifetime.

  5. Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen☆

    PubMed Central

    Ko, Rinkei; Bennett, Samuel E.

    2011-01-01

    Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl2. The functional significance of the UNG2·PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30–810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-3H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl2. The stimulatory effect of PCNA was increased by the addition of MgCl2; however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl2 and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA. PMID:16216562

  6. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.

  7. Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

    DOE PAGES

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

    2015-06-02

    Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

  8. Effect of C5-Methylation of Cytosine on the UV-Induced Reactivity of Duplex DNA: Conformational and Electronic Factors.

    PubMed

    Banyasz, Akos; Esposito, Luciana; Douki, Thierry; Perron, Marion; Lepori, Clément; Improta, Roberto; Markovitsi, Dimitra

    2016-05-12

    C5-methylation of cytosines is strongly correlated with UV-induced mutations detected in skin cancers. Mutational hot-spots appearing at TCG sites are due to the formation of pyrimidine cyclobutane dimers (CPDs). The present study, performed for the model DNA duplex (TCGTA)3·(TACGA)3 and the constitutive single strands, examines the factors underlying the effect of C5-methylation on pyrimidine dimerization at TCG sites. This effect is quantified for the first time by quantum yields ϕ. They were determined following irradiation at 255, 267, and 282 nm and subsequent photoproduct analysis using HPLC coupled to mass spectrometry. C5-methylation leads to an increase of the CPD quantum yield up to 80% with concomitant decrease of that of pyrimidine(6-4) pyrimidone adducts (64PPs) by at least a factor of 3. The obtained ϕ values cannot be explained only by the change of the cytosine absorption spectrum upon C5-methylation. The conformational and electronic factors that may affect the dimerization reaction are discussed in light of results obtained by fluorescence spectroscopy, molecular dynamics simulations, and quantum mechanical calculations. Thus, it appears that the presence of an extra methyl on cytosine affects the sugar puckering, thereby enhancing conformations of the TC step that are prone to CPD formation but less favorable to 64PPs. In addition, C5-methylation diminishes the amplitude of conformational motions in duplexes; in the resulting stiffer structure, ππ* excitations may be transferred from initially populated exciton states to reactive pyrimidines giving rise to CPDs.

  9. Fluorescence of 5-arylvinyl-5'-methyl-2,2'-bipyridyl ligands and their zinc complexes.

    PubMed

    Younes, Ali H; Zhang, Lu; Clark, Ronald J; Zhu, Lei

    2009-11-20

    The photophysical properties of 5-arylvinyl-5'-methyl-2,2'-bipyridyls (AVMBs, 1-9, 11) and their zinc complexes were studied. Similar 2,2'-bipyridyl-based ligands have been applied as optical sensors for metal ions and sensitizers for solar energy conversion. The goal of this investigation is to reveal the factors that determine the emission band shift and fluorescence quantum yield change of the title ligand system upon zinc binding. The outcome of this study will not only advance the fundamental understanding of the coordination-driven photophysical processes embodied in the AVMB platform but facilitate the rational design of fluorescent probes for metal ions, particularly zinc. The AVMB ligands were synthesized using the Horner-Wadsworth-Emmons reaction. AVMBs containing electron-donating aryl groups show absorption and emission in the visible region, which can be assigned to charge-transfer transitions as supported by solvent-dependency and computational studies. The binding between AVMB ligands and zinc ion in acetonitrile was studied using isothermal titration calorimetry (ITC). A multicomponent equilibrium model is suggested that explains the multiple transitions evidenced in fluorescence titration isotherms. Coordination to zinc ion stabilizes the charge-transfer excited state of an AVMB ligand with an electron-donating aryl substituent, consequently results in bathochromic shifts in both absorption and emission. However, unlike the emission band shift, the fluorescence quantum yield change upon zinc complex formation does not have an intuitive correlation with the electronic nature of the aryl group. Lifetime measurements using the Time-Correlated Single Photon Counting method enabled the determination of nonradiative and radiative decay rate constants. Both rates of an AVMB ligand decrease upon zinc binding. The collective effect gives rise to the change in fluorescence quantum yield with the apparent lack of correlation with the electronic property of

  10. Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

    PubMed Central

    Xia, Bo; Liu, Yinling; Guevara, Jose; Li, Jing; Jilich, Celeste; Yang, Ye; Wang, Liangjiang; Dominy, Brian N.; Cao, Weiguo

    2017-01-01

    Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function.

  11. Single nucleotide polymorphisms in uracil-processing genes, intake of one-carbon nutrients and breast cancer risk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Objectives: The misincorporation of uracil into DNA leads to genomic instability. In a previous study, some of us identified four common single nucleotide polymorphisms (SNPs) in uracil-processing genes (rs2029166 and rs7296239 in SMUG1, rs34259 in UNG and rs4775748 in DUT) that were asso...

  12. Barrier-free intermolecular proton transfer in the uracil-glycine complex induced by excess electron attachment

    NASA Astrophysics Data System (ADS)

    Gutowski, M.; Dąbkowska, I.; Rak, J.; Xu, S.; Nilles, J. M.; Radisic, D.; Bowen, K. H., Jr.

    2002-09-01

    The photoelectron spectra (PES) of anions of uracil-glycine and uracil-phenylalanine complexes reveal broad features with maxima at 1.8 and 2.0 eV. The results of ab initio density functional B3LYP and second order Møller-Plesset theory calculations indicate that the excess electron occupies a π^* orbital localized on uracil. The excess electron attachment to the complex can induce a barrier-free proton transfer (BFPT) from the carboxylic group of glycine to the O8 atom of uracil. As a result, the four most stable structures of the anion of uracil-glycine complex can be characterized as the neutral radical of hydrogenated uracil solvated by the anion of deprotonated glycine. The similarity between the PES spectra for the uracil complexes with glycine and phenylalanine suggests that the BFPT is also operative in the case of the latter anionic species. The BFPT to the O8 atom of uracil may be related to the damage of nucleic acid bases by low energy electrons because the O8 atom is involved in a hydrogen bond with adenine in the standard Watson-Crick pairing scheme.

  13. Ferrocene-bis(thymine/uracil) conjugates: base pairing directed, spacer dependent self-assembly and supramolecular packing.

    PubMed

    Patwa, Amit N; Gonnade, Rajesh G; Kumar, Vaijayanti A; Bhadbhade, Mohan M; Ganesh, Krishna N

    2010-12-17

    X-ray crystallographic studies of methylene linked Ferrocene-bis(thymine/uracil) conjugates Fc(T:T)(M) and Fc(U:U)(M) reveal base dependent 2-D supramolecular assemblies generated via wobble self-pairing for bis-thymine and reverse wobble self-pairing for bis-uracil conjugates, differing in architecture from the corresponding butylene spacer linked conjugates.

  14. Molecularly Imprinted Polymers (MIPs) Against Uracils : Functional Monomer Design, Monomer-Template Interactions in Solution and MIP Performance in Chromatography

    DTIC Science & Technology

    2002-04-05

    UNCLASSIFIED Defense Technical Information Center Compilation Part Notice ADP013598 TITLE: Molecularly Imprinted Polymers [ MIPs ] Against Uracils...Research Society MI.3 Molecularly Imprinted Polymers ( MIPs ) Against Uracils : Functional Monomer Design, Monomer-Template Interactions In Solution And MIP ... polymers ( MIPs ) has burgeoned in recent years [1]. The imprinting of nucleic acid bases and related compounds has attracted particular interest [2]. A

  15. Pulsed field ionization electron spectroscopy and molecular structure of aluminum uracil.

    PubMed

    Krasnokutski, Serge A; Yang, Dong-Sheng

    2007-10-25

    Al-uracil (Al-C4H4N2O2) was synthesized in a laser-vaporization supersonic molecular beam source and studied with pulsed field ionization-zero electron kinetic energy (ZEKE) photoelectron spectroscopy and density functional theory (DFT). The DFT calculations predicted several low-energy Al-uracil isomers with Al binding to the diketo, keto-enol, and dienol tautomers of uracil. The ZEKE spectroscopic measurements of Al-uracil determined the ionization energy of 43 064(5) cm-1 [or 5.340(6) eV] and a vibrational mode of 51 cm-1 for the neutral complex and several vibrational modes of 51, 303, 614, and 739 cm-1 for the ionized species. Combination of the ZEEK spectrum with the DFT and Franck-Condon factor calculations determined the preferred isomeric structure and electronic states of the Al-uracil complex. This isomer is formed by Al binding to the O4 atom of the diketo tautomer of uracil and has a planar Cs symmetry. The ground electronic states of the neutral and ionized species are 2A' ' and 1A', respectively. The 2A' ' neutral state has a slightly shorter Al-O4 distance than the 1A' ion state. However, the 1A' ion state has stronger metal-ligand binding compared to the 2A' ' state. The increased Al-O4 distance from the 2A' ' state to the 1A' state is attributed to the loss of the pi binding interaction between Al and O4 in the singlet ion state, whereas the increased metal-ligand binding strength is due to the additional charge-dipole interaction in the ion that surpasses the loss of the pi orbital interaction.

  16. Synthesis, photophysical and electroluminescent properties of novel iridium (III) complexes based on 5-methyl-2-phenylbenzo[d]oxazole derivatives

    NASA Astrophysics Data System (ADS)

    Li, Xiao; Chi, Hai-Jun; Dong, Yan; Xiao, Guo-Yong; Lei, Peng; Zhang, Dong-Yu; Cui, Zheng

    2013-12-01

    A new series of phosphorescent iridium (III) complexes based on 5-methyl-2-phenylbenzo[d]oxazole derivatives as main ligands, i.e. bis(5-methyl-2- phenylbenzo[d]oxazole-N,C2‧)iridium(acetylacetonate) [(mpbo)2Ir(acac)], bis(2-(4-fluorophenyl)-5-methylbenzo[d]oxazole-N,C2‧)iridium(acetylacetonate) [(fmbo)2Ir(acac)] and bis(5-methyl-2-p-tolylbenzo[d]oxazole-N,C2‧) iridium(acetylacetonate) [(mtbo)2Ir(acac)], were synthesized for organic light-emitting diodes (OLEDs), and their photophysical, electroluminescent properties were investigated. All complexes have high thermal stability and emit intense phosphorescence from green to yellow at room temperature with high quantum efficiencies and relatively short lifetimes. The OLED based on (fmbo)2Ir(acac) as dopant emitter showed very high luminance of 26,004 cd m-2 and luminance efficiency of 18.5 cd A-1. The evidences indicated that this series of iridium (III) complexes were potential candidates for applications in organic electroluminescent devices.

  17. Excited-state deactivation pathways in uracil versus hydrated uracil: solvatochromatic shift in the (1)nπ* state is the key.

    PubMed

    Zhang, Xing; Herbert, John M

    2014-07-17

    Excited-state deactivation mechanisms of uracil are investigated using spin-flip time-dependent density functional theory. Two important minimum-energy crossing points are located, for both gas-phase and hydrated uracil, and optimized relaxation pathways connecting the most important critical points on the (1)nπ* and (1)ππ* potential energy surfaces are determined. An ultrafast decay time constant, measured via femtosecond spectroscopy, is assigned to direct (1)ππ* → S0 deactivation, while a slower decay component is assigned to indirect (1)ππ* → (1)nπ* → S0 deactivation. The shorter lifetime of the dark (1)nπ* state in aqueous solution is attributed to a decrease in the energy barrier along the pathway connecting the (1)nπ* minimum to a (1)ππ*/S0 conical intersection. This barrier arises due to hydrogen bonding between uracil and water, leading to a blue-shift in the S0 → (1)nπ* excitation energy and considerable modification of energy barriers on the (1)nπ* potential surface. These results illustrate how hydrogen bonding to the chromophore can significantly impact excited-state dynamics and also highlight that relaxation pathways can be elucidated using low-cost methods based on density functional theory.

  18. Folate supplementation differently affects uracil content in DNA in the mouse colon and liver

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High folate intake may increase the risk of cancer, especially in the elderly. The present study examined the effects of ageing and dietary folate on uracil misincorporation into DNA, which has a mutagenic effect, in the mouse colon and liver. Old (18 months; n 42) and young (4 months; n 42) male C5...

  19. The alpha/beta fold uracil DNA glycosylases: a common origin with diverse fates

    PubMed Central

    Aravind, L; Koonin, Eugene V

    2000-01-01

    Background: Uracil DNA glycosylases (UDGs) are major repair enzymes that protect DNA from mutational damage caused by uracil incorporated as a result of a polymerase error or deamination of cytosine. Four distinct families of UDGs have been identified, which show very limited sequence similarity to each other, although two of them have been shown to possess the same structural fold. The structural and evolutionary relationships between the rest of the UDGs remain uncertain. Results: Using sequence profile searches, multiple alignment analysis and protein structure comparisons, we show here that all known UDGs possess the same fold and must have evolved from a common ancestor. Although all UDGs catalyze essentially the same reaction, significant changes in the configuration of the catalytic residues were detected within their common fold, which probably results in differences in the biochemistry of these enzymes. The extreme sequence divergence of the UDGs, which is unusual for enzymes with the same principal activity, is probably due to the major role of the uracil-flipping caused by the conformational strain enacted by the enzyme on uracil-containing DNA, as compared with the catalytic action of individual polar residues. We predict two previously undetected families of UDGs and delineate a hypothetical scenario for their evolution. Conclusions: UDGs form a single protein superfamily with a distinct structural fold and a common evolutionary origin. Differences in the catalytic mechanism of the different families combined with the construction of the catalytic pocket have, however, resulted in extreme sequence divergence of these enzymes. PMID:11178247

  20. DNA base excision repair of uracil residues in reconstituted nucleosome core particles

    PubMed Central

    Nilsen, Hilde; Lindahl, Tomas; Verreault, Alain

    2002-01-01

    The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase β and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase β on nucleosome cores. PMID:12411511

  1. Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil.

    PubMed

    Richardson, Tomas T; Wu, Xiaohua; Keith, Brian J; Heslop, Pauline; Jones, Anita C; Connolly, Bernard A

    2013-02-01

    Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3'-5' proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase-DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.

  2. Inhibition of uracil-DNA glycosylase increases SCEs in BrdU-treated and visible light-irradiated cells

    SciTech Connect

    Maldonado, A.; Hernandez, P.; Gutierrez, C.

    1985-11-01

    The authors have approached the study of the ability of different types of lesions produced by DNA-damaging agents to develop sister-chromatid exchanges (SCEs) by analyzing SCE levels observed in Allium cepa L cells with BrdU-substituted DNA and exposed to visible light (VL), an irradiation which produces uracil residues in DNA after debromination of bromouracil and enhances SCE levels but only above a certain dose. They have partially purified an uracil-DNA glycosylase activity from A. cepa L root meristem cells, which removes uracil from DNA, the first step in the excision repair of this lesion. This enzyme was inhibited in vitro by 6-amino-uracil and uracil but not by thymine. When cells exposed to VL, at a dose that did not produce per se an SCE increase, were immediately post-treated with these inhibitors of uracil-DNA glycosylase, a significant increase in SCE levels was obtained. Moreover, SCE levels in irradiated cells dropped to control level when a short holding time elapsed between exposure to VL and the beginning of post-treatment with the inhibitor. Thus, our results showed that inhibitors of uracil-DNA glycosylase enhanced SCE levels in cells with unifilarly BrdU-substituted DNA exposed to visible light; and indicated the existence of a very rapid repair of SCE-inducing lesions produced by visible light irradiation of cells with unifilarly BrdU-containing DNA.

  3. Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.

    PubMed

    Tubeleviciute, Agne; Skirgaila, Remigijus

    2010-08-01

    The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

  4. Effects of protonation and C5 methylation on the electrophilic addition reaction of cytosine: a computational study.

    PubMed

    Jin, Lingxia; Wang, Wenliang; Hu, Daodao; Min, Suotian

    2013-01-10

    The mechanism for the effects of protonation and C5 methylation on the electrophilic addition reaction of Cyt has been explored by means of CBS-QB3 and CBS-QB3/PCM methods. In the gas phase, three paths, two protonated paths (N3 and O2 protonated paths B and C) as well as one neutral path (path A), were mainly discussed, and the calculated results indicate that the reaction of the HSO(3)(-) group with neutral Cyt is unlikely because of its high activation free energy, whereas O2-protonated path (path C) is the most likely to occur. In the aqueous phase, path B is the most feasible mechanism to account for the fact that the activation free energy of path B decreases compared with the corresponding path in the gas phase, whereas those of paths A and C increase. The main striking results are that the HSO(3)(-) group directly interacts with the C5═C6 bond rather than the N3═C4 bond and that the C5 methylation, compared with Cyt, by decreasing values of global electrophilicity index manifests that C5 methylation forms are less electrophilic power as well as by decreasing values of NPA charges on C5 site of the intermediates make the trend of addition reaction weaken, which is in agreement with the experimental observation that the rate of 5-MeCyt reaction is approximately 2 orders of magnitude slower than that of Cyt in the presence of bisulfite. Apart from cis and trans isomers, the rare third isomer where both the CH(3) and SO(3) occupy axial positions has been first found in the reactions of neutral and protonated 5-MeCyt with the HSO(3)(-) group. Furthermore, the transformation of the third isomer from the cis isomer can occur easily.

  5. 3-Cyano-6-(5-methyl-3-pyrazoloamino) pyridines (Part 2): A dual inhibitor of Aurora kinase and tubulin polymerization.

    PubMed

    Morioka, Masahiko

    2016-12-15

    A new class of a dual inhibitor of Aurora kinase and tubulin polymerization was created by introducing various substituted phenoxyethylamino or pyridyloxyethylamino groups to the 2-position of 3-cyano-4-methyl-6-(5-methyl-3-pyrazoloamino)-pyridine. Compound 3g exhibited Aurora kinase inhibition, excellent protein kinase selectivity to Aurora kinase in comparison with 66 other kinases, inhibition of phosphorylation of Ser10 of histone H3 as an Aurora kinase inhibitor, inhibition of tubulin polymerization in vitro, good cell membrane permeability, and a good PK profile. Therefore compound 3g was effective in some antitumor mouse models at a dose of 30mg/kgpoqd.

  6. Crystal thickness dependence of the photoinduced crystal bending of 1-(5-methyl-2-(4-(p-vinylbenzoyloxymethyl)phenyl)-4-thiazolyl)-2-(5-methyl-2-phenyl-4-thiazolyl)perfluorocyclopentene.

    PubMed

    Kitagawa, Daichi; Kobatake, Seiya

    2014-05-01

    Rod-like crystals of 1-(5-methyl-2-(4-(p-vinylbenzoyloxymethyl)phenyl)-4-thiazolyl)-2-(5-methyl-2-phenyl-4-thiazolyl)perfluorocyclopentene with lengths of over 1 mm showed photoreversible bending over 100 cycles upon irradiation with alternating ultraviolet (UV) and visible light. The crystals bent toward the incident light due to a contraction of the crystal length and a gradient of the crystal thickness, which depend on the extent of photoisomerization. It was observed that the bending speed depends on the crystal thickness, and the curvature change on changing the crystal thickness agrees well with Timoshenko's bimetal model, as well as with the observation that crystals of 1,2-bis(2-methyl-5-(4-(1-naphthoyloxymethyl)phenyl)-3-thienyl)perfluorocyclopentene bend away from the incident light due to an expansion of the crystal length and a gradient of the crystal thickness, which depend on the extent of photoisomerization. It was revealed that Timoshenko's bimetal model can be applied to photoinduced crystal bending behaviors caused by both contraction and expansion. These findings are very useful for evaluating and designing photomechanical actuators.

  7. Sorption of organic molecules on surfaces of a microporous polymer adsorbent modified with different quantities of uracil

    NASA Astrophysics Data System (ADS)

    Gus'kov, V. Yu.; Ganieva, A. G.; Kudasheva, F. Kh.

    2016-11-01

    The sorption of organic molecules on the surfaces of a number of adsorbents based on a microporous copolymer of styrene and divinylbenzene modified with different quantities of uracil is studied by means of inverse gas chromatography at infinite dilution. Samples containing 10-6, 10-5, 10-4, 10-3, 10-2, and 0.5 × 10‒1 weight parts of uracil (the pC of uracil ranges from 1.3 to 6) are studied. The contributions from different intermolecular interactions to the Helmholtz energy of sorption are calculated via the linear free energy relationship. It is found that as the concentration of uracil on the surface of the polymer adsorbent grows, the contributions from different intermolecular interactions and the conventional polarity of the surface have a bend at pC = 3, due probably to the formation of a supramolecular structure of uracil. Based on the obtained results, it is concluded that the formation of the supramolecular structure of uracil on the surface of the polymer adsorbent starts when pC < 3.

  8. The Photochemistry of Pyrimidine in Pure H2O Ice Subjected to Different Radiation Environments and the Formation of Uracil

    NASA Technical Reports Server (NTRS)

    Nuevo, M.; Chen, Y.-J.; Materese. C. K..; Hu, W.-J.; Qiu, J.-M.; Wu, S.-R.; Fung, H.-S.; Sandford, S. A.; Chu, C.-C.; Yih, T.-S.; Wu, R.; Ip, W.-H.

    2013-01-01

    Nucleobases are N-heterocycles which are the informational subunits of DNA and RNA. They include pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in several meteorites, although no Nheterocycles have been observed in space to data. Laboratory experiments showed that the ultraviolet (UV) irradiation of pyrimidine in pure H2O ice at low temperature (<=20 K) leads to the formation of pyrimidine derivatives including the nucleobase uracil and its precursor 4(3H)-pyrimidone. These results were confirmed by quantum chemical calculations. When pyrimidine is mixed with combinations of H2O, NH3, CH3OH, and CH4 ices under similar conditions, uracil and cytosine are formed. In the present work we study the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in H2O ice with high-energy UV photons (Lyman , He I, and He II lines) provided by a synchrotron source. The photo-destruction of pyrimidine in these H2O ices as well as the formation yields for 4(3H)-pyrimidone and uracil are compared with our previous results in order to study the photo-stability of pyrimidine and the production efficiency of uracil as a function of the photon energy.

  9. Crystal structures of Toxoplasma gondii uracil phosphoribosyltransferase reveal the atomic basis of pyrimidine discrimination and prodrug binding.

    PubMed Central

    Schumacher, M A; Carter, D; Scott, D M; Roos, D S; Ullman, B; Brennan, R G

    1998-01-01

    Uracil phosphoribosyltransferase (UPRTase) catalyzes the transfer of a ribosyl phosphate group from alpha-D-5-phosphoribosyl-1-pyrophosphate to the N1 nitrogen of uracil. The UPRTase from the opportunistic pathogen Toxoplasma gondii is a rational target for antiparasitic drug design. To aid in structure-based drug design studies against toxoplasmosis, the crystal structures of the T.gondii apo UPRTase (1.93 A resolution), the UPRTase bound to its substrate, uracil (2.2 A resolution), its product, UMP (2.5 A resolution), and the prodrug, 5-fluorouracil (2.3 A resolution), have been determined. These structures reveal that UPRTase recognizes uracil through polypeptide backbone hydrogen bonds to the uracil exocyclic O2 and endocyclic N3 atoms and a backbone-water-exocyclic O4 oxygen hydrogen bond. This stereochemical arrangement and the architecture of the uracil-binding pocket reveal why cytosine and pyrimidines with exocyclic substituents at ring position 5 larger than fluorine, including thymine, cannot bind to the enzyme. Strikingly, the T. gondii UPRTase contains a 22 residue insertion within the conserved PRTase fold that forms an extended antiparallel beta-arm. Leu92, at the tip of this arm, functions to cap the active site of its dimer mate, thereby inhibiting the escape of the substrate-binding water molecule. PMID:9628859

  10. Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts.

    PubMed Central

    Kuhnlein, U; Lee, B; Linn, S

    1978-01-01

    Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblasts lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent Km for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal. PMID:643602

  11. Electron-induced hydrogen loss in uracil in a water cluster environment

    SciTech Connect

    Smyth, M.; Kohanoff, J.; Fabrikant, I. I.

    2014-05-14

    Low-energy electron-impact hydrogen loss due to dissociative electron attachment (DEA) to the uracil and thymine molecules in a water cluster environment is investigated theoretically. Only the A{sup ′}-resonance contribution, describing the near-threshold behavior of DEA, is incorporated. Calculations are based on the nonlocal complex potential theory and the multiple scattering theory, and are performed for a model target with basic properties of uracil and thymine, surrounded by five water molecules. The DEA cross section is strongly enhanced when the attaching molecule is embedded in a water cluster. This growth is due to two effects: the increase of the resonance lifetime and the negative shift in the resonance position due to interaction of the intermediate negative ion with the surrounding water molecules. A similar effect was earlier found in DEA to chlorofluorocarbons.

  12. Functional changes in a novel uracil-DNA glycosylase determined by mutational analyses.

    PubMed

    Im, E K; Han, Y S; Chung, J H

    2008-01-01

    Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in bacteria and eukaryotes, which removes uracil residues from DNA strands. Methanococcus jannaschii UDG (MjUDG), a novel monofunctional glycosylase, contains a helix-hairpin-helix (HhH) motif and Gly/Pro rich loop (GPD region), which is important for catalytic activity; it shares these features with other glycosylases such as endonuclease III. First, to examine the role of two conserved amino acid residues (Asp150 and Tyr152) in the HhH-GPD region of MjUDG, mutant MjUDG proteins were constructed, in which Asp 150 was replaced with either Glu or Trp (D150E and D150W), Tyr152 was replaced with either Glu or Asn (Y152E and Y152N). Mutant D150W completely lacked DNA glycosylase activity, whereas D150E displayed reduced activity of about 70% of the wild type value. However, the mutants Y152E and Y152N retained unchanged levels of UDG activity. We also replaced Glu132 in the HhH motif with a lysine residue equivalent to Lys120 in endonuclease III. This mutation converted the enzyme into a bifunctional glycosylase/AP lyase capable of both removing uracil at a glycosylic bond and cleaving the phosphodiester backbone at an AP site. Mutant E132K catalyzes a beta-elimination reaction at the AP site via uracil excision and forms a Schiff base intermediate in the form of a protein-DNA complex.

  13. Quantum chemical predictions of the vibrational spectra of polyatomic molecules. The uracil molecule and two derivatives.

    PubMed

    Palafox, Mauricio Alcolea; Rastogi, V K

    2002-02-01

    This work describes the different scaling procedures used to correct the quantum-chemical theoretical predictions of the IR and Raman vibrational wavenumbers. Examples of each case are shown, with special attention to the uracil molecule and some derivatives. The results obtained with different semiempirical and ab initio methods, and basis sets, are compared and discussed. A comprehensive compendium of the main scale factors and scaling equations available to obtain the scaled wavenumbers is also shown.

  14. Folate deficiency induces neurodegeneration and brain dysfunction in mice lacking uracil DNA glycosylase.

    PubMed

    Kronenberg, Golo; Harms, Christoph; Sobol, Robert W; Cardozo-Pelaez, Fernando; Linhart, Heinz; Winter, Benjamin; Balkaya, Mustafa; Gertz, Karen; Gay, Shanna B; Cox, David; Eckart, Sarah; Ahmadi, Michael; Juckel, Georg; Kempermann, Gerd; Hellweg, Rainer; Sohr, Reinhard; Hörtnagl, Heide; Wilson, Samuel H; Jaenisch, Rudolf; Endres, Matthias

    2008-07-09

    Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung-/-) versus wild-type controls. Folate depletion increased nuclear mutation rates in Ung-/- embryonic fibroblasts, and conferred death of cultured Ung-/- hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung-/- but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in Ung-/- mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency.

  15. Analogues of uracil nucleosides with intrinsic fluorescence (NIF-analogues): synthesis and photophysical properties.

    PubMed

    Segal, Meirav; Fischer, Bilha

    2012-02-28

    Uridine cannot be utilized as fluorescent probe due to its extremely low quantum yield. For improving the uracil fluorescence characteristics we extended the natural chromophore at the C5 position by coupling substituted aromatic rings directly or via an alkenyl or alkynyl linker to create fluorophores. Extension of the uracil base was achieved by treating 5-I-uridine with the appropriate boronic acid under the Suzuki coupling conditions. Analogues containing an alkynyl linker were obtained from 5-I-uridine and the suitable boronic acid in a Sonogashira coupling reaction. The uracil fluorescent analogues proposed here were designed to satisfy the following requirements: a minimal chemical modification at a position not involved in base-pairing, resulting in relatively long absorption and emission wavelengths and high quantum yield. 5-((4-Methoxy-phenyl)-trans-vinyl)-2'-deoxy-uridine, 6b, was found to be a promising fluorescent probe. Probe 6b exhibits a quantum yield that is 3000-fold larger than that of the natural chromophore (Φ 0.12), maximum emission (478 nm) which is 170 nm red shifted as compared to uridine, and a Stokes shift of 143 nm. In addition, since probe 6b adopts the anti conformation and S sugar puckering favored by B-DNA, it makes a promising nucleoside analogue to be incorporated in an oligonucleotide probe for detection of genetic material.

  16. Isomeric luminescent Zn(II) coordination polymers based on pyridinecarboxylate and 5-methyl-1H-tetrazole ligands

    NASA Astrophysics Data System (ADS)

    An, Zhe; Gao, Jing; Zhu, Ling

    2013-12-01

    Two new metal-organic frameworks, namely [Zn(nic)(mtz)]n (1) and [Zn(isonic)(mtz)]n (2) (Hnic = nicotinic acid, Hisonic = isonicotinic acid, Hmtz = 5-methyl-1H-tetrazole), have been obtained through the solvothermal reactions of Zn(NO3)2, Htmz and Hnic or Hisonic. Single crystal X-ray diffraction analysis reveals that compound 1 features a 2D layered structure with sql topology, which is further extended into a 3D supramolecular framework via weak CH…π interactions, and compound 2 is 2-fold interpenetrated 3D framework with dia topology. Luminescent investigation shows that both of them emit blue luminescence at room temperature.

  17. Diaqua-(5-methyl-1H-pyrazole-3-carboxyl-ato)(4-nitro-benzoato)copper(II).

    PubMed

    Hu, Fei-Long; Yin, Xian-Hong; Feng, Yu; Mi, Yan; Zhang, Shan-Shan

    2009-01-23

    In the title complex, [Cu(C(7)H(4)NO(4))(C(5)H(5)N(2)O(2))(H(2)O)(2)], the Cu(II) ion is coordinated in a slightly distorted square-pyramidal enviroment. The basal plane is formed by an N atom and an O atom from a 5-methyl-1H-pyrazole-3-carboxyl-ate ligand and by two O atoms from two water ligands. The apical position is occupied by a carboxylate O atom from a 4-nitro-benzoate ligand. In the crystal structure, inter-molecular O-H⋯O and N-H⋯O hydrogen bonds link complex moleclues, forming extended chains parallel to the a axis.

  18. The thermodynamic contribution of the 5-methyl group of thymine in the two- and three-stranded complexes formed by poly(dU) and poly(dT) with poly(dA).

    PubMed

    Ross, Philip D; Howard, Frank B

    2003-02-01

    To assess the thermodynamic contribution of the 5-methyl group of thymine, we have studied the two-stranded helical complexes poly(dA).poly(dU) and poly(dA).poly(dT) and the three-stranded complexes--poly(dA).2poly(dU), poly(dA).poly(dT).poly(dU) and poly(dA).2poly(dT)--by differential scanning calorimetry, and uv optical melting experiments. The thermodynamic quantities associated with the 3 --> 2, 2 --> 1, and 3 --> 1 melting transitions are found to vary with salt concentration and temperature in a more complex manner than commonly believed. The transition temperatures, T(m), are generally not linear in the logarithm of concentration or activity of NaCl. The change in enthalpy and in entropy upon melting varies with salt concentration and temperature, and a change in heat capacity accompanies each transition. The poly(dA).2poly(dU) triple helix is markedly different from poly(dA).2poly(dT) in both its CD spectrum and thermodynamic behavior, while the poly(dA).poly(dT).poly(dU) triple helix resembles poly(dA).2poly(dT) in these properties. In comparing poly(dA).2poly(dT) with either the poly(dA).poly(dT).poly(dU) or the poly(dA).2poly(dU) triplexes, the substitution of thymine for uracil in the third strand results in an enhancement of stability against the 3 --> 2 dissociation of deltadeltaG degrees = -135 +/- 85 cal (mol A)(-1) at 37 degrees C. This represents a doubling of the absolute stability toward dissociation compared to the triplexes with poly(dU) as the third strand. The poly (dA).poly (dT) duplex is more stable than poly(dA).poly(dU) by deltadeltaG degrees = -350 +/- 60 cal (mol base pair)(-1) at 37 degrees C. Poly(dA).poly(dT) has 50% greater stability than poly(dA).poly(dU) as a result of the dT for dU substitution in the duplex.

  19. A multiancestry study identifies novel genetic associations with CHRNA5 methylation in human brain and risk of nicotine dependence

    PubMed Central

    Hancock, Dana B.; Wang, Jen-Chyong; Gaddis, Nathan C.; Levy, Joshua L.; Saccone, Nancy L.; Stitzel, Jerry A.; Goate, Alison; Bierut, Laura J.; Johnson, Eric O.

    2015-01-01

    Nicotine dependence is influenced by chromosome 15q25.1 single nucleotide polymorphisms (SNPs), including the missense SNP rs16969968 that alters function of the α5 nicotinic acetylcholine receptor (CHRNA5) and noncoding SNPs that regulate CHRNA5 mRNA expression. We tested for cis-methylation quantitative trait loci (cis-meQTLs) using SNP genotypes and DNA methylation levels measured across the IREB2-HYKK-PSMA4-CHRNA5-CHRNA3-CHRNB4 genes on chromosome 15q25.1 in the BrainCloud and Brain QTL cohorts [total N = 175 European-Americans and 65 African-Americans (AAs)]. We identified eight SNPs that were significantly associated with CHRNA5 methylation in prefrontal cortex: P ranging from 6.0 × 10−10 to 5.6 × 10−5. These SNP–methylation associations were also significant in frontal cortex, temporal cortex and pons: P ranging from 4.8 × 10−12 to 3.4 × 10−3. Of the eight cis-meQTL SNPs, only the intronic CHRNB4 SNP rs11636753 was associated with CHRNA5 methylation independently of the known SNP effects in prefrontal cortex, and it was the most significantly associated SNP with nicotine dependence across five independent cohorts (total N = 7858 European ancestry and 3238 AA participants): P = 6.7 × 10−4, odds ratio (OR) [95% confidence interval (CI)] = 1.11 (1.05–1.18). The rs11636753 major allele (G) was associated with lower CHRNA5 DNA methylation, lower CHRNA5 mRNA expression and increased nicotine dependence risk. Haplotype analyses showed that rs11636753-G and the functional rs16969968-A alleles together increased risk of nicotine dependence more than each variant alone: P = 3.1 × 10−12, OR (95% CI) = 1.32 (1.22–1.43). Our findings identify a novel regulatory SNP association with nicotine dependence and connect, for the first time, previously observed differences in CHRNA5 mRNA expression and nicotine dependence risk to underlying DNA methylation differences. PMID:26220977

  20. DNA uracil repair initiated by the archaeal ExoIII homologue Mth212 via direct strand incision

    PubMed Central

    Schomacher, Lars; Chong, James P. J.; McDermott, Paul; Kramer, Wilfried; Fritz, Hans-Joachim

    2009-01-01

    No genes for any of the known uracil DNA glycosylases of the UDG superfamily are present in the genome of Methanothermobacter thermautotrophicus ΔH, making it difficult to imagine how DNA-U repair might be initiated in this organism. Recently, Mth212, the ExoIII homologue of M. thermautotrophicus ΔH has been characterized as a DNA uridine endonuclease, which suggested the possibility of a novel endonucleolytic entry mechanism for DNA uracil repair. With no system of genetic experimentation available, the problem was approached biochemically. Assays of DNA uracil repair in vitro, promoted by crude cellular extracts, provide unequivocal confirmation that this mechanism does indeed operate in M. thermautotrophicus ΔH. PMID:19240141

  1. VizieR Online Data Catalog: Line Frequencies for Uracil (Bruenken+, 2006)

    NASA Astrophysics Data System (ADS)

    Bruenken, S.; McCarthy, M. C.; Thaddeus, P.; Godfrey, P. D.; Brown, R. D.

    2006-08-01

    We report new laboratory spectroscopic data on the nucleic acid uracil. The dataset has been extended both into the microwave and towards mm-wavelengths with the aim to provide accurate transition rest frequencies for astrophysical searches. The complete experimental linelist used for the analysis of the data is compiled in Table 2. Lines marked with an asterisk are taken from Brown et al. (1988, J. Mol. Struct. 190, 185 and J. Am. Chem. Soc. 110, 2329), all other lines have been measured in this work. (1 data file).

  2. CD spectra of 5-methyl-2-thiouridine in tRNA-Met-f from an extreme thermophile.

    PubMed Central

    Watanabe, K; Oshima, T; Nishimura, S

    1976-01-01

    5-Methyl-2-thiouridine (S) in tRNA-Met-f from an extreme thermophile is located in the TpsiC region, replacing T, and has a positive CD band centered at 310 nm. Upon heating, the profiles of the change in this band were similar to the UV melting profiles of the change monitored at 260 nm. This strongly suggests a close relation between heat denaturation of the tRNA and the conformation of the S base. Oligonucleotides containing S showed negative CD bands at 320-330 nm, like the monomer S itself, but when the 3'-2/5 fragment containing S formed a complex with the complementary 5'-3/5 fragment, a positive CD band appeared at 310 nm. These results suggest that combination of the TpsiC loop containing S with the hU loop is necessary for the positive band of S at 310 nm. S may serve to strengthen the association of the TpsiC loop with the hU loop in tRNA of the thermophile. PMID:967669

  3. Non-classical mechanism of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor channel block by fluoxetine.

    PubMed

    Barygin, Oleg I; Komarova, Margarita S; Tikhonova, Tatiana B; Tikhonov, Denis B

    2015-04-01

    Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca(2+)-permeable and Ca(2+)-impermeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca(2+)-impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC50 value for the inhibition of Ca(2+)-permeable AMPA receptors in giant striatal interneurons was 43 ± 7 μM. The inhibition of Ca(2+)-permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open-channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane-impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open-channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.

  4. Field induced changes in the ring/chain equilibrium of hydrogen bonded structures: 5-methyl-3-heptanol

    NASA Astrophysics Data System (ADS)

    Young-Gonzales, Amanda R.; Richert, Ranko

    2016-08-01

    Using non-linear dielectric techniques, we have measured the dynamics of 5-methyl-3-heptanol at a temperature at which the Kirkwood correlation factor gK indicates the coexistence of ring- and chain-like hydrogen-bonded structures. Steady state permittivity spectra recorded in the presence of a high dc bias electric field (17 MV/m) reveal that both the amplitude and the time constant are increased by about 10% relative to the low field limit. This change is attributed to the field driven conversion from ring-like to the more polar chain-like structures, and a direct observation of its time dependence shows that the ring/chain structural transition occurs on a time scale that closely matches that of the dielectric Debye peak. This lends strong support to the picture that places fluctuations of the end-to-end vector of hydrogen bonded structures at the origin of the Debye process, equivalent to fluctuations of the net dipole moment or gK. Recognizing that changes in the ring/chain equilibrium constant also impact the spectral separation between Debye and α-process may explain the difference in their temperature dependence whenever gK is sensitive to temperature, i.e., when the structural motifs of hydrogen bonding change considerably.

  5. FT-IR, Laser-Raman spectra and computational analysis of 5-Methyl-3-phenylisoxazole-4-carboxylic acid.

    PubMed

    Sert, Yusuf; Mahendra, M; Keskinoğlu, S; Chandra; Srikantamurthy, N; Umesha, K B; Çırak, Ç

    2015-03-15

    In this study the experimental and theoretical vibrational frequencies of a newly synthesized anti-tumor, antiviral, hypoglycemic, antifungal and anti-HIV agent namely, 5-Methyl-3-phenylisoxazole-4-carboxylic acid has been investigated. The experimental FT-IR (4000-400 cm(-1)) and Laser-Raman spectra (4000-100 cm(-1)) of the molecule in solid phase have been recorded. The theoretical vibrational frequencies and optimized geometric parameters (bond lengths, bond angles and torsion angles) have been calculated by using density functional theory (DFT/B3LYP: Becke, 3-parameter, Lee-Yang-Parr and DFT/M06-2X: highly parametrized, empirical exchange correlation function) with 6-311++G(d,p) basis set by Gaussian 09W software, for the first time. The assignments of the vibrational frequencies have been done by potential energy distribution (PED) analysis by using VEDA 4 software. The theoretical optimized geometric parameters and vibrational frequencies have been found to be in good agreement with the corresponding experimental data and results in the literature. In addition, the highest occupied molecular orbital (HOMO) energy, the lowest unoccupied molecular orbital (LUMO) energy and the other related molecular energy values of the compound have been investigated by using the same theoretical calculations.

  6. FT-IR, Laser-Raman spectra and computational analysis of 5-Methyl-3-phenylisoxazole-4-carboxylic acid

    NASA Astrophysics Data System (ADS)

    Sert, Yusuf; Mahendra, M.; Keskinoğlu, S.; Chandra; Srikantamurthy, N.; Umesha, K. B.; Çırak, Ç.

    2015-03-01

    In this study the experimental and theoretical vibrational frequencies of a newly synthesized anti-tumor, antiviral, hypoglycemic, antifungal and anti-HIV agent namely, 5-Methyl-3-phenylisoxazole-4-carboxylic acid has been investigated. The experimental FT-IR (4000-400 cm-1) and Laser-Raman spectra (4000-100 cm-1) of the molecule in solid phase have been recorded. The theoretical vibrational frequencies and optimized geometric parameters (bond lengths, bond angles and torsion angles) have been calculated by using density functional theory (DFT/B3LYP: Becke, 3-parameter, Lee-Yang-Parr and DFT/M06-2X: highly parametrized, empirical exchange correlation function) with 6-311++G(d,p) basis set by Gaussian 09W software, for the first time. The assignments of the vibrational frequencies have been done by potential energy distribution (PED) analysis by using VEDA 4 software. The theoretical optimized geometric parameters and vibrational frequencies have been found to be in good agreement with the corresponding experimental data and results in the literature. In addition, the highest occupied molecular orbital (HOMO) energy, the lowest unoccupied molecular orbital (LUMO) energy and the other related molecular energy values of the compound have been investigated by using the same theoretical calculations.

  7. Bis­(2-amino-5-methyl­pyridinium) fumarate–fumaric acid (1/1)

    PubMed Central

    Hemamalini, Madhukar; Fun, Hoong-Kun

    2010-01-01

    In the crystal structure of the title compound, C6H9N2 +·0.5C4H2O4 2−·0.5C4H6O4, the fumarate dianion and fumaric acid mol­ecule are located on inversion centres. The 2-amino-5-methyl­pyrimidinium cation inter­acts with the carboxyl­ate group of the fumarate anion through a pair of N—H⋯O hydrogen bonds, forming an R 2 2(8) ring motif. These motifs are centrosymmetrically paired via N—H⋯O hydrogen bonds, forming a complementary DDAA array. The carboxyl groups of the fumaric acid mol­ecules and the carboxyl­ate groups of the fumarate anions are hydrogen bonded through O—H⋯O hydrogen bonds, leading to a supra­molecular chain along [101]. The crystal structure is further stabilized by weak C—H⋯O hydrogen bonds. PMID:21588388

  8. The calculated free energy effects of 5-methyl cytosine on the B to Z transition in DNA.

    PubMed

    Pearlman, D A; Kollman, P A

    We have examined the free energy effects of 5-methylation of cytosine on the B in equilibrium Z conformational equilibrium in DNA. Free energy differences were calculated using the free energy perturbation approach, which uses an easily derived equation from classical statistical mechanics to relate the free energy difference between two states to the ensemble average of the potential energy difference between the states. Calculations were carried both in explicit solvent and (for comparison) in vacuo. The free energy values obtained for the explicit solvent systems are total free energies, with contributions from all parts of the system (solvent + solute), and so are relevant to the B in equilibrium Z transitions observed under real (physiological) conditions. We calculate that in solution, methylation makes the B in equilibrium Z transition more favorable by about -0.4 kcal/mole base pair (bp) in free energy. This value compares well with approximate experimentally derived values of about -0.3 kcal/mole-bp. We also discuss a method for determining the free energy difference between conformational states poorly maintained by a potential energy model. Finally, the effects of methylation on the melting temperature of DNA are examined.

  9. Inhibition of uracil DNA glycosylase sensitizes cancer cells to 5-fluorodeoxyuridine through replication fork collapse-induced DNA damage

    PubMed Central

    Yan, Yan; Han, Xiangzi; Qing, Yulan; Condie, Allison G.; Gorityala, Shashank; Yang, Shuming; Xu, Yan; Zhang, Youwei; Gerson, Stanton L.

    2016-01-01

    5-fluorodeoxyuridine (5-FdU, floxuridine) is active against multiple cancers through the inhibition of thymidylate synthase, which consequently introduces uracil and 5-FU incorporation into the genome. Uracil DNA glycosylase (UDG) is one of the main enzymes responsible for the removal of uracil and 5-FU. However, how exactly UDG mediates cellular sensitivity to 5-FdU, and if so whether it is through its ability to remove uracil and 5-FU have not been well characterized. In this study, we report that UDG depletion led to incorporation of uracil and 5-FU in DNA following 5-FdU treatment and significantly enhanced 5-FdU's cytotoxicity in cancer cell lines. Co-treatment, but not post-treatment with thymidine prevented cell death of UDG depleted cells by 5-FdU, indicating that the enhanced cytotoxicity is due to the retention of uracil and 5-FU in genomic DNA in the absence of UDG. Furthermore, UDG depleted cells were arrested at late G1 and early S phase by 5-FdU, followed by accumulation of sub-G1 population indicating cell death. Mechanistically, 5-FdU dramatically reduced DNA replication speed in UDG depleted cells. UDG depletion also greatly enhanced DNA damage as shown by γH2AX foci formation. Notably, the increased γH2AX foci formation was not suppressed by caspase inhibitor treatment, suggesting that DNA damage precedes cell death induced by 5-FdU. Together, these data provide novel mechanistic insights into the roles of UDG in DNA replication, damage repair, and cell death in response to 5-FdU and suggest that UDG is a target for improving the anticancer effect of this agent. PMID:27517750

  10. Solvent effects on the steady-state absorption and fluorescence spectra of uracil, thymine and 5-fluorouracil.

    PubMed

    Gustavsson, Thomas; Sarkar, Nilmoni; Bányász, Akos; Markovitsi, Dimitra; Improta, Roberto

    2007-01-01

    We report a comparison of the steady-state absorption and fluorescence spectra of three representative uracil derivatives (uracil, thymine and 5-fluorouracil) in alcoholic solutions. The present results are compared with those from our previous experimental and computational studies of the same compounds in water and acetonitrile. The effects of solvent polarity and hydrogen bonding on the spectra are discussed in the light of theoretical predictions. This comparative analysis provides a more complete picture of the solvent effects on the absorption and fluorescence properties of pyrimidine nucleobases, with special emphasis on the mechanism of the excited state deactivation.

  11. N-[(E)-(5-Methyl-thio-phen-2-yl)methyl-idene]-1H-1,2,4-triazol-3-amine.

    PubMed

    Chohan, Zahid H; Hanif, Muhammad; Tahir, M Nawaz

    2008-12-13

    In the title Schiff base, C(8)H(8)N(4)S, a condensation product of 5-methyl-thio-phene-2-carboxaldehyde and 3-amino-1,2,4-triazole, the dihedral angle between the triazolyl and thienyl rings is 6.44 (14)°. The compound exists as a polymeric chain arising from inter-molecular N-H⋯N bonding.

  12. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 1 2013-04-01 2013-04-01 false 4- -2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one. 73.3122 Section 73.3122 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... exceed the minimum reasonably required to accomplish the intended coloring effect. (2) Authorization...

  13. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 1 2012-04-01 2012-04-01 false 4- -2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one. 73.3122 Section 73.3122 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... exceed the minimum reasonably required to accomplish the intended coloring effect. (2) Authorization...

  14. Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

    PubMed Central

    Bouhamdan, M; Benichou, S; Rey, F; Navarro, J M; Agostini, I; Spire, B; Camonis, J; Slupphaug, G; Vigne, R; Benarous, R; Sire, J

    1996-01-01

    The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr. PMID:8551605

  15. Optical properties of organically functionalized silicon surfaces: Uracil-like nucleobases on Si(001)

    NASA Astrophysics Data System (ADS)

    Molteni, Elena; Cappellini, Giancarlo; Onida, Giovanni; Fratesi, Guido

    2017-02-01

    We predict UV reflectance anisotropy spectra (RAS) of the organically functionalized silicon (001) surface covered by pyrimidinic uracil-like nucleobases. First-principles results based on density functional theory show characteristic spectral features appearing in the UV range between 3 and 7 eV, besides the expected quench in the well-known two-minima RAS signal of clean Si(001). Nucleobase adsorption in the energetically favored "dimer bridge" configuration gives rise to a characteristic RAS line shape, common to thymine, uracil, and 5-fluorouracil. We trace back the origin of such spectral features by singling out RAS structures induced by relaxation and passivation effects on the Si surface, and those directly associated with molecular excitations. The former turn out to be the same for the three nucleobases, and are totally unaffected by molecular tilting. The sign and position of the latter RAS peaks at higher energy exhibit a moderate nucleobase dependence, and can be fully rationalized in terms of the molecular orbitals involved. The present theoretical results call for a RAS experimental study in the UV region extending up to ≃6 -7 eV.

  16. Escherichia coli-Derived Uracil Increases the Antibacterial Activity and Growth Rate of Lactobacillus plantarum.

    PubMed

    Ha, Eun-Mi

    2016-05-28

    Lactobacillus plantarum (L. plantarum) is a representative probiotic. In particular, L. plantarum is the first commensal bacterium to colonize the intestine of infants. For this reason, the initial settlement of L. plantarum can play an important role in determining an infant's health as well as their eventual health status as an adult. In addition, L. plantarum combats pathogenic infections (such as Escherichia coli (E. coli), one of the early pathogenic colonizers in an unhealthy infant gut) by secreting antimicrobial substances. The aim of this research was to determine how L. plantarum combats E. coli infection and why it is a representative probiotic in the intestine. Consequently, this research observed that E. coli releases uracil. L. plantarum specifically recognizes E. coli-derived uracil, which increases the growth rate and production of antimicrobial substance of L. plantarum. In addition, through the inhibitory activity test, this study postulates that the antimicrobial substance is a protein and can be considered a bacteriocin-like substance. Therefore, this research assumes that L. plantarum exerts its antibacterial ability by recognizing E. coli and increasing its growth rate as a result, and this phenomenon could be one of the reasons for L. plantarum settling in the intestine of infants as a beneficial bacterium.

  17. The peculiar spectral properties of amino-substituted uracils: a combined theoretical and experimental study.

    PubMed

    Bányász, Akos; Karpati, Szilvia; Mercier, Yannick; Reguero, Mar; Gustavsson, Thomas; Markovitsi, Dimitra; Improta, Roberto

    2010-10-07

    A detailed experimental and computational study of the absorption and fluorescence spectra of 5-aminouracil (5 AU) and 6-aminouracil (6 AU) in aqueous solution is reported. The lowest energy band of the steady-state absorption spectra of 5 AU is considerably red-shifted, noticeably less intense, and broader than its counterpart in uracil (U). On the contrary, the 6 AU lowest energy absorption peak is close in energy to that of U, but it is much narrower and the transition is much more intense. The emission properties of 5 AU, 6 AU, and U are also very different. Both amino-substituted compounds exhibit indeed a much larger Stokes shift as compared to U, and the emission band of 5 AU is much narrower than that of 6 AU. Those features are fully rationalized with the help of PCM/TD-PBE0 calculations in aqueous solution and MS-CASPT2/CASSCF calculations in the gas phase. A stable minimum on the potential energy surface of the lowest energy bright state is found for 5 AU, both in the gas phase and in aqueous solution. For 6 AU a barrierless path leads to the conical intersection with the ground electronic state, but a nonplanar plateau region is predicted in aqueous solution, which is responsible for the very large Stokes shift. Some general considerations on the excited-state dynamics of uracil derivatives are also reported.

  18. Simulation of the resonance Raman spectra for 5-halogenated (F, Cl, and Br) uracils.

    PubMed

    Sun, Shuai; Brown, Alex

    2015-04-30

    The resonance Raman spectra of the 5-halogenated (F, Cl, and Br) uracils are simulated via the Herzberg-Teller (HT) short-time dynamics formalism. The gradient of the S1 excited state is computed at the CAMB3LYP/aug-cc-pVTZ level of theory in the conductor-like polarizable continuum model for water (C-PCM, H2O), based on the equilibrium geometry determined using PBE0/aug-cc-pVTZ in H2O (C-PCM). The simulated resonance Raman spectra show good agreement with the experimental spectra in terms of both peak positions and intensities. The differences between the resonance Raman spectra of the three 5-halogenated uracils, caused by the effect of halogen substitution, are examined in terms of ground-state normal-mode eigenvectors and excited-state Cartesian gradients, according to the HT formalism. The differences in the normal-mode eigenvectors and excited-state Cartesian gradients between 5-fluorouracil and 5-chlorouracil are used to interpret the dissimilarity between their resonance Raman spectra. Meanwhile, the similarity between the spectra of 5-chlorouracil and 5-bromouracil is explained by the correspondence between their normal modes and excited-state gradients.

  19. Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxycytidine.

    PubMed

    Ohshima, Motohiro; Tadakuma, Tomomi; Hayashi, Hideki; Inoue, Kazuyuki; Itoh, Kunihiko

    2010-01-01

    We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly(4)-Ser)(3). The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m(5)dCyd and weakly with 5-methylcytidine (m(5)Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC(50) 0.054 microg/ml) was approximately 80 times higher than that of the parent MoAb (IC(50) 4.27 microg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m(5)dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained approximately 1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

  20. Eimeria tenella: parasite-specific incorporation of /sup 3/H-uracil as a quantitative measure of intracellular development

    SciTech Connect

    Schmatz, D.M.; Crane, M.S.; Murray, P.K.

    1986-02-01

    An assay has been developed using parasite-specific incorporation of /sup 3/H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, /sup 3/H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional /sup 3/H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of /sup 3/H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.

  1. Epithelia hyperplasia in the renal papilla and pelvis but not the urinary bladder of male F344 rats associated with dietary sodium phosphates after uracil exposure.

    PubMed

    Shibata, M A; Sano, M; Shirai, T; Imaida, K; Fukushima, S

    1993-08-01

    Effects of the bladder tumor promoter Na3PO4 and the non-bladder-tumor promoter NaH2PO4 on development of hyperplastic lesions of urinary bladder and renal papilla/pelvis were investigated after exposure of male F344 rats to the nongenotoxic carcinogen uracil. Animals were administered with 3.0% uracil in the diet for 4 weeks and thereafter fed 3.0% Na3PO4 or 3.0% NaH2PO4 for 32 weeks. No enhancing effect of either phosphate salt on uracil-induced proliferative lesions of urinary bladder was observed. However, the sequential treatments gave rise to enhanced development of hyperplastic lesions in the renal papilla/pelvis compared to the case with uracil alone. In addition, a small number of renal pelvic papillomas were observed in the group given Na3PO4 after uracil. These phosphate salts also induced nephrocalcinosis in the papilla/pelvis concomitant with development of renal hyperplastic lesions in this location. A sequential study revealed calculus formation and proliferative lesions in both the urinary bladder and renal papilla/pelvis after 4 weeks dietary application of uracil. After cessation, calculi disappeared and the majority of hyperplastic lesions regressed, consistent with a decrease in DNA synthesis levels. Persistence of uracil-induced epithelial hyperplasia in renal papilla/pelvis under the influence of phosphate salts might have been directly due to chronic stimulation by nephrocalcinosis in these sites.

  2. Hydrogen-bonding and the dissolution mechanism of uracil in an acetate ionic liquid: new insights from NMR spectroscopy and quantum chemical calculations.

    PubMed

    Araújo, João M M; Pereiro, Ana B; Canongia Lopes, José N; Rebelo, Luís P N; Marrucho, Isabel M

    2013-04-18

    The dissolution of uracil-a pyrimidine nucleic acid base-in the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][CH3COO]) has been investigated by methods of (1)H and (13)C NMR spectroscopy, (1)H-(1)H NOESY NMR spectroscopy, and quantum chemical calculations. The uracil-[C2mim][CH3COO] interactions that define the dissolution mechanism comprise the hydrogen bonds between the oxygen atoms of the acetate anion and the hydrogen atoms of the N1-H and N3-H groups of uracil and also the hydrogen bonds between the most acidic aromatic hydrogen atom (H2) of the imidazolium cation and the oxygen atoms of the carbonyl groups of uracil. The bifunctional solvation nature of the ionic liquid can be inferred from the presence of interactions between both ions of the ionic liquid and the uracil molecule. The location of such interaction sites was revealed using NMR data ((1)H and (13)C chemical shifts both in the IL and in the uracil molecule), complemented by DFT calculations. NOESY experiments provided additional evidence concerning the cation-uracil interactions.

  3. The Role of Sarcosine, Uracil, and Kynurenic Acid Metabolism in Urine for Diagnosis and Progression Monitoring of Prostate Cancer

    PubMed Central

    Gkotsos, Georgios; Virgiliou, Christina; Lagoudaki, Ioanna; Sardeli, Chrysanthi; Raikos, Nikolaos; Theodoridis, Georgios; Dimitriadis, Georgios

    2017-01-01

    The aim of this pilot study is to evaluate sarcosine, uracil, and kynurenic acid in urine as potential biomarkers in prostate cancer detection and progression monitoring. Sarcosine, uracil, and kynurenic acid were measured in urine samples of 32 prostate cancer patients prior to radical prostatectomy, 101 patients with increased prostate-specific antigen prior to ultrasonographically-guided prostatic biopsy collected before and after prostatic massage, and 15 healthy volunteers (controls). The results were related to histopathologic data, Gleason score, and PSA (Prostate Specific Antigen). Metabolites were measured after analysis of urine samples with Ultra-High Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) instrumentation. Multivariate, nonparametric statistical tests including receiver operating characteristics analyses, one-way analysis of variance (Kruskal–Wallis test), parametric statistical analysis, and Pearson correlation, were performed to evaluate diagnostic performance. Decreased median sarcosine and kynurenic acid and increased uracil concentrations were observed for patients with prostate cancer compared to participants without malignancy. Results showed that there was no correlation between the concentration of the studied metabolites and the cancer grade (Gleason score <7 vs. ≥7) and the age of the patients. Evaluation of biomarkers by ROC (Receiving Operating Characteristics) curve analysis showed that differentiation of prostate cancer patients from participants without malignancy was not enhanced by sarcosine or uracil levels in urine. In contrast to total PSA values, kynurenic acid was found a promising biomarker for the detection of prostate cancer particularly in cases where collection of urine samples was performed after prostatic massage. Sarcosine and uracil in urine samples of patients with prostate cancer were not found as significant biomarkers for the diagnosis of prostate cancer. None of the

  4. Theoretical structural and vibrational study of 5-trifluoromethyluracil. A comparison with uracil

    SciTech Connect

    Rudyk, Roxana; Ramos, María E.; Checa, María A.; Brandán, Silvia A.; Chamorro, Eduardo E.

    2014-10-06

    In the present work, a comparative study on the structural and vibrational properties of the 5-trifluoromethyluracil (TFMU) derivative with those corresponding to uracil in gas and aqueous solution phases was performed combining the available H{sup 1}-NMR, C{sup 13}-NMR, F{sup 19}-NMR and FTIR spectra with Density Functional Theory (DFT) calculations. Three stable conformers were theoretically determined in both media by using the hybrid B3LYP/6-31G* method. The solvent effects were simulated by means of the self-consistent reaction field (SCRF) method employing the integral equation formalism variant (IEFPCM). Complete assignments of the vibrational spectra in both phases were performed combining the internal coordinates analysis and the DFT calculations with the Scaled Quantum Mechanics Force Field (SQMFF) methodology. The atomic charges, bond orders, solvation energies, dipole moments, molecular electrostatic potentials and force constants parameters were calculated for the three conformers of TFMU in gas phase and aqueous solution.

  5. Surface-Enhanced Hyper-Raman Spectra of Adenine, Guanine, Cytosine, Thymine, and Uracil

    PubMed Central

    2016-01-01

    Using picosecond excitation at 1064 nm, surface-enhanced hyper-Raman scattering (SEHRS) spectra of the nucleobases adenine, guanine, cytosine, thymine, and uracil with two different types of silver nanoparticles were obtained. Comparing the SEHRS spectra with SERS data from the identical samples excited at 532 nm and with known infrared spectra, the major bands in the spectra are assigned. Due to the different selection rules for the one- and two-photon excited Raman scattering, we observe strong variation in relative signal strengths of many molecular vibrations obtained in SEHRS and SERS spectra. The two-photon excited spectra of the nucleobases are found to be very sensitive with respect to molecule–nanoparticle interactions. Using both the SEHRS and SERS data, a comprehensive vibrational characterization of the interaction of nucleobases with silver nanostructures can be achieved. PMID:28077982

  6. Ferrocene-linked thymine/uracil conjugates: base pairing directed self-assembly and supramolecular packing.

    PubMed

    Patwa, Amit N; Gupta, Susmita; Gonnade, Rajesh G; Kumar, Vaijayanti A; Bhadbhade, Mohan M; Ganesh, Krishna N

    2008-02-15

    Ferrocene-linked bis(nucleobase) (1a-c) and chimeric nucleobase (1d) conjugates have been synthesized from mono- and bis(hydroxybutyl)ferrocene 6 via Mitsunobu reaction as the key step. X-ray crystallographic studies of ferrocene bis(nucleobase) conjugates reveal two-dimensional supramolecular organizations of backbones through self-assembled Watson-Crick and reverse Watson-Crick type pairs. Ferrocene-bis(thymine) conjugate self-assembles by reverse Watson-Crick pairing, while the corresponding bis(uracil) conjugate self-assembles by alternating WC and reverse WC type pairing. Such continuous assemblies are not seen in monosubstituted ferrocene nucleobase conjugates which form only planar sheets. The results are interesting from the point of understanding and engineering supramolecular assemblies through rational design of base pairing patterns.

  7. Planar versus non-planar: The important role of weak C-H⋯O hydrogen bonds in the crystal structure of 5-methyl-salicyl-aldehyde.

    PubMed

    Baisch, Ulrich; Scicluna, Marie Christine; Näther, Christian; Vella-Zarb, Liana

    2017-02-01

    The crystal structure of 5-methyl-salicyl-aldehyde (5-MSA; systematic name 2-hy-droxy-5-methyl-benzaldehyde), C8H8O2, was discovered to be a textbook example of the drastic structural changes caused by just a few weak C-H⋯O inter-actions due to the additional methyl-ation of the aromatic ring compared to salicyl-aldehyde SA. This weak inter-molecular hydrogen bonding is observed between aromatic or methyl carbon donor atoms and hydroxyl or aldehyde acceptor oxygen atoms with d(D⋯A) = 3.4801 (18) and 3.499 (11) Å. The mol-ecule shows a distorted geometry of the aromatic ring with elongated bonds in the vicinity of substituted aldehyde and hydroxyl carbon atoms. The methyl hydrogen atoms are disordered over two sets of sites with occupancies of 0.69 (2) and 0.31 (2).

  8. Crystal structures of (S)-(+)-5-(3-bromo/chloro-4-isopropoxyphen­yl)-5-methyl­imidazolidine-2,4-dione

    PubMed Central

    Ohba, Shigeru; Koura, Minoru; Sumida, Hisashi; Shibuya, Kimiyuki

    2016-01-01

    In (S)-(+)-5-(3-bromo-4-isopropoxyphen­yl)-5-methyl­imidazolidine-2,4-dione, C13H15BrN2O3, (I), the hydantoin groups are connected via inter­molecular N—H⋯O hydrogen bonds, forming a terraced sheet structure. In the chloro analogue, (S)-(+)-5-(3-chloro-4-isopropoxyphen­yl)-5-methyl­imidazolidine-2,4-dione, C13H15ClN2O3, (II), the inter­molecular N—H⋯O hydrogen-bonding network forms a flat sheet. Comparison of the crystal structures reveals that (II) is more loosely packed than (I). PMID:26958383

  9. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

    PubMed

    Dmitrochenko, A E; Turiianskaia, O M; Gilep, A A; Usanov, S A; Iantsevich, A V

    2014-01-01

    An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

  10. Increased uracil insertion in DNA is cytotoxic and increases the frequency of mutation, double strand break formation and VSG switching in Trypanosoma brucei.

    PubMed

    Castillo-Acosta, Víctor M; Aguilar-Pereyra, Fernando; Bart, Jean-Mathieu; Navarro, Miguel; Ruiz-Pérez, Luis M; Vidal, Antonio E; González-Pacanowska, Dolores

    2012-12-01

    Deoxyuridine 5'-triphosphate pyrophosphatase (dUTPase) and uracil-DNA glycosylase (UNG) are key enzymes involved in the control of the presence of uracil in DNA. While dUTPase prevents uracil misincorporation by removing dUTP from the deoxynucleotide pool, UNG excises uracil from DNA as a first step of the base excision repair pathway (BER). Here, we report that strong down-regulation of dUTPase in UNG-deficient Trypanosoma brucei cells greatly impairs cell viability in both bloodstream and procyclic forms, underscoring the extreme sensitivity of trypanosomes to uracil in DNA. Depletion of dUTPase activity in the absence of UNG provoked cell cycle alterations, massive dUTP misincorporation into DNA and chromosomal fragmentation. Overall, trypanosomatid cells that lack dUTPase and UNG activities exhibited greater proliferation defects and DNA damage than cells deficient in only one of these activities. To determine the mutagenic consequences of uracil in DNA, mutation rates and spectra were analyzed in dUTPase-depleted cells in the presence of UNG activity. These cells displayed a spontaneous mutation rate 9-fold higher than the parental cell line. Base substitutions at A:T base pairs and deletion frequencies were both significantly enhanced which is consistent with the generation of mutagenic AP sites and DNA strand breaks. The increase in strand breaks conveyed a concomitant increase in VSG switching in vitro. The low tolerance of T. brucei to uracil in DNA emphasizes the importance of uracil removal and regulation of intracellular dUTP pool levels in cell viability and genetic stability and suggests potential strategies to compromise parasite survival.

  11. Helix–hairpin–helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues

    PubMed Central

    Schomacher, Lars; Smolorz, Sabine; Ciirdaeva, Elena; Ber, Svetlana; Kramer, Wilfried; Fritz, Hans-Joachim

    2010-01-01

    The mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of Δung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard. PMID:20410075

  12. Microsolvation effect, hydrogen-bonding pattern, and electron affinity of the uracil-water complexes U-(H2O)n (n = 1, 2, 3).

    PubMed

    Bao, Xiaoguang; Sun, Huai; Wong, Ning-Bew; Gu, Jiande

    2006-03-30

    To achieve a systematic understanding of the influence of microsolvation on the electron accepting behaviors of nucleobases, the reliable theoretical method (B3LYP/DZP++) has been applied to a comprehensive conformational investigation on the uracil-water complexes U-(H(2)O)(n) (n = 1, 2, 3) in both neutral and anionic forms. For the neutral complexes, the conformers of hydration on the O2 of uracil are energetically favored. However, hydration on the O4 atom of uracil is more stable for the radical anions. The electron structure analysis for the H-bonding patterns reveal that the CH...OH(2) type H-bond exists only for di- and trihydrated uracil complexes in which a water dimer or trimer is involved. The electron density structure analysis and the atoms-in-molecules (AIM) analysis for U-(H(2)O)(n) suggest a threshold value of the bond critical point (BCP) density to justify the CH...OH(2) type H-bond; that is, CH...OH(2) could be considered to be a H-bond only when its BCP density value is equal to or larger than 0.010 au. The positive adiabatic electron affinity (AEA) and vertical detachment energy (VDE) values for the uracil-water complexes suggest that these hydrated uracil anions are stable. Moreover, the average AEA and VDE of U-(H(2)O)(n) increase as the number of the hydration waters increases.

  13. Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases.

    PubMed

    Handa, P; Acharya, N; Varshney, U

    2001-05-18

    Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.

  14. Visual and light scattering spectrometric method for the detection of melamine using uracil 5'-triphosphate sodium modified gold nanoparticles.

    PubMed

    Liang, Lijiao; Zhen, Shujun; Huang, Chengzhi

    2017-02-15

    A highly selective method was presented for colorimetric determination of melamine using uracil 5'-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

  15. Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene.

    PubMed Central

    Andersen, P S; Frees, D; Fast, R; Mygind, B

    1995-01-01

    Mutants defective in utilization of uracil at low concentrations have been isolated and characterized. The mutations in question (uraA) map close to the upp gene encoding uracil phosphoribosyltransferase. By complementation analysis, a plasmid that complements the uraA mutation has been isolated. The uraA gene was shown to be the second gene in a bicistronic operon with upp as the promoter proximal gene. The nucleotide sequence of the gene was determined, and the gene encodes a hydrophobic membrane protein with a calculated Mr of 45,030. The UraA protein has been identified in sodium dodecyl sulfate-polyacrylamide gels in the membrane fraction of minicells harboring the uraA plasmids. PMID:7721693

  16. Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication.

    PubMed

    Magri, Andrea; Ozerov, Alexander A; Tunitskaya, Vera L; Valuev-Elliston, Vladimir T; Wahid, Ahmed; Pirisi, Mario; Simmonds, Peter; Ivanov, Alexander V; Novikov, Mikhail S; Patel, Arvind H

    2016-07-12

    Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N(3)-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N(1) position.

  17. pH effect on structural character of uracil in aqueous solution studied by single-shot CARS

    NASA Astrophysics Data System (ADS)

    Qin, Wenhong; Zhao, Dong; Guo, Chu

    1991-10-01

    A preliminary study is reported on the structure of uracil, a base residue of nucleic acid, in aqueous solutions of different pH values by coherent anti-Stokes Raman scattering (CARS). The results obtained reveal that Raman lines associated with the stretching and bending vibration modes of the uracil ring around 1000 and 1240 cm -1 are sensitive to the pH of the solutions. All thes findings, however, are difficult to observe by laser resonance Raman spectroscopy, which is used widely in practice at present. In addition, we conclude that CARS measurement seems to be a powerful tool capable of offering structural specificity associated with molecular vibrational frequencies of base residues of nucleic acids and their interactions with the surrounding media.

  18. A DNA machine-based fluorescence amplification strategy for sensitive detection of uracil-DNA glycosylase activity.

    PubMed

    Wu, Yushu; Wang, Lei; Zhu, Jing; Jiang, Wei

    2015-06-15

    Sensitive detection of uracil-DNA glycosylase (UDG) activity is critical for function study of UDG and clinical diagnosis. Here, we developed a novel fluorescent strategy for sensitive detection of UDG activity based on the signal amplification by a label-free and enzyme-free DNA machine. A double-strand DNA (dsDNA) probe P1-P2 with uracil bases and trigger sequence was designed for UDG recognition and signal transduction. Two hairpin probes H1 and H2 which were partially complementary were employed to construct the label-free and enzyme-free DNA machine. Under the action of UDG, uracil bases were removed from the P1-P2 dsDNA probe, and then a strand P2' with abasic sites was released. Subsequently, the liberated P2' activated the DNA machine and generated numerous H1-H2 complexes containing G-quadruplex (G4) structures in the end. Finally, the G4 structures could bind with N-methylmesoporphyrin IX (NMM) to form G4-NMM complexes with the enhanced fluorescence responses. This strategy could detect UDG activity as low as 0.00044 U/mL. In addition, the strategy was also applied for the analysis of UDG activity in HeLa cells lysate with low effect of cellular components. Moreover, this strategy was successfully applied for assaying the inhibition of UDG using uracil glycosylase inhibitor (UGI). This strategy provided a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis.

  19. Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ϕ29 DNA mimic protein p56

    PubMed Central

    Baños-Sanz, José Ignacio; Mojardín, Laura; Sanz-Aparicio, Julia; Lázaro, José M.; Villar, Laurentino; Serrano-Heras, Gemma; González, Beatriz; Salas, Margarita

    2013-01-01

    Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme. PMID:23671337

  20. Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage Φ29 DNA mimic protein p56.

    PubMed

    Baños-Sanz, José Ignacio; Mojardín, Laura; Sanz-Aparicio, Julia; Lázaro, José M; Villar, Laurentino; Serrano-Heras, Gemma; González, Beatriz; Salas, Margarita

    2013-07-01

    Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage 29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.

  1. Extending the molecular size in accurate quantum-chemical calculations: the equilibrium structure and spectroscopic properties of uracil.

    PubMed

    Puzzarini, Cristina; Barone, Vincenzo

    2011-04-21

    The equilibrium structure of uracil has been investigated using both theoretical and experimental data. With respect to the former, quantum-chemical calculations at the coupled-cluster level in conjunction with a triple-zeta basis set have been carried out. Extrapolation to the basis set limit, performed employing the second-order Møller-Plesset perturbation theory, and inclusion of core-correlation and diffuse-function corrections have also been considered. Based on the available rotational constants for various isotopic species together with corresponding computed vibrational corrections, the semi-experimental equilibrium structure of uracil has been determined for the first time. Theoretical and semi-experimental structures have been found in remarkably good agreement, thus pointing out the limitations of previous experimental determinations. Molecular and spectroscopic properties of uracil have then been studied by means of the composite computational approach introduced for the molecular structure evaluation. Among the results achieved, we mention the revision of the dipole moment. On the whole, it has been proved that the computational procedure presented is able to provide parameters with the proper accuracy to support experimental investigations of large molecules of biological interest.

  2. Role of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor regulation in stress-induced pain chronification

    PubMed Central

    Liu, Sufang; Tao, Feng

    2017-01-01

    Persistent postsurgical pain is a serious issue in public health, which has received increased interest in recent years. Previous studies have reported that psychological factors promote the development of chronic postsurgical pain. However, it is unclear how chronification of postsurgical pain occurs. The α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPA) phosphorylation in the central nervous system plays a critical role in synaptic plasticity and contributes to central sensitization and chronic pain development. Here, we discuss the role of AMPA receptor regulation in stress-induced pain chronification after surgery. PMID:28289513

  3. Trading in cooperativity for specificity to maintain uracil-free DNA

    PubMed Central

    Szabó, Judit E.; Takács, Enikő; Merényi, Gábor; Vértessy, Beáta G.; Tóth, Judit

    2016-01-01

    Members of the dUTPase superfamily play an important role in the maintenance of the pyrimidine nucleotide balance and of genome integrity. dCTP deaminases and the bifunctional dCTP deaminase-dUTPases are cooperatively regulated by dTTP. However, the manifestation of allosteric behavior within the same trimeric protein architecture of dUTPases, the third member of the superfamily, has been a question of debate for decades. Therefore, we designed hybrid dUTPase trimers to access conformational states potentially mimicking the ones observed in the cooperative relatives. We studied how the interruption of different steps of the enzyme cycle affects the active site cross talk. We found that subunits work independently in dUTPase. The experimental results combined with a comparative structural analysis of dUTPase superfamily enzymes revealed that subtile structural differences within the allosteric loop and the central channel in these enzymes give rise to their dramatically different cooperative behavior. We demonstrate that the lack of allosteric regulation in dUTPase is related to the functional adaptation to more efficient dUTP hydrolysis which is advantageous in uracil-DNA prevention. PMID:27063406

  4. Interactions of Some Divalent Metal Ions with Thymine and Uracil Thiosemicarbazide Derivatives.

    PubMed

    Hammud, Hassan H; El-Dakdouki, Mohammad H; Sonji, Nada; Sonji, Ghassan; Bouhadir, Kamal H

    2016-05-03

    The study of interactions between metal ions and nucleobases, nucleosides, nucleotides, or nucleic acids has become an active research area in chemical, biological, and therapeutic fields. In this respect, the coordination behavior of nucleobase derivatives to transition metals was studied in order to get a better understanding about DNA-metal interactions in in vitro and in vivo systems. Two nucleobase derivatives, 3-benzoyl-1-[3-(thymine-1-yl)propamido]thiourea and 3-benzoyl-1-[3-(uracil-1-yl)propamido]thiourea, were synthesized and their dissociation constants were determined at different temperatures and 0.3 ionic strength. Potentiometric studies were carried out on the interaction of the derivatives towards some divalent metals in 50% v/v ethanol-water containing 0.3 mol.dm(-3) KCl, at five different temperatures. The formation constants of the metal complexes for both ligands follow the order: Cu(2+) > Ni(2+) > Co(2+) > Zn(2+) > Pb(2+) > Cd(2+) > Mn(2+). The thermodynamic parameters were estimated; the complexation process has been found to be spontaneous, exothermic, and entropically favorable.

  5. Tegafur-uracil (UFT) plus folinic acid in advanced rectal cancer.

    PubMed

    Sanchiz, F; Milla, A

    1994-12-01

    We previously reported positive results to Tegafur-Uracil (UFT) chemotherapy in a group of patients with advanced rectal cancer. We have continued the study and now report the effectiveness of UFT plus folinic acid (FA) in 52 patients with advanced rectal cancer. The therapeutic schedule was UFT, 600 mg/m2/day x 14 days p.o. + FA, 90 mg/m2/day x 14 days p.o. Fifty-two out of a total of 56 patients were evaluated for response and toxicity. A higher incidence of positive responses in patients without previous chemotherapy was appreciated. Twenty-one of the 52 evaluated patients showed a partial response (PR). Responses were strongly correlated with previous chemotherapy (14/20; 70% PR of cases without previous chemotherapy vs 7/32; 22% of cases with previous chemotherapy). All responding patients came forward with a median time to progression of 8.2 months (19.6 months for patients without previous chemotherapy vs 7.7 months for patients with previous chemotherapy, P < 0.01). We concluded that the UFT plus FA could be a treatment of choice for patients with advanced rectal cancer.

  6. Oral tegafur-uracil as metronomic therapy following intravenous FOLFOX for stage III colon cancer

    PubMed Central

    Huang, Wen-Yen; Ho, Ching-Liang; Lee, Chia-Cheng; Hsiao, Cheng-Wen; Wu, Chang-Chieh; Jao, Shu-Wen; Yang, Jen-Fu; Lo, Cheng-Hsiang; Chen, Jia-Hong

    2017-01-01

    The purpose of this study was to estimate the impact of metronomic therapy with oral tegafur-uracil (UFUR) following an intravenous FOLFOX regimen as surgical adjuvant chemotherapy on the overall survival (OS) and disease-free survival (DFS) of stage III colon cancer patients. From the retrospective database of patients who underwent a surgical resection for colorectal cancer at the Tri-Service General Hospital from October 2008 through December 2014, stage III colon carcinomas treated with radical R0 resection were reviewed. One hundred thirty two patients were treated with a FOLFOX regimen (comparison group), and 113 patients were treated with the same regimen followed by additional oral UFUR (UFUR group). The clinical characteristics and mean age of the comparison and UFUR groups were similar. Furthermore, for all study patients, DFS was not significantly different between the two groups. However, 5-year OS rates were 86.8% and 68.5% in the UFUR and comparison groups, respectively (p = 0.0107). Adding UFUR to a FOLFOX regimen was found to significantly improve the OS in patients with stage III colon cancer. UFUR as a maintenance therapy following FOLFOX regimen as an alternative therapeutic option for the treatment of stage III colon cancer patients. PMID:28328969

  7. SHMT1 knockdown induces apoptosis in lung cancer cells by causing uracil misincorporation

    PubMed Central

    Paone, A; Marani, M; Fiascarelli, A; Rinaldo, S; Giardina, G; Contestabile, R; Paiardini, A; Cutruzzolà, F

    2014-01-01

    Reprogramming of cellular metabolism towards de novo serine production fuels the growth of cancer cells, providing essential precursors such as amino acids and nucleotides and controlling the antioxidant and methylation capacities of the cell. The enzyme serine hydroxymethyltransferase (SHMT) has a key role in this metabolic shift, and directs serine carbons to one-carbon units metabolism and thymidilate synthesis. While the mitochondrial isoform of SHMT (SHMT2) has recently been identified as an important player in the control of cell proliferation in several cancer types and as a hot target for anticancer therapies, the role of the cytoplasmic isoform (SHMT1) in cancerogenesis is currently less defined. In this paper we show that SHMT1 is overexpressed in tissue samples from lung cancer patients and lung cancer cell lines, suggesting that, in this widespread type of tumor, SHMT1 plays a relevant role. We show that SHMT1 knockdown in lung cancer cells leads to cell cycle arrest and, more importantly, to p53-dependent apoptosis. Our data demonstrate that the induction of apoptosis does not depend on serine or glycine starvation, but is because of the increased uracil accumulation during DNA replication. PMID:25412303

  8. Cadmium(II) inhibition of human uracil-DNA glycosylase by catalytic water supplantation

    NASA Astrophysics Data System (ADS)

    Gokey, Trevor; Hang, Bo; Guliaev, Anton B.

    2016-12-01

    Toxic metals are known to inhibit DNA repair but the underlying mechanisms of inhibition are still not fully understood. DNA repair enzymes such as human uracil-DNA glycosylase (hUNG) perform the initial step in the base excision repair (BER) pathway. In this work, we showed that cadmium [Cd(II)], a known human carcinogen, inhibited all activity of hUNG at 100 μM. Computational analyses based on 2 μs equilibrium, 1.6 μs steered molecular dynamics (SMD), and QM/MM MD determined that Cd(II) ions entered the enzyme active site and formed close contacts with both D145 and H148, effectively replacing the catalytic water normally found in this position. Geometry refinement by density functional theory (DFT) calculations showed that Cd(II) formed a tetrahedral structure with D145, P146, H148, and one water molecule. This work for the first time reports Cd(II) inhibition of hUNG which was due to replacement of the catalytic water by binding the active site D145 and H148 residues. Comparison of the proposed metal binding site to existing structural data showed that D145:H148 followed a general metal binding motif favored by Cd(II). The identified motif offered structural insights into metal inhibition of other DNA repair enzymes and glycosylases.

  9. Cadmium(II) inhibition of human uracil-DNA glycosylase by catalytic water supplantation

    PubMed Central

    Gokey, Trevor; Hang, Bo; Guliaev, Anton B.

    2016-01-01

    Toxic metals are known to inhibit DNA repair but the underlying mechanisms of inhibition are still not fully understood. DNA repair enzymes such as human uracil-DNA glycosylase (hUNG) perform the initial step in the base excision repair (BER) pathway. In this work, we showed that cadmium [Cd(II)], a known human carcinogen, inhibited all activity of hUNG at 100 μM. Computational analyses based on 2 μs equilibrium, 1.6 μs steered molecular dynamics (SMD), and QM/MM MD determined that Cd(II) ions entered the enzyme active site and formed close contacts with both D145 and H148, effectively replacing the catalytic water normally found in this position. Geometry refinement by density functional theory (DFT) calculations showed that Cd(II) formed a tetrahedral structure with D145, P146, H148, and one water molecule. This work for the first time reports Cd(II) inhibition of hUNG which was due to replacement of the catalytic water by binding the active site D145 and H148 residues. Comparison of the proposed metal binding site to existing structural data showed that D145:H148 followed a general metal binding motif favored by Cd(II). The identified motif offered structural insights into metal inhibition of other DNA repair enzymes and glycosylases. PMID:27974818

  10. Vibrational spectroscopic investigations, molecular dynamic simulations and molecular docking studies of N‧-diphenylmethylidene-5-methyl-1H-pyrazole-3-carbohydrazide

    NASA Astrophysics Data System (ADS)

    Pillai, Renjith Raveendran; Menon, Vidya V.; Mary, Y. Shyma; Armaković, Stevan; Armaković, Sanja J.; Panicker, C. Yohannan

    2017-02-01

    FT-IR and FT-Raman spectra of N‧-diphenylmethylidene-5-methyl-1H-pyrazole-3-carbohydrazide were recorded and analyzed. Due to the industrial and biological importance of pyrazole derivatives, we have carried out an extensive quantum chemical study on N‧-diphenylmethylidene-5-methyl-1H-pyrazole-3-carbohydrazide. The theoretical ground state geometry and electronic structure of the title molecule were optimized by DFT/B3LYP/6-311G++(d,p) method and compared with those of the crystal data. The wave numbers obtained are assigned by potential energy distribution. The ring breathing modes of the benzene rings are assigned theoretically at 1009 cm-1 for the mono substituted phenyl rings. The first order hyperpolarizability is comparable with that of similar derivatives and 16 times that of the standard NLO material urea. Conformational analysis was conducted in order to locate all possible conformations of the title compound, followed by investigation of local reactivity properties by MEP and ALIE surfaces. Natural bond orbital analysis has been carried out to analyse the stability of the molecule arising from hyper-conjugative interactions and charge delocalization. Further, reactive properties via autoxidation and hydrolysis mechanisms have been assessed through calculations of bond dissociation energies and radial distribution functions. Docking results confirmed that the compound was a potential inhibitor of CDK2s and were in agreement with the previous reported studies.

  11. Planar versus non-planar: The important role of weak C—H⋯O hydrogen bonds in the crystal structure of 5-methyl­salicyl­aldehyde

    PubMed Central

    Baisch, Ulrich; Scicluna, Marie Christine; Näther, Christian; Vella-Zarb, Liana

    2017-01-01

    The crystal structure of 5-methyl­salicyl­aldehyde (5-MSA; systematic name 2-hy­droxy-5-methyl­benzaldehyde), C8H8O2, was discovered to be a textbook example of the drastic structural changes caused by just a few weak C—H⋯O inter­actions due to the additional methyl­ation of the aromatic ring compared to salicyl­aldehyde SA. This weak inter­molecular hydrogen bonding is observed between aromatic or methyl carbon donor atoms and hydroxyl or aldehyde acceptor oxygen atoms with d(D⋯A) = 3.4801 (18) and 3.499 (11) Å. The mol­ecule shows a distorted geometry of the aromatic ring with elongated bonds in the vicinity of substituted aldehyde and hydroxyl carbon atoms. The methyl hydrogen atoms are disordered over two sets of sites with occupancies of 0.69 (2) and 0.31 (2). PMID:28217332

  12. Dual targeting of tumor angiogenesis and chemotherapy by endostatin-cytosine deaminase-uracil phosphoribosyltransferase.

    PubMed

    Chen, Chun-Te; Yamaguchi, Hirohito; Lee, Hong-Jen; Du, Yi; Lee, Heng-Huan; Xia, Weiya; Yu, Wen-Hsuan; Hsu, Jennifer L; Yen, Chia-Jui; Sun, Hui-Lung; Wang, Yan; Yeh, Edward T H; Hortobagyi, Gabriel N; Hung, Mien-Chie

    2011-08-01

    Several antiangiogenic drugs targeting VEGF/VEGF receptor (VEGFR) that were approved by the Food and Drug Administration for many cancer types, including colorectal and lung cancer, can effectively reduce tumor growth. However, targeting the VEGF signaling pathway will probably influence the normal function of endothelial cells in maintaining homeostasis and can cause unwanted adverse effects. Indeed, emerging experimental evidence suggests that VEGF-targeting therapy induced less tumor cell-specific cytotoxicity, allowing residual cells to become more resistant and eventually develop a more malignant phenotype. We report an antitumor therapeutic EndoCD fusion protein developed by linking endostatin (Endo) to cytosine deaminase and uracil phosphoribosyltransferase (CD). Specifically, Endo possesses tumor antiangiogenesis activity that targets tumor endothelial cells, followed by CD, which converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the cytotoxic antitumor drug 5-fluorouracil (5-FU) in the local tumor area. Moreover, selective targeting of tumor sites allows an increasing local intratumoral concentration of 5-FU, thus providing high levels of cytotoxic activity. We showed that treatment with EndoCD plus 5-FC, compared with bevacizumab plus 5-FU treatment, significantly increased the 5-FU concentration around tumor sites and suppressed tumor growth and metastasis in human breast and colorectal orthotropic animal models. In addition, in contrast to treatment with bevacizumab/5-FU, EndoCD/5-FC did not induce cardiotoxicity leading to heart failure in mice after long-term treatment. Our results showed that, compared with currently used antiangiogenic drugs, EndoCD possesses potent anticancer activity with virtually no toxic effects and does not increase tumor invasion or metastasis. Together, these findings suggest that EndoCD/5-FC could become an alternative option for future antiangiogenesis therapy.

  13. Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

    PubMed

    Wu, Yushu; Wang, Lei; Jiang, Wei

    2017-03-15

    Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10(-5)U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.

  14. Following Ultrafast Radiationless Relaxation Dynamics With Strong Field Dissociative Ionization: A Comparison Between Adenine, Uracil, and Cytosine

    SciTech Connect

    Kotur, Marija; Weinacht, Thomas C.; Zhou, Congyi; Matsika, Spiridoula

    2011-03-22

    We present the application of ultrafast time- and mass-resolved ion yield laser spectroscopy in conjunction with ab initio electronic structure calculations to track molecular excited-state dynamics. We discuss how molecular fragment ions can be associated with conformations the molecule assumes during its relaxation, and how various features of the pump-probe signal for those fragments can be used to infer details of the excited state dynamics. We present results for radiationless relaxation in DNA and RNA bases adenine, cytosine, and uracil in the gas phase, pumped near a one-photon resonance transition to an excited state, and probed via strong-field near-IR dissociative ionization.

  15. Crystallization and preliminary X-ray diffraction analysis of three recombinant mutants of Vaccinia virus uracil DNA glycosylase.

    PubMed

    Sartmatova, Darika; Nash, Taishayla; Schormann, Norbert; Nuth, Manunya; Ricciardi, Robert; Banerjee, Surajit; Chattopadhyay, Debasish

    2013-03-01

    Amino-acid residues located at a highly flexible area in the uracil DNA glycosylase of Vaccinia virus were mutated. In the crystal structure of wild-type D4 these residues lie at the dimer interface. Specifically, three mutants were generated: (i) residue Arg167 was replaced with an alanine (R167AD4), (ii) residues Glu171, Ser172 and Pro173 were substituted with three glycine residues (3GD4) and (iii) residues Glu171 and Ser172 were deleted (Δ171-172D4). Mutant proteins were expressed, purified and crystallized in order to investigate the effects of these mutations on the structure of the protein.

  16. Double proton transfer mechanism in the adenine-uracil base pair and spontaneous mutation in RNA duplex

    NASA Astrophysics Data System (ADS)

    Cerón-Carrasco, José P.; Requena, Alberto; Perpète, Eric A.; Michaux, Catherine; Jacquemin, Denis

    2009-12-01

    We study the mechanism of double proton transfer (DPT) in the adenine-uracil (AU) base pair, both in gas phase and under the influence of surrounding water molecules. According to our ab initio calculations, no stable proton transfer product exists in gas phase, while in solution, the DPT process may occur only through the catalysis of water molecules. Nevertheless, a thermodynamic analysis confirms that AU does not contribute to spontaneous mutation in RNA duplex, and thus guanine-cytosine (GC) would be the only base pair contributing to spontaneous mutation.

  17. 5-Methylation of cytosine in CG:CG base-pair steps: a physicochemical mechanism for the epigenetic control of DNA nanomechanics.

    PubMed

    Yusufaly, Tahir I; Li, Yun; Olson, Wilma K

    2013-12-27

    van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile "opening" mode and two shear "sliding" and "tearing" modes in the orthogonal plane. The stacking interactions of the methyl groups globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. Additionally, the indirect effects of the methyl groups on possible base-pair steps neighboring CG:CG are observed to be of comparable importance to their direct effects on CG:CG. The results have implications for the epigenetic control of DNA mechanics.

  18. Synthesis and crystal structure of copper complex of 7,16-bis(2-hydroxy-5-methyl-3-nitrobenzyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane

    NASA Astrophysics Data System (ADS)

    Ma, Shu-lan; Zhu, Wen-xiang

    2002-12-01

    A lariat crown ether compound 7,16-bis(2-hydroxy-5-methylbenzyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane has been prepared via one-pot Mannich reaction. A copper(II) complex with the ligand 7,16-bis(2-hydroxy-5-methyl-3-nitrobenzyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane was unexpectedly synthesized and characterized by elemental analysis, IR and UV spectra. The crystal structure of the complex has been determined by X-ray diffraction. Both crystal structure analysis and spectroscopy study indicated that the side-arm phenols of lariat crown ether are nitrated while complexing with Cu(NO 3) 2. Structure shows that the copper(II) ion is coordinated to two nitrogen and four oxygen atoms, two from the crown ether and other two from the deprotonated phenolate groups. The coordination polyhedron is a distorted octahedron.

  19. Crystal structure of (Z)-ethyl 3-[2-(5-methyl-7-nitro-1H-indole-2-carbon-yl)hydrazinyl-idene]butano-ate.

    PubMed

    Errossafi, Amal; El Kihel, Abdellatif; Guesmi, Salaheddine; Saadi, Mohamed; El Ammari, Lahcen

    2015-09-01

    The reaction of 5-methyl-7-nitro-1H-indole-2-carbohydrazide with ethyl aceto-acetate yielded the title mol-ecule, C16H18N4O5, in which the indole ring is almost planar, with the greatest deviation from the mean plane being 0.006 (2) Å. The nine atoms of the indole ring are almost perpendicular to the mean plane through the ethyl acetate group, as indicated by the dihedral angle of 87.02 (4)° between them. In the crystal, centrosymmetric supra-molecular dimers are formed via N-H⋯O hydrogen bonds and eight-membered amide {⋯HNCO}2 synthons. These are consolidated into a three-dimensional architecture by C-H⋯O contacts, and by π-π inter-actions between six-membered rings [inter-centroid distance = 3.499 (2) Å].

  20. 5-Methylation of Cytosine in CG:CG Base-Pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir; Olson, Wilma; Li, Yun

    2014-03-01

    Van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile ``opening'' mode and two shear ``sliding'' and ``tearing'' modes in the orthogonal plane. The stacking interactions of the methyl groups are observed to globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. The results have implications for the epigenetic control of DNA mechanics.

  1. Synthesis, HPLC Enantioresolution, and X-ray Analysis of a New Series of C5-methyl Pyridazines as N-Formyl Peptide Receptor (FPR) Agonists

    PubMed Central

    Cilibrizzi, Agostino; Crocetti, Letizia; Giovannoni, Maria Paola; Graziano, Alessia; Vergelli, Claudia; Bartolucci, Gianluca; Soldani, Giacomo; Quinn, Mark T.; Schepetkin, Igor A.; Faggi, Cristina

    2015-01-01

    The synthesis of three racemates and the corresponding non chiral analogues of a C5-methyl pyridazine series is described here, as well as the isolation of pure enantiomers and their absolute configuration assignment. In order to obtain optically active compounds, direct chromatographic methods of separation by HPLC-UV were investigated using four chiral stationary phases (CSPs: Lux Amylose-2®, Lux Cellulose-1®, Lux Cellulose-2® and Lux Cellulose-3®). The best resolution was achieved using amylose tris(5-chloro-2-methylphenylcarbamate) (Lux Amylose-2®), and single enantiomers were isolated on a semipreparative scale with high enantiomeric excess, suitable for biological assays. The absolute configuration of optically active compounds was unequivocally established by X-ray crystallographic analysis and comparative chiral HPLC-UV profile. All compounds of the series were tested for formyl peptide receptor (FPR) agonist activity, and four were found to be active, with EC50 values in the micromolar range. PMID:23744588

  2. High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.

    PubMed

    Fraga, Mario F; Uriol, Esther; Borja Diego, L; Berdasco, María; Esteller, Manel; Cañal, María Jesús; Rodríguez, Roberto

    2002-06-01

    A new approach to the evaluation of the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides is proposed. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA has been performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation degree and also to be faster than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolyses notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues.

  3. Gas-phase reactions of nopinone, 3-isopropenyl-6-oxo-heptanal, and 5-methyl-5-vinyltetrahydrofuran-2-ol with OH, NO{sub 3}, and ozone

    SciTech Connect

    Calogirou, A.; Jensen, N.R.; Nielsen, C.J.; Kotzias, D.; Hjorth, J.

    1999-02-01

    In the troposphere, {alpha}-pinene, {beta}-pinene, limonene, and linalool are mainly oxidized to pinonaldehyde, nopinone, 3-isopropenyl-6-oxoheptanal (IPOH), and 5-methyl-5-vinyltetrahydrofuran-2-ol (MVT), respectively. The rate constant of the reactions of nopinone, IPOH, and MVT with OH, NO{sub 3}, and O{sub 3} were determined by long path FT-IR spectroscopy, and the oxidation products from the reactions between the OH radical and pinonaldehyde, nopinone, IPOH, and MVT were investigated using GC-MS and HPLC. The reaction rate constants (k) for the reactions have been determined at 740 {+-} 5 Torr and 298 {+-} 5 K, and a number of reaction products were identified. From the results obtained in this investigation and previous studies, it was concluded that a typical atmospheric lifetime with respect to chemical reactions was only a few hours for pinonaldehyde, IPOH, and MVT but was much longer for nopinone with a lifetime of about 10 h.

  4. Cell cycle regulation as a mechanism for functional separation of the apparently redundant uracil DNA glycosylases TDG and UNG2

    PubMed Central

    Hardeland, Ulrike; Kunz, Christophe; Focke, Frauke; Szadkowski, Marta; Schär, Primo

    2007-01-01

    Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin–proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin–proteasome pathway constitutes the underlying regulatory system. PMID:17526518

  5. Exploration of acetanilide derivatives of 1-(ω-phenoxyalkyl)uracils as novel inhibitors of Hepatitis C Virus replication

    PubMed Central

    Magri, Andrea; Ozerov, Alexander A.; Tunitskaya, Vera L.; Valuev-Elliston, Vladimir T.; Wahid, Ahmed; Pirisi, Mario; Simmonds, Peter; Ivanov, Alexander V.; Novikov, Mikhail S.; Patel, Arvind H.

    2016-01-01

    Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC50 values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N3-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N1 position. PMID:27406141

  6. Antihypertensive Effect of a Combination of Uracil and Glycerol Derived from Lactobacillus plantarum Strain TWK10-Fermented Soy Milk.

    PubMed

    Liu, Yi-Yen; Zeng, Shih-Yu; Leu, Yann-Lii; Tsai, Tsung-Yu

    2015-08-26

    We previously demonstrated that angiotensin-converting enzyme (ACE) could be inhibited by soy milk that had been fermented with the Lactobacillus plantarum strain TWK10, suggesting great potential for the development of antihypertensive products. In this work, the bioactive ACE inhibitors in TWK10-fermented soy milk water extracts were isolated, and a combination of uracil and glycerol (CUG) was identified as one of the ACE inhibitors. We then examined the physiological effects of CUG treatment in short-term and long-term studies using spontaneously hypertensive rats (SHRs) as an experimental model. The results revealed that the fermented soy milk extracts and CUG decreased blood pressure by 11.97 ± 3.71 to 19.54 ± 9.54 mmHg, 8 h after oral administration, and exhibited antihypertensive effects in SHRs in a long-term study. In addition, CUG was shown to decrease blood pressure by suppressing either the renin activity or the ACE activity and, thus, decreasing the downstream vasoconstricting peptide angiotensin II and the hormone aldosterone. CUG also promoted nitric oxide production, resulting in vasodilation and further improvement to hypertension. This important finding suggests that TWK10-fermented soy milk and its functional ingredients, uracil and glycerol, exhibit antihypertensive effects via multiple pathways and provide a healthier and more natural antihypertensive functional food.

  7. Molecular recognition and interaction between uracil and urea in solid-state studied by terahertz time-domain spectroscopy.

    PubMed

    Yang, Jingqi; Li, Shaoxian; Zhao, Hongwei; Song, Bo; Zhang, Guoxin; Zhang, Jianbing; Zhu, Yiming; Han, Jiaguang

    2014-11-20

    Using terahertz time-domain spectroscopy characterization, we observe that urea is able to recognize and interact with uracil efficiently even in the solid phase without involving water or solvents. A cocrystal configuration linked by a pair of hydrogen bonds between uracil and urea was formed. The terahertz absorption spectrum of the cocrystal shows a distinct new absorption at 0.8 THz (26.7 cm(-1)), which originates from the intermolecular hydrogen bonding. Both mechanical milling and heating can accelerate the reaction efficiently. Density functional theory was adopted to simulate the vibrational modes of the cocrystal, and the results agree well with the experimental observation. Multiple techniques, including powder X-ray diffraction, scanning electron microscopy, and Fourier transform infrared spectroscopy, were performed to investigate the reaction process, and they presented supportive evidence. This work enables in-depth understanding of recognition and interaction of urea with nucleobases and comprehension of the denaturation related to RNA. We also demonstrate that terahertz spectroscopy is an effective and alternative tool for online measurement and quality control in pharmaceutical and chemical industries.

  8. A DFT analysis of the molecular structure, vibrational spectra and other molecular properties of 5-nitrouracil and comparison with uracil

    NASA Astrophysics Data System (ADS)

    Kattan, D.; Alcolea Palafox, M.; Rathor, S. K.; Rastogi, V. K.

    2016-02-01

    The four unit cells found in the crystals of the biomolecule 5-Nitrouracil were simulated as tetramer forms by density functional calculations. Four tetramer forms were fully optimized. Specific scale factors and scaling equations deduced from uracil molecule were employed in the predicted wavenumbers of 5-nitrouracil. The experimental FT-Raman and FT-IR spectra were recorded in the solid state. Comprehensive interpretation of the experimental FT-IR and FT-Raman spectra of the compound under study in the solid state is based on potential energy distribution. A good reproduction of the experimental wavenumbers is obtained and the % error is very small in the majority of cases. A complete vibrational assignment in the isolated state was also carried out aided by the theoretical harmonic frequency analysis and the results compared with those reported in Ar matrix. The scaled wavenumbers were used in the reassignment of several experimental bands. A comparison between the molecular structure and charge distribution of 5-Nitrouracil with related 5-uracil derivatives was presented. The effect of the nitro substitution in the 5th position of the pyrimidine ring was evaluated.

  9. Adjuvant Oral Uracil-Tegafur with Leucovorin for Colorectal Cancer Liver Metastases: A Randomized Controlled Trial

    PubMed Central

    Takayama, Tadatoshi; Miyagawa, Shinichi; Yamamoto, Junji; Ijichi, Masayoshi; Teruya, Masanori; Yoshimi, Fuyo; Kawasaki, Seiji; Koyama, Hiroto; Oba, Masaru; Takahashi, Michiro; Mizunuma, Nobuyuki; Watanabe, Toshiaki

    2016-01-01

    Background The high recurrence rate after surgery for colorectal cancer liver metastasis (CLM) remains a crucial problem. The aim of this trial was to evaluate the efficacy of adjuvant therapy with uracil-tegafur and leucovorin (UFT/LV). Methods In the multicenter, open-label, phase III trial, patients undergoing curative resection of CLM were randomly assigned in a 1:1 ratio to either the UFT/LV group or surgery alone group. The UFT/LV group orally received 5 cycles of adjuvant UFT/LV (UFT 300mg/m2 and LV 75mg/day for 28 days followed by a 7-day rest per cycle). The primary endpoint was recurrence-free survival (RFS). Secondary endpoints included overall survival (OS). Results Between February 2004 and December 2010, 180 patients (90 in each group) were enrolled into the study. Of these, 3 patients (2 in the UFT/LV group and 1 in the surgery alone group) were excluded from the efficacy analysis. Median follow-up was 4.76 (range, 0.15–9.84) years. The RFS rate at 3 years was higher in the UFT/LV group (38.6%, n = 88) than in the surgery alone group (32.3%, n = 89). The median RFS in the UFT/LV and surgery alone groups were 1.45 years and 0.70 years, respectively. UFT/LV significantly prolonged the RFS compared with surgery alone with the hazard ratio of 0.56 (95% confidence interval, 0.38–0.83; P = 0.003). The hazard ratio for death of the UFT/LV group against the surgery alone group was not significant (0.80; 95% confidence interval, 0.48–1.35; P = 0.409). Conclusion Adjuvant therapy with UFT/LV effectively prolongs RFS after hepatic resection for CLM and can be recommended as an alternative choice. Trial Registration UMIN Clinical Trials Registry C000000013 PMID:27588959

  10. Comparison of the redox chemistry of sulfur- and selenium-containing analogs of uracil.

    PubMed

    Payne, N Connor; Geissler, Andrew; Button, Aileen; Sasuclark, Alexandru R; Schroll, Alayne L; Ruggles, Erik L; Gladyshev, Vadim N; Hondal, Robert J

    2017-03-01

    Selenium is present in proteins in the form of selenocysteine, where this amino acid serves catalytic oxidoreductase functions. The use of selenocysteine in nature is strongly associated with redox catalysis. However, selenium is also found in a 2-selenouridine moiety at the wobble position of tRNA(Glu), tRNA(Gln) and tRNA(Lys). It is thought that the modifications of the wobble position of the tRNA improves the selectivity of the codon-anticodon pair as a result of the physico-chemical changes that result from substitution of sulfur and selenium for oxygen. Both selenocysteine and 2-selenouridine have widespread analogs, cysteine and thiouridine, where sulfur is used instead. To examine the role of selenium in 2-selenouridine, we comparatively analyzed the oxidation reactions of sulfur-containing 2-thiouracil-5-carboxylic acid (s(2)c(5)Ura) and its selenium analog 2-selenouracil-5-carboxylic acid (se(2)c(5)Ura) using (1)H-NMR spectroscopy, (77)Se-NMR spectroscopy, and liquid chromatography-mass spectrometry. Treatment of s(2)c(5)Ura with hydrogen peroxide led to oxidized intermediates, followed by irreversible desulfurization to form uracil-5-carboxylic acid (c(5)Ura). In contrast, se(2)c(5)Ura oxidation resulted in a diselenide intermediate, followed by conversion to the seleninic acid, both of which could be readily reduced by ascorbate and glutathione. Glutathione and ascorbate only minimally prevented desulfurization of s(2)c(5)Ura, whereas very little deselenization of se(2)c(5)Ura occurred in the presence of the same antioxidants. In addition, se(2)c(5)Ura but not s(2)c(5)Ura showed glutathione peroxidase activity, further suggesting that oxidation of se(2)c(5)Ura is readily reversible, while oxidation of s(2)c(5)Ura is not. The results of the study of these model nucleobases suggest that the use of 2-selenouridine is related to resistance to oxidative inactivation that otherwise characterizes 2-thiouridine. As the use of selenocysteine in proteins also

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

    PubMed

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

    1995-09-12

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

  12. Synthesis and Potency of Novel Uracil Nucleotides and Derivatives as P2Y2 and P2Y6 Receptor Agonists

    PubMed Central

    Ko, Hyojin; Carter, Rhonda L.; Cosyn, Liesbet; Petrelli, Riccardo; de Castro, Sonia; Besada, Pedro; Zhou, Yixing; Cappellacci, Loredana; Franchetti, Palmarisa; Grifantini, Mario; Van Calenbergh, Serge; Harden, T. Kendall; Jacobson, Kenneth A.

    2008-01-01

    The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y2, P2Y4, and P2Y6 receptors. The 2-thio modification, found previously to enhance P2Y2 receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y2 receptor, in the form of Up4-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include: 9, α,β-methylene-UDP, a P2Y6 receptor agonist; 30, Up4-phenyl ester and 34, Up4-[1]glucose, selective P2Y2 receptor agonists; 43, the 2-thio analogue of INS37217 (P1-(uridine 5′)-P4- (2′-deoxycytidine 5′) tetraphosphate), a potent and selective P2Y2 receptor agonist. PMID:18514530

  13. Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination

    PubMed Central

    Bregenhorn, Stephanie; Kallenberger, Lia; Artola-Borán, Mariela; Peña-Diaz, Javier; Jiricny, Josef

    2016-01-01

    During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and –deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR. PMID:26743004

  14. Poly[[di-aqua-deca-μ2-cyanido-κ(20) C:N-hexa-cyanido-κ(6) C-bis-(μ2-5-methyl-pyrimidine-κ(2) N:N')bis-(5-methyl-pyrimidine-κN)tricopper(II)ditungstate(V)] dihydrate].

    PubMed

    Tsunobuchi, Yoshihide; Kaneko, Souhei; Nakabayashi, Koji; Ohkoshi, Shin-Ichi

    2014-02-01

    In the title complex, {[Cu3[W(CN)8]2(C5H6N2)4(H2O)2]·2H2O} n , the coordination polyhedron of the eight-coordinated W(V) atom is a bicapped trigonal prism, in which five CN groups are bridged to Cu(II) ions, and the other three CN groups are terminally bound. Two of the Cu(II) ions lie on a centre of inversion and each of the three independent Cu(II) cations is pseudo-octahedrally coordinated. In the crystal structure, cyanido-bridged-Cu-W-Cu layers are linked by pillars involving the third independent Cu(II) ion, generating a three-dimensional network with non-coordinating water mol-ecules and 5-methyl-pyrimidine mol-ecules. O-H⋯O and O-H⋯N hydrogen bonds involve the coordinating and non-coordin-ating water mol-ecules, the CN groups and the 5-methyl-pyrimidine mol-ecules.

  15. FT-IR and Raman spectra, ab initio and density functional computations of the vibrational spectra, molecular geometries and atomic charges of uracil and 5-methyluracil (thymine).

    PubMed

    Singh, J S

    2015-02-25

    FT-IR (400-4000 cm(-1)) and Raman spectra (200-4000 cm(-1)) of uracil and 5-methyluracil (thymine) have been recorded and analyzed. The optimized molecular geometries, atomic polar tensor (APT) charges and vibrational characteristics have been studied theoretically using restricted Hartree-Fock (RHF) and density functional theory (DFT) methods. Using the Becke's exchange in conjunction with Lee-Yang-Parr's correlation functional and Becke's three-parameter hybrid method (B3LYP), the ab initio and DFT calculations were carried out to study the optimized molecular fundamental vibrational frequencies for uracil and 5-methyluracil (thymine) by employing Gaussian-03 program. The fundamental vibrational frequencies along with their corresponding intensities in IR and Raman activities and depolarization ratios of the Raman lines have also been calculated using the RHF and DFT methods employing different basis sets. In quantum chemical calculations, most of the B3LYP/6-311++G(∗∗) vibrational frequencies are in excellent agreement with the available experimental assignments and helped to propose in the reassignments of some missing frequencies in experimental study. Assuming under the Cs point group for both molecules, the distribution of normal mode of vibrations between the two species as planar (a') and non-planar (a″) for all 39 normal vibrational modes of 5-methyluracil are given by 26a'+13a″, of which 30 modes (21a'+9a″) correspond to the uracil moiety and 9 modes (5a'+4a″) to the CH3 group. Consistent assignments have been made for the internal modes of CH3 group, especially for the anti-symmetric CH3 stretching and bending modes. A possible explanation could be the planarity of pyrimidine ring and non-planarity at carbon site of methyl group which might cause the splitting of frequencies including three components due to the substitution of CH3 group at the site of C5 atom on pyrimidine ring of uracil. The three non-equivalent CH bonds of CH3 group are

  16. Crystal structure of (Z)-ethyl 3-[2-(5-methyl-7-nitro-1H-indole-2-carbon­yl)hydrazinyl­idene]butano­ate

    PubMed Central

    Errossafi, Amal; El Kihel, Abdellatif; Guesmi, Salaheddine; Saadi, Mohamed; El Ammari, Lahcen

    2015-01-01

    The reaction of 5-methyl-7-nitro-1H-indole-2-carbohydrazide with ethyl aceto­acetate yielded the title mol­ecule, C16H18N4O5, in which the indole ring is almost planar, with the greatest deviation from the mean plane being 0.006 (2) Å. The nine atoms of the indole ring are almost perpendicular to the mean plane through the ethyl acetate group, as indicated by the dihedral angle of 87.02 (4)° between them. In the crystal, centrosymmetric supra­molecular dimers are formed via N—H⋯O hydrogen bonds and eight-membered amide {⋯HNCO}2 synthons. These are consolidated into a three-dimensional architecture by C—H⋯O contacts, and by π–π inter­actions between six-membered rings [inter-centroid distance = 3.499 (2) Å]. PMID:26396904

  17. Synthesis and properties of oligonucleotides containing 4-thiothymidine, 5-methyl-2-pyrimidinone-1-beta-D(2'-deoxyriboside) and 2-thiothymidine.

    PubMed Central

    Connolly, B A; Newman, P C

    1989-01-01

    Methods are given for the synthesis of derivatives of 4-thiothymidine (4ST), 5-methyl-2-pyrimidinone-1-beta-D(2'-deoxyriboside) (4HT) and 2-thiothymidine (2ST) suitable for incorporation into oligodeoxynucleotides by the cyanoethyl phosphoramidite method. 4HT and 2ST are incorporated with no base protection but the sulphur atom in 4ST is protected with an S-sulphenylmethyl (-SCH3) function. This can be removed with dithiothreitol after synthesis. These T analogues have been incorporated into GACGATATCGTC, a self-complementary dodecamer containing the Eco RV recognition site (underlined), in place of the two T residues within this site. Although pure dodecamers are obtained in each case the syntheses are not as efficient as those seen when normal unmodified bases are used mainly due to the chemical reactivity of 4ST, 4HT and 2ST. Some of the chemical properties of oligonucleotides containing these bases (reactivity towards NH3) as well as their physical properties (melting temperatures, U.V., fluorescence and circular dichroism spectra) have been determined and are discussed. PMID:2762115

  18. Purification and biochemical characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors of pig brain.

    PubMed Central

    Chang, Y C; Wu, T Y; Li, B F; Gao, L H; Liu, C I; Wu, C L

    1996-01-01

    Two preparations of glutamate receptors were purified from the synaptic junctions of pig brain by a combination of detergent solubilization, anion-exchange chromatography, wheat-germ agglutinin affinity chromatography and sedimentation through sucrose gradients. These preparations were enriched in specific L-[3H]glutamate binding activity (> 5000 pmol of glutamate binding sites/mg of protein), and the rank order of ligand affinity for binding to these preparations was: quisqualate > 6-cyano-7- nitroquinoxaline-2,3-dione > alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) > L-glutamate > kainate > > N-methyl-D-aspartate approximately L-2-amino-4-phosphonobutyrate. SDS/PAGE analysis revealed that more than 80% of the protein in either of these preparations appeared as a single protein band of 106 kDa. Two-dimensional gel electrophoresis further revealed that these 106 kDa proteins consisted of a series of acidic proteins which were recognized by antibodies against rat AMPA receptor subunits. These 106 kDa proteins were also recognized by wheatgerm agglutinin and concanavalin A; in addition, peptide N-glycosidase F treatment of these preparations decreased their size to 99 kDa. Our results suggest that the putative glutamate receptors isolated here are likely to belong to the AMPA subtype of glutamate receptors in pig brain. Using the purification procedure reported here, 5 micrograms of AMPA receptor proteins can be isolated from 250 g of pig brain tissue. PMID:8870648

  19. Mercury(II) complexes with 5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione: Synthesis, structural characterization, and theoretical studies

    NASA Astrophysics Data System (ADS)

    Sabounchei, Seyyed Javad; Shahriary, Parisa; Salehzadeh, Sadegh; Gholiee, Yasin; Khavasi, Hamid Reza

    2013-11-01

    New Hg(II) complexes with 5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione (L) and various halogen ions were synthesized. Based on elemental analysis and flame atomic absorption spectroscopy, complexes have general formula HgL2X2 (X = Cl- (1), Br- (2), and I- (3)). These compounds have been studied by IR, 1H and 13C NMR spectroscopy at room temperature. According to X-ray diffraction analysis, complex 2 crystallizes in monoclinic system. Hg(II) ion has been surrounded by a distorted tetrahedral arrangement of two monodentate ligands (each one coordinating by a Npyridine ring atom) and two bromine atoms. Based on crystal packing findings, intermolecular classical H-bonds of the type Nsbnd H⋯O and non-classical H-bonds of the type Csbnd H⋯O and Csbnd H⋯Br, as an important member of noncovalent interaction family, are driving forces for the formation of a very distorted structure. Theoretical studies showed that neither the size of the halide anion nor the intramolecular interactions between two ligands are the reason for the very small Nsbnd Hgsbnd N bond angle, observed in complex 2.

  20. Modulation of DL-. alpha. -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/quisqualate receptors by phospholipase A sub 2 : A necessary step in long-term potentiation

    SciTech Connect

    Massicotte, G.; Baudry, M. ); Vanderklish, P.; Lynch, G. )

    1991-03-01

    The effects of kainate (KA)-induced epileptic seizures on the binding properites of hippocampal glutamate receptors, on the modulation of DL-{alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/quisqualate receptor by phospholipase A{sub 2} (PLA{sub 2}), and on the formation of long-term potentiation (LTP) were studied in hippocampal membranes and hippocampal slices. Systemic administration of KA produced specific changes in the binding properties of the AMPA/quisqualate receptors and its regulation. Whereas the binding of various ligands to the N-methyl-D-aspartate receptors was not modified by KA treatment, there was a singificant decrease in the maximal number of binding sites for ({sup 3}H)AMPA. The loss of LTP was not due to changes in postsynaptic responses elicited by the bursts that trigger the potentiation effect, thus suggesting that KA treatment disrupts processes that follow N-methyl-D-aspartate receptor activation. Systemic administration of KA was associated with calpain activation as the amount of spectrin breakdown products was increased severalfold in hippocampus but not in cerebellum. Pretreatment of telencephalic membranes with calpain greatly reduced the PLA{sub 2}-induced increase in ({sup 3}H)AMPA binding. The results provide evidence in favor of an essential role of PLA{sub 2} in the development of LTP and suggest that the order of activation of different calcium-dependent processes is critical for producing the final changes underlying LTP.

  1. The role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in depression: central mediators of pathophysiology and antidepressant activity?

    PubMed

    Freudenberg, Florian; Celikel, Tansu; Reif, Andreas

    2015-05-01

    Depression is a major psychiatric disorder affecting more than 120 million people worldwide every year. Changes in monoaminergic transmitter release are suggested to take part in the pathophysiology of depression. However, more recent experimental evidence suggests that glutamatergic mechanisms might play a more central role in the development of this disorder. The importance of the glutamatergic system in depression was particularly highlighted by the discovery that N-methyl-D-aspartate (NMDA) receptor antagonists (particularly ketamine) exert relatively long-lasting antidepressant like effects with rapid onset. Importantly, the antidepressant-like effects of NMDA receptor antagonists, but also other antidepressants (both classical and novel), require activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Additionally, expression of AMPA receptors is altered in patients with depression. Moreover, preclinical evidence supports an important involvement of AMPA receptor-dependent signaling and plasticity in the pathophysiology and treatment of depression. Here we summarize work published on the involvement of AMPA receptors in depression and discuss a possible central role for AMPA receptors in the pathophysiology, course and treatment of depression.

  2. The chemokine growth-related gene product β protects rat cerebellar granule cells from apoptotic cell death through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors

    PubMed Central

    Limatola, Cristina; Ciotti, Maria Teresa; Mercanti, Delio; Vacca, Fabrizio; Ragozzino, Davide; Giovannelli, Aldo; Santoni, Angela; Eusebi, Fabrizio; Miledi, Ricardo

    2000-01-01

    Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K+. We have found that the growth-related gene product β (GROβ) partially prevents the K+-depletion-induced cell death, and that the neuroprotective action of GROβ on granule cells is mediated through the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GROβ-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-d-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 μM) treatment. Moreover, GROβ-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GROβ is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors. PMID:10811878

  3. In vitro and in vivo antiherpetic effects of (1R,2R)-1-(5'-methylful-3'-yl)propane-1,2,3-triol.

    PubMed

    Sasaki, Kohei; Hayashi, Kyoko; Matsuya, Yuji; Sugimoto, Kenji; Lee, Jung-Bum; Kurosaki, Fumiya; Hayashi, Toshimitsu

    2016-04-01

    In this study, we demonstrated the in vitro and in vivo antiherpetic activities of a stable furan derivative, (1R,2R)-1-(5'-methylful-3'-yl)propane-1,2,3-triol (MFPT), which had originally been isolated from Streptomyces sp. strain FV60. In the present study, we synthesized MFPT from (5-methylfuran-3-yl)methanol in 6 steps for use in the experiments. MFPT showed potent in vitro antiviral activities against two acyclovir (ACV)-sensitive (KOS and HF) strains and an ACV-resistant (A4-3) strain of herpes simplex virus type 1 (HSV-1) and an ACV-sensitive HSV type 2 (HSV-2) UW 268 strain, their selectivity indices ranging from 310 to 530. By intravaginal application of MFPT to mice, the virus yields decreased dose-dependently against the three strains of HSV-1 and HSV-2. When MFPT was applied at a dose of 1.0 mg/day, the lesion scores, as clinical signs manifested by viral infection, were extensively suppressed in HSV-1-infected mice, whereas the lesion scores in HSV-2-infected mice were not markedly decreased. Interestingly, MFPT exerted an inhibitory effect against ACV-resistant HSV-1 in mice to a similar degree as in ACV-sensitive HSV-1-infected mice. Therefore, the compound might have potential for developing a topical antiviral agent that could be also applied to the infections caused by ACV-resistant viruses.

  4. Monitoring Non-Adiabatic Dynamics of the RNA Base Uracil by UV-Pump-IR-Probe Spectroscopy

    PubMed Central

    Fingerhut, Benjamin P.; Dorfman, Konstantin E.; Mukamel, Shaul

    2013-01-01

    Resolving the excited state dynamics of DNA- and RNA- nucleobases has attracted considerably attention. UV irradiation of the isolated nucleobases leads to the population of an electronic excited state which is quenched by internal conversion mediated by conical intersections on an ultrafast timescale. We present non-adiabatic on-the-fly molecular dynamics simulations of the UV-pump-IR-probe signal of the pyrimidine nucleobase uracil using a novel semiclassical protocol which takes into account the path integral over the excited state vibrational dynamics and properly describes the joint temporal and spectral resolution of the technique. Simulations of vibrational motions of carbonyl fingerprint modes in the electronically excited states reveal clear signatures of different relaxation pathways on a timescale of hundreds of femtoseconds which arise from an ultrafast branching in the excited state. We show that the inherent temporal and spectral resolution of the technique is not purely instrumental but also depends on the vibrational fluctuation timescale. PMID:23914288

  5. Internal conversion and intersystem crossing pathways in UV excited, isolated uracils and their implications in prebiotic chemistry.

    PubMed

    Yu, Hui; Sanchez-Rodriguez, Jose A; Pollum, Marvin; Crespo-Hernández, Carlos E; Mai, Sebastian; Marquetand, Philipp; González, Leticia; Ullrich, Susanne

    2016-07-27

    The photodynamic properties of molecules determine their ability to survive in harsh radiation environments. As such, the photostability of heterocyclic aromatic compounds to electromagnetic radiation is expected to have been one of the selection pressures influencing the prebiotic chemistry on early Earth. In the present study, the gas-phase photodynamics of uracil, 5-methyluracil (thymine) and 2-thiouracil-three heterocyclic compounds thought to be present during this era-are assessed in the context of their recently proposed intersystem crossing pathways that compete with internal conversion to the ground state. Specifically, time-resolved photoelectron spectroscopy measurements evidence femtosecond to picosecond timescales for relaxation of the singlet (1)ππ* and (1)nπ* states as well as for intersystem crossing to the triplet manifold. Trapping in the excited triplet state and intersystem crossing back to the ground state are investigated as potential factors contributing to the susceptibility of these molecules to ultraviolet photodamage.

  6. Imaging [18F]FAU [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl) uracil] in dogs.

    PubMed

    Sun, Haihao; Collins, Jerry M; Mangner, Thomas J; Muzik, Otto; Shields, Anthony F

    2003-01-01

    We have studied the biodistribution of [(18)F]FAU [(1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)uracil], which previous work has shown is incorporated into DNA and functions as an inhibitor of DNA synthesis. It is being tested as a potential antineoplastic agent and imaging agent for PET. We have produced [(18)F]FAU and injected the tracer into 3 normal dogs and imaged them for up to 4 hours and removed tissues along with blood and urine samples for HPLC and activity analysis. The results showed that [(18)F]FAU evenly distributed to most of organs. In sharp contrast to our prior experience with thymidine and its analogs, marrow had less retention of [(18)F]FAU than the non-proliferating tissues.

  7. Automated quantum chemistry based molecular dynamics simulations of electron ionization induced fragmentations of the nucleobases Uracil, Thymine, Cytosine, and Guanine.

    PubMed

    Grimme, Stefan; Bauer, Christopher Alexander

    2015-01-01

    The gas-phase decomposition pathways of electron ionization (EI)-induced radical cations of the nucleobases uracil, thymine, cytosine, and guanine are investigated by means of mixed quantum-classical molecular dynamics. No preconceived fragmentation channels are used in the calculations. The results compare well to a plethora of experimental and theoretical data for these important biomolecules. With our combined stochastic and dynamic approach, one can access in an unbiased way the energetically available decomposition mechanisms. Additionally, we are able to separate the EI mass spectra of different tautomers of cytosine and guanine. Our method (previously termed quantum chemistry electron ionization mass spectra) reproduces free nucleobase experimental mass spectra well and provides detailed mechanistic in-sight into high-energy unimolecular decomposition processes.

  8. Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

    PubMed

    Yousif, Ashraf S; Stanlie, Andre; Begum, Nasim A; Honjo, Tasuku

    2014-10-01

    Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR.

  9. A facile route to 5-methyl-5H-indeno[1,2-c]quinolones via palladium-catalyzed cyclization of 2-alkynylbromobenzenes with N,N-dimethyl-2-alkynylanilines.

    PubMed

    Pan, Xiaolin; Luo, Yong; Kuang, Yunyan; Li, Guangming

    2014-08-21

    A tandem reaction catalyzed by palladium is developed to provide a facile and simple route for the synthesis of 5-methyl-5H-indeno[1,2-c]quinolones, which can introduce diversity and complexity into the products from readily available starting materials. This transformation proceeds well with good functional group tolerance.

  10. Multiple Decay Mechanisms and 2D‐UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine‐Uracil Monophosphate

    PubMed Central

    Li, Quansong; Giussani, Angelo; Segarra‐Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A.; Mukamel, Shaul; Roca‐Sanjuán, Daniel

    2016-01-01

    Abstract The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D‐UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine 1La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine 1Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter‐base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long‐lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the 1Lb, S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D‐UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm−1 in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D‐UV spectroscopy to disentangle the photophysics of multichromophoric systems. PMID:27113273

  11. Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity.

    PubMed

    Fox, Barbara A; Sanders, Kiah L; Rommereim, Leah M; Guevara, Rebekah B; Bzik, David J

    2016-07-01

    Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the

  12. Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity

    PubMed Central

    Fox, Barbara A.; Sanders, Kiah L.; Rommereim, Leah M.; Bzik, David J.

    2016-01-01

    Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the

  13. Reactions of 5-methylcytosine cation radicals in DNA and model systems: thermal deprotonation from the 5-methyl group vs. excited state deprotonation from sugar

    PubMed Central

    Adhikary, Amitava; Kumar, Anil; Palmer, Brian J.; Todd, Andrew D.; Heizer, Alicia N.; Sevilla, Michael D.

    2014-01-01

    Purpose To study the formation and subsequent reactions of the 5-methyl-2′-deoxycytidine cation radical (5-Me-2′-dC•+) in nucleosides and DNA-oligomers and compare to one electron oxidized thymidine. Materials and methods Employing electron spin resonance (ESR), cation radical formation and its reactions were investigated in 5-Me-2′-dC, thymidine (Thd) and their derivatives, in fully double stranded (ds) d[GC*GC*GC*GC*]2 and in the 5-Me-C/A mismatched, d[GGAC*AAGC:CCTAATCG], where C* = 5-Me-C. Results We report 5-Me-2′-dC•+ production by one-electron oxidation of 5-Me-2′-dC by Cl2•− via annealing in the dark at 155 K. Progressive annealing of 5-Me-2′-dC•+ at 155 K produces the allylic radical (C-CH2•). However, photoexcitation of 5-Me-2′-dC•+ by 405 nm laser or by photoflood lamp leads to only C3′• formation. Photoexcitation of N3-deprotonated thyminyl radical in Thd and its 5′-nucleotides leads to C3′• formation but not in 3′-TMP which resulted in the allylic radical (U-CH2•) and C5′• production. For excited 5-Me-2′,3′-ddC•+, absence of the 3′-OH group does not prevent C3′• formation. For d[GC*GC*GC*GC*]2 and d[GGAC*AAGC:CCTAATCG], intra-base paired proton transferred form of G cation radical (G(N1-H)•:C(+H+)) is found with no observable 5-Me-2′-dC•+ formation. Photoexcitation of (G(N1-H)•:C(+H+)) in d[GC*GC*GC*GC*]2 produced only C1′• and not the expected photoproducts from 5-Me-2′-dC•+. However, photoexcitation of (G(N1-H)•:C(+H+)) in d[GGAC*AAGC:CCTAATCG] led to C5′• and C1′• formation. Conclusions C-CH2• formation from 5-Me-2′-dC•+ occurs via ground state deprotonation from C5-methyl group on the base. In the excited 5-Me-2′-dC•+ and 5-Me-2′,3′-ddC•+, spin and charge localization at C3′ followed by deprotonation leads to C3′• formation. Thus, deprotonation from C3′ in the excited cation radical is kinetically controlled and sugar C-H bond energies are

  14. Synthesis, structural characterization and theoretical approach of 3-(2,6-dichlorobenzyl)-5-methyl-N-nitro-1,3,5-oxadiazinan-4-imine.

    PubMed

    Ni, Haiwei; Zhang, Yu; Zhang, Fang; Zhao, Jianying; Wu, Liubi; Chu, Xiaozhong

    2015-03-05

    3-(2,6-Dichlorobenzyl)-5-methyl-N-nitro-1,3,5-oxadiazinan-4-imine (DNOI) was synthesized and characterized by X-ray diffraction, FT-IR, FT-Raman and UV-Vis spectra. The X-ray diffraction study showed that DNOI has a one dimensional configuration, due to the intermolecular C9H⋯O1 and N4H⋯O2 hydrogen bonds. The benzene ring and the oxadiazine rings are tilted with respect to each other by 63.07° (C3N1C5C6). Vibrational spectra and electronic spectra measurements were made for the compound. Optimized geometrical structure and harmonic vibrational frequencies were computed with DFT (B3LYP, B3P86, and M062X) methods using 6-311++G(d,p) basis set. Assignments of the observed spectra were proposed. The equilibrium geometries computed by all of the methods were compared with X-ray diffraction results. The absorption spectra of the title compound were computed both in gas phase and in CH3OH solution using TD-B3LYP/6-311++G(d,p) and PCM-B3LYP/6-311++G(d,p) approaches, respectively. The calculated results provide a good description of positions of the bands maxima in the observed electronic spectrum. Temperature dependence of thermodynamic parameters in the range of 100-1000K were determined, entropy, heat capacity and enthalpy changes were increasing with temperature increasing, while for Gibbs free energy is decreasing with temperature increasing. The bond orbital occupancies, contribution from parent natural bond orbital (NBO), the natural atomic hybrids was calculated and discussed.

  15. Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

    PubMed Central

    Optiz, Thoralf; Grooms, Sonja Y.; Bennett, Michael V. L.; Zukin, R. Suzanne

    2000-01-01

    Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca2+ influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca2+-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons. PMID:11087875

  16. A novel diterpene skeleton: identification of a highly aromatic, cytotoxic and antioxidant 5-methyl-10-demethyl-abietane-type diterpene from Premna serratifolia.

    PubMed

    Habtemariam, Solomon; Varghese, George K

    2015-01-01

    Premna serratifolia Linn. (syn: . P. corymbosa (Burm. f.) Merr., P. integrifolia L. and P. obtusifolia R. Br.) is a member of the Verbenaceae family that is extensively used in the Ayurvedic system of medicine in India. As part of our continuous pharmacological and phytochemical studies on medicinal plants, we have screened the methanolic extracts of leaves, root bark (RB) and root wood of P. serratifolia for cytotoxic activity against two cancer cell lines: SHSY-5Y neuroblastoma and B16 melanoma cells. The RB extract that showed promising activity was fractionated using solvents of increasing polarity followed by a combination of Sephadex LH-20 column and Combiflash chromatography as well as HPLC to afford the active principle. Comprehensive spectroscopic analysis including 1D and 2D NMR (COSY, HMQC, HMBC, NOESY) and MS analysis revealed the identity of the isolated compound as 11,12,16-trihydroxy-2-oxo-5-methyl-10-demethyl-abieta-1[10],6,8,11,13-pentene that appears to be a novel compound based on a new diterpene skeleton. The cytotoxic activity of the isolated compound was 21 and 23 times higher than the crude extract against the SHSY-5Y and B16 cells, respectively. The novel compound also possesses in vitro antioxidant effects as evidenced by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging effect where an IC50 value of 20.4 ± 1.3 μM was obtained. In comparison, the positive control, caffeic acid, showed an IC50 value of 14.4 ± 1.6 μM.

  17. Anxiolytic- and antidepressant-like effects of the methadone metabolite 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP).

    PubMed

    Forcelli, Patrick A; Turner, Jill R; Lee, Bridgin G; Olson, Thao T; Xie, Teresa; Xiao, Yingxian; Blendy, Julie A; Kellar, Kenneth J

    2016-02-01

    The enhancement of GABAergic and monoaminergic neurotransmission has been the mainstay of pharmacotherapy and the focus of drug-discovery for anxiety and depressive disorders for several decades. However, the significant limitations of drugs used for these disorders underscores the need for novel therapeutic targets. Neuronal nicotinic acetylcholine receptors (nAChRs) may represent one such target. For example, mecamylamine, a non-competitive antagonist of nAChRs, displays positive effects in preclinical tests for anxiolytic and antidepressant activity in rodents. In addition, nicotine elicits similar effects in rodent models, possibly by receptor desensitization. Previous studies (Xiao et al., 2001) have identified two metabolites of methadone, EMDP (2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline) and EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine), which are considered to be inactive at opiate receptors, as relatively potent noncompetitive channel blockers of rat α3β4 nAChRs. Here, we show that these compounds are likewise highly effective blockers of human α3β4 and α4β2 nAChRs. Moreover, we show that they display relatively low affinity for opiate binding sites labeled by [(3)H]-naloxone. We then evaluated these compounds in rats and mice in preclinical behavioral models predictive of potential anxiolytic and antidepressant efficacy. We found that EMDP, but not EDDP, displayed robust effects predictive of anxiolytic and antidepressant efficacy without significant effects on locomotor activity. Moreover, EMDP at behaviorally active doses, unlike mecamylamine, did not produce eyelid ptosis, suggesting it may produce fewer autonomic side effects than mecamylamine. Thus, the methadone metabolite EMDP may represent a novel therapeutic avenue for the treatment of some affective disorders.

  18. Anxiolytic- and Antidepressant-like Effects of the Methadone Metabolite 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP)

    PubMed Central

    Lee, Bridgin G.; Olson, Thao T.; Xie, Teresa; Xiao, Yingxian; Blendy, Julie A.; Kellar, Kenneth J.

    2015-01-01

    The enhancement of GABAergic and monoaminergic neurotransmission has been the mainstay of pharmacotherapy and the focus of drug-discovery for anxiety and depressive disorders for several decades. However, the significant limitations of drugs used for these disorders underscores the need for novel therapeutic targets. Neuronal nicotinic acetylcholine receptors (nAChRs) may represent one such target. For example, mecamylamine, a non-competitive antagonist of nAChRs, displays positive effects in preclinical tests for anxiolytic and antidepressant activity in rodents. In addition, nicotine elicits similar effects in rodent models, possibly by receptor desensitization. Previous studies (Xiao et al., 2001) have identified two metabolites of methadone, EMDP (2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline) and EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine), which are considered to be inactive at opiate receptors, as relatively potent noncompetitive channel blockers of rat α3β4 nAChRs. Here, we show that these compounds are likewise highly effective blockers of human α3β4 and α4β2 nAChRs. Moreover, we show that they display relatively low affinity for opiate binding sites labeled by [3H]-naloxone. We then evaluated these compounds in rats and mice in preclinical behavioral models predictive of potential anxiolytic and antidepressant efficacy. We found that EMDP, but not EDDP, displayed robust effects predictive of anxiolytic and antidepressant efficacy without significant effects on locomotor activity. Moreover, EMDP at behaviorally active doses, unlike mecamylamine, did not produce eyelid ptosis, suggesting it may produce fewer autonomic side effects than mecamylamine. Thus, the methadone metabolite EMDP may represent a novel therapeutic avenue for the treatment of some affective disorders. PMID:26365569

  19. Studies of the environmental fate and effect of aircraft deicing fluids: Detection of 5-methyl-1H-benzotriazole in the fathead minnow (Pimephales promelas)

    USGS Publications Warehouse

    Cancilla, Devon A.; Baird, J.C.; Geis, S.W.; Corsi, Steven R.

    2003-01-01

    This paper presents the results of a number of field and laboratory studies to evaluate the environmental impact of aircraft deicing and anti-icing fluids (ADAFs) on aquatic systems. Both 5-methyl-1H-benzotriazole (5-MeBt) and 4-methyl-1H-benzotriazole (4-MeBt), known additives to ADAFs, were found in whole-tissue extracts from minnows placed downstream of an effluent outfall that receives ADAF contaminated runoff from General Mitchell International Airport (Milwaukee, WI, USA). Neither of these compounds was detected in tissues from minnows placed upstream from the airport. A toxicity assessment of water collected during the minnow exposure studies utilizing Hyalella azteca, Pimephales promelas, and Ceriodaphnia dubia showed greater toxicity in a secondary airport outfall containing ADAFs when compared to upstream non-ADAF-contaminated samples. In two 28-d static renewal tests using 5-MeBt laboratory-fortified waters, 5-MeBt was detected in whole-tissue extracts of minnows at all concentrations tested. In studies using laboratory water fortified with 5-MeBt, the median lethal concentration (LC50) of 5-MeBt for P. promelas was found to be 22.0 mg/L. The LC50 for C. dubia to 5-MeBt laboratory-fortified water was found to be 81.3 mg/L. The 25% inhibition concentration (IC25) of 5-MeBt for the green alga Selenastrum capricornutum was 23.2 mg/L, and the average median effective concentration (EC50) for Microtox was 4.25 mg/L. The results of these field and lab studies indicate that additives, other than glycols, used in aircraft deicing fluids can be found in aquatic systems and may be of greater risk than previously believed.

  20. Study of dynamics and crystallization kinetics of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile at ambient and elevated pressure

    NASA Astrophysics Data System (ADS)

    Adrjanowicz, K.; Kaminski, K.; Paluch, M.; Ngai, K. L.; Yu, Lian

    2012-06-01

    The organic liquid ROY, i.e., 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile, has been a subject of detailed study in the last few years. One interest in ROY lies in its polymorph-dependent fast crystal growth mode below and above the glass transition temperature. This growth mode is not diffusion controlled, and the possibility that it is enabled by secondary relaxation had been suggested. However, a previous study by dielectric relaxation spectroscopy had not been able to find any resolved secondary relaxation. The present paper reports new dielectric measurements of ROY in the liquid and glassy states at ambient pressure and elevated pressure, which were performed to provide more insight into the molecular dynamics as well as the crystallization tendency of ROY. In the search of secondary relaxation, a special glassy state of ROY was prepared by applying high pressure to the liquid state, from which secondary relaxation was possibly resolved. Thus, the role of secondary relaxation in crystallization of ROY remains to be clarified. Notwithstanding, the secondary relaxation present is not necessarily the sole enabler of crystallization. In an effort to search for possible cause of crystallization other than secondary relaxation, we also performed crystallization kinetics studies of ROY at different T and P combinations while keeping the structural relaxation time constant. The results show that crystallization of ROY speeds up with pressure, opposite to the trend found in the crystallization of ibuprofen studied up to 1 GPa. The dielectric relaxation and thermodynamic properties of ROY with phenolphthalein dimethylether (PDE) are similar in many respects, but PDE does not crystallize. Taking all the above into account, besides the secondary relaxation, the specific chemical structure, molecular interactions and packing of the molecules are additional factors that could affect the kinetics of crystallization found in ROY.

  1. Positive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators have different impact on synaptic transmission in the thalamus and hippocampus.

    PubMed

    Xia, Yan-Fang; Kessler, Markus; Arai, Amy C

    2005-04-01

    Earlier studies showed that positive modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors enhance synaptic responses and facilitate synaptic plasticity. Those studies focused mainly on hippocampal functions. However, AMPA receptors have regionally distinct subunit compositions and thus potencies and efficacies of modulators may vary across the brain. The present study compared the effects of CX546 [1-(1,4-benzodioxan-6-ylcarbonyl) piperidine], a benzamide-type modulator, on synaptic transmission in neurons of the reticular thalamic nucleus (RTN), which regulates the firing mode of relay cells in other thalamic nuclei, and on hippocampal CA1 pyramidal cells. CX546 greatly prolonged synaptic responses in CA1 pyramidal cells, but at the same concentration it had only weak modulatory effects in RTN neurons. Effects on miniature excitatory postsynaptic currents (EPSCs) were similar to those on EPSCs in both regions, suggesting that variations in neuronal morphology and transmitter release kinetics do not account for the differences. Relay cells in the ventrobasal thalamus also exhibited weak modulatory effects that were comparable with those in RTN neurons. Regionally different effects on response duration were also observed with CX516 [BDP-12, 1-(quinoxalin-6-ylcarbonyl)piperidine], a second benzamide drug. In contrast, 100 microM cyclothiazide produced comparable synaptic enhancements in hippocampus and RTN. The regional selectivity of benzamide drugs (ampakines) may be explained, at least in part, by a lower potency at thalamic AMPA receptors, perhaps due to the prevalence of the subunits GluR3 and 4. Although regional preferences of the ampakines were modest in their extent, they may be sufficient to be of relevance when considering future therapeutic applications of such compounds.

  2. Glutamate regulates intracellular calcium and gene expression in oligodendrocyte progenitors through the activation of DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

    PubMed Central

    Pende, M; Holtzclaw, L A; Curtis, J L; Russell, J T; Gallo, V

    1994-01-01

    Oligodendrocytes and their progenitors (O-2A) express functional kainate- and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring glutamate receptors. The physiological consequences of activation of these receptors were studied in purified rat cortical O-2A progenitors and in the primary oligodendrocyte cell line CG-4. Changes in the mRNA levels of a set of immediate early genes were studied and were correlated to intracellular Ca2+ concentration, as measured by fura-2 Ca2+ imaging. Both in CG-4 and in cortical O-2A progenitors, basal mRNA levels of NGFI-A were much higher than c-fos, c-jun, or jun-b. Glutamate, kainate, and AMPA greatly increased NGFI-A mRNA and protein by activation of membrane receptors in a Ca(2+)-dependent fashion. Agonists at non-N-methyl-D-aspartate receptors promoted transmembrane Ca2+ influx through voltage-dependent channels as well as kainate and/or AMPA channels. The influx of Ca2+ ions occurring through glutamate-gated channels was sufficient by itself to increase the expression of NGFI-A mRNA. AMPA receptors were found to be directly involved in intracellular Ca2+ and NGFI-A mRNA regulation, because the effects of kainate were greatly enhanced by cyclothiazide, an allosteric modulator that selectively suppresses desensitization of AMPA but not kainate receptors. Our results indicate that glutamate acting at AMPA receptors regulates immediate early gene expression in cells of the oligodendrocyte lineage by increasing intracellular calcium. Consequently, modulation of these receptor channels may have immediate effects at the genomic level and regulate oligodendrocyte development at critical stages. Images PMID:8159727

  3. Generation of AcGFP fusion with single-chain Fv selected from a phage display library constructed from mice hyperimmunized against 5-methyl 2'-deoxycytidine.

    PubMed

    Ohshima, Motohiro; Inoue, Kazuyuki; Hayashi, Hideki; Tsuji, Daiki; Mizugaki, Michinao; Itoh, Kunihiko

    2010-11-01

    DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

  4. Inroads into base excision repair II. The discovery of DNA glycosylases. "An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues," Proc. Nat. Acad. Sci. USA, 1974.

    PubMed

    Friedberg, Errol C; Lindahl, Tomas

    2004-11-02

    The discovery of a DNA glycosylase that specifically removes uracil from DNA, opened the door for uncovering a large class of such enzymes that are fundamental to the process of base excision repair of DNA.

  5. Comment on ‘To stack or not to stack: Performance of a new density functional for the uracil and thymine dimers’ [Chem. Phys. Lett. 459 (2008) 164

    NASA Astrophysics Data System (ADS)

    van Mourik, Tanja

    2009-04-01

    A Letter by Gu et al. [J. Gu, J. Wang, J. Leszczynski, Y. Xie, H.F. Schaefer III, Chem. Phys. Lett. 459 (2008) 164] presented MP2/6-31+G(d) and MP2/TZVPP stacking energies for the uracil and thymine dimers, with the aim to assess the performance of the new M06-2X density functional. However, the stacking energies were not corrected for the basis set superposition error (BSSE). Here we show that correction for this error dramatically changes the results. BSSE correction severely reduces the stacking energy of the thymine dimer, whereas the stacked uracil dimer structure considered by Gu et al. is not even a minimum on the MP2/6-31+G(d) potential energy surface.

  6. Visual and light scattering spectrometric method for the detection of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Liang, Lijiao; Zhen, Shujun; Huang, Chengzhi

    2017-02-01

    A highly selective method was presented for colorimetric determination of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

  7. [125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

    PubMed

    Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

    2002-07-01

    The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway

  8. Identification of ozonation by-products of 4- and 5-methyl-1H-benzotriazole during the treatment of surface water to drinking water.

    PubMed

    Müller, Alexander; Weiss, Stefan C; Beisswenger, Judith; Leukhardt, H Georg; Schulz, Wolfgang; Seitz, Wolfram; Ruck, Wolfgang K L; Weber, Walter H

    2012-03-01

    During the treatment of surface water to drinking water, ozonation is often used for disinfection and to remove organic trace substances, whereby oxidation by-products can be formed. Here we use the example of tolyltriazole to describe an approach for identifying relevant oxidation by-products in the laboratory and subsequently detecting them in an industrial-scale process. The identification process involves ozonation experiments with pure substances at laboratory level (concentration range mg L(-1)). The reaction solutions from different ozone contact times were analyzed by high performance liquid chromatography - quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) in full scan mode. Various approaches were used to detect the oxidation by-products: (i) target searches of postulated oxidation by-products, (ii) comparisons of chromatograms (e.g., UV/VIS) of the different samples, and (iii) color-coded abundance time courses (kinetic) of all detected compounds were illustrated in a kind of a heat map. MS/MS, H/D exchange, and derivatization experiments were used for structure elucidation for the detected by-product. Due to the low contaminant concentrations (ng L(-1)-range) of contaminants in the untreated water, the conversion of results from laboratory experiments to an industrial-scale required the use of HPLC-MS/MS with sample enrichment (e.g., solid phase extraction.) In cases where reference substances were not available or oxidation by-products without clear structures were detected, reaction solutions from laboratory experiments were used to optimize the analytical method to detect ng L(-1) in the samples of the industrial processes. We exemplarily demonstrated the effectiveness of the methodology with the industrial chemicals 4- and 5-methyl-1H-benzotriazole (4- and 5-MBT) as an example. Moreover, not only did we identify several oxidation by-products in the laboratory experiments tentatively, but also detected three of the eleven reaction

  9. Substituent effects on the regioselectivity of the Paternò-Büchi reaction of 5- or/and 6-methyl substituted uracils with 4,4'-disubstituted benzophenones.

    PubMed

    Kong, Feng-Feng; Zhai, Bao-Chang; Song, Qin-Hua

    2008-11-01

    The [2 + 2] photochemical cycloadditions (the Paternò-Büchi reaction) of 5- or/and 6-methyl substituted uracil derivatives with 4,4'-disubstituted benzophenones generate two series of regioisomeric oxetanes, 3 and 4. The regioselectivity (3/4) and the photochemical efficiency are strongly dependent on methyl substituent(s) at the C5-C6 double bond of the uracils. The more the methyl groups at the C5-C6 double bond, the higher the efficiency. The regioselectivity (3/4) ranges from ca. 20 : 80 for 1,3,6-trimethyluracil (1C) to ca. 80 : 20 for 1,3,6-trimethylthymine (1D). The substituent effects would derive from the hyperconjugation of the methyl group(s) at C5 or/and C6 of the uracils, which influences the stability of intermediary triplet 1,4-biradicals and the nucleophilicity of C5 and C6 sites of the double bond. Computational studies reveal that potential energies of triplet 1,4-biradicals forming oxetanes 3, BR5, are 1-4 kcal mol(-1) higher than those of 1,4-biradicals forming oxetanes 4, BR6, and electron densities at C5 are 0.1-0.3 unit higher than those at C6, of the double bond of the uracils, i.e. BR5 are less stable than BR6, and BR5 form more easily than BR6. Temperature effects on the regioselectivity of the Paternò-Büchi reaction of 1D with three benzophenones (2a-c) reveal that efficiencies of oxetanes 3 are higher at a lower reaction temperature, and efficiencies of 4 are higher at a higher temperature. Therefore, triplet biradicals BR5 and BR6 would be regarded as intermediates of kinetic control and thermodynamic control, respectively.

  10. VUV and mid-UV photoabsorption cross sections of thin films of guanine and uracil: application on their photochemistry in the solar system.

    PubMed

    Saïagh, Kafila; Cottin, Hervé; Aleian, Aicha; Fray, Nicolas

    2015-04-01

    We present a photostability study of two nucleobases, guanine and uracil. For the first time, the photoabsorption cross-section spectra of these molecules in the solid phase were measured in the VUV and mid-UV domain (115≤λ≤300 nm). They show a quite similar absorption level throughout this wavelength range, highlighting the importance of considering the whole VUV and UV domain during photolysis experiments in the laboratory. Their photolysis constant (J) can be estimated from those measurements as follows: 2.2×10(-2) s(-1)±11% for guanine and 5.3×10(-2) s(-1)±14% for uracil. This work shows that (i) measuring kinetic constants from a direct and "traditional" photolysis of a thin sample in the laboratory suffers strong limitations and (ii) achieving this measurement requires comprehensive modeling of the radiative transfer that occurs in any sample not optically thin (i.e.,≤2 nm). Moreover, this work has provided other data of interest: the refractive index of solid guanine and of uracil at 650 nm are 1.52 (±0.01) and 1.39 (±0.02), respectively, and the integrated IR band strengths (A) of solid guanine between 3700 and 2120 cm(-1) (3.4×10(-16) cm·molecule(-1)±13%) and of solid uracil between 3400 and 1890 cm(-1) (2.1×10(-16) cm·molecule(-1)±21%).

  11. Synthesis of 5-[3-(2-aminopyrimidin-4-yl)aminopropyn-1-yl]uracil derivative that recognizes Ade-Thy base pairs in double-stranded DNA.

    PubMed

    Ito, Yu; Masaki, Yoshiaki; Kanamori, Takashi; Ohkubo, Akihiro; Seio, Kohji; Sekine, Mitsuo

    2016-01-01

    5-[3-(2-Aminopyrimidin-4-yl)aminopropyn-1-yl]uracil (Ura(Pyr)) was designed as a new nucleobase to recognize Ade-Thy base pair in double-stranded DNA. We successfully synthesized the dexoynucleoside phosphoramidite having Ura(Pyr) and incorporated it into triplex forming oligonucleotides (TFOs). Melting temperature analysis revealed that introduction of Ura(Pyr) into TFOs could effectively stabilize their triplex structures without loss of base recognition capabilities.

  12. In silico studies to explore the mutagenic ability of 5-halo/oxy/li-oxy-uracil bases with guanine of DNA base pairs.

    PubMed

    Jana, Kalyanashis; Ganguly, Bishwajit

    2014-10-16

    DNA nucleobases are reactive in nature and undergo modifications by deamination, oxidation, alkylation, or hydrolysis processes. Many such modified bases are susceptible to mutagenesis when formed in cellular DNA. The mutagenesis can occur by mispairing with DNA nucleobases by a DNA polymerase during replication. We have performed a study of mispairing of DNA bases with unnatural bases computationally. 5-Halo uracils have been studied as mispairs in mutagenesis; however, the reports on their different forms are scarce in the literature. The stability of mispairs with keto form, enol form, and ionized form of 5-halo-uracil has been computed with the M06-2X/6-31+G** level of theory. The enol form of 5-halo-uracil showed remarkable stability toward DNA mispair compared to the corresponding keto and ionized forms. (F)U-G mispair showed the highest stability in the series and (Halo)(U(enol/ionized)-G mispair interactions energies are more stable than the natural G-C basepair of DNA. To enhance the stability of DNA mispairs, we have introduced the hydroxyl group in the place of halogen atoms, which provides additional hydrogen-bonding interactions in the system while forming the 5-membered ring. The study has been further extended with lithiated 5-hydroxymethyl-uracil to stabilize the DNA mispair. (CH2OLi)U(ionized)-G mispair has shown the highest stability (ΔG = -32.4 kcal/mol) with multi O-Li interactions. AIM (atoms in molecules) and EDA (energy decomposition analysis) analysis has been performed to examine the nature of noncovalent interactions in such mispairs. EDA analysis has shown that electrostatic energy mainly contributes toward the interaction energy of mispairs. The higher stability achieved in these studied mispairs can play a pivotal role in the mutagenesis and can help to attain the mutation for many desired biological processes.

  13. Influence of methylation on the properties of uracil and its noncovalent interactions with alkali metal ions: Threshold collision-induced dissociation and theoretical studies

    NASA Astrophysics Data System (ADS)

    Yang, Zhibo; Rodgers, M. T.

    2005-03-01

    The influence of methylation on the properties of uracil and its noncovalent interactions with alkali metal ions is investigated both experimentally and theoretically. Threshold collision-induced dissociation (CID) of M+(xMeU) with Xe is studied in a guided ion beam mass spectrometer. M+ include the following alkali metal ions: Li+, Na+, and K+. Five methylated uracils are examined, xMeU = 1-methyluracil, 3-methyluracil, 6-methyluracil, 1,3-dimethyluracil, and 5,6-dimethyluracil. In all cases endothermic loss of the intact nucleobase is the dominant reaction pathway, while ligand exchange to produce MXe+ is observed as a minor reaction pathway. The threshold regions of the cross sections are interpreted to extract 0 and 298 K bond dissociation energies (BDEs) for M+xMeU after accounting for the effects of multiple ion-neutral collisions, kinetic and internal energies of the reactants, and dissociation lifetimes. Ab initio calculations at the MP2(full)/6-31G* level of theory are used to determine the structures of these complexes and provide molecular constants required for the thermochemical analysis of the experimental data. Theoretical bond dissociation energies are determined from single point energy calculations at the MP2(full)/6-311+G(2d,2p) level using the MP2(full)/6-31G* geometries. Excellent agreement between theory and experiment is found for the Na+ and K+ systems, while theory systematically underestimates the strength of binding in the Li+ systems. Theoretical calculations are also performed to examine the influence of methylation on the acidities, proton affinities, and Watson-Crick base pairing energies. The present results are compared to earlier studies of uracil and 5-methyluracil to more fully elucidate the influence of methylation on the properties of uracil, its noncovalent interactions with alkali metal ions, and nucleic acid stability.

  14. Genotyping of a family with a novel deleterious DPYD mutation supports the pretherapeutic screening of DPD deficiency with dihydrouracil/uracil ratio.

    PubMed

    Thomas, F; Hennebelle, I; Delmas, C; Lochon, I; Dhelens, C; Garnier Tixidre, C; Bonadona, A; Penel, N; Goncalves, A; Delord, J P; Toulas, C; Chatelut, E

    2016-02-01

    Despite the growing evidence that dihydropyrimidine dehydrogenase deficiency (DPD, encoded by the DPYD gene) confers a higher risk of developing severe toxicity, most patients are not screened for DPD deficiency before fluoropyrimidine treatment. We report here the genetic and phenotypic analyses of DPD in a family related to a patient who died after a first cycle of 5-fluorouracil and in 15 additional retrospective patients having a partial DPD deficiency (as measured by plasma dihydrouracil/uracil ratio). The patient with lethal toxicity was found to be a compound heterozygote for two DPYD mutations: a novel 8-bp duplication (c.168_175dupGAATAATT, p.Phe59Ter) and c.1679T>G (Ile560Ser). The patient's dihydrouracil/uracil ratio indicates complete DPD deficiency. The novel mutation was found in two members of the patient's family. Deleterious DPYD mutations were identified in 9 out of the 15 patients. The relationship between genotype and dihydrouracil/uracil values in the 22 patients of the present study was significant (P = 0.01).

  15. Using structural-based protein engineering to modulate the differential inhibition effects of SAUGI on human and HSV uracil DNA glycosylase.

    PubMed

    Wang, Hao-Ching; Ho, Chun-Han; Chou, Chia-Cheng; Ko, Tzu-Ping; Huang, Ming-Fen; Hsu, Kai-Cheng; Wang, Andrew H-J

    2016-05-19

    Uracil-DNA glycosylases (UDGs) are highly conserved proteins that can be found in a wide range of organisms, and are involved in the DNA repair and host defense systems. UDG activity is controlled by various cellular factors, including the uracil-DNA glycosylase inhibitors, which are DNA mimic proteins that prevent the DNA binding sites of UDGs from interacting with their DNA substrate. To date, only three uracil-DNA glycosylase inhibitors, phage UGI, p56, and Staphylococcus aureus SAUGI, have been determined. We show here that SAUGI has differential inhibitory effects on UDGs from human, bacteria, Herpes simplex virus (HSV; human herpesvirus 1) and Epstein-Barr virus (EBV; human herpesvirus 4). Newly determined crystal structures of SAUGI/human UDG and a SAUGI/HSVUDG complex were used to explain the differential binding activities of SAUGI on these two UDGs. Structural-based protein engineering was further used to modulate the inhibitory ability of SAUGI on human UDG and HSVUDG. The results of this work extend our understanding of DNA mimics as well as potentially opening the way for novel therapeutic applications for this kind of protein.

  16. Mechanism for the abiotic synthesis of uracil via UV-induced oxidation of pyrimidine in pure H{sub 2}O ices under astrophysical conditions

    SciTech Connect

    Bera, Partha P.; Nuevo, Michel; Sandford, Scott A.; Lee, Timothy J.; Milam, Stefanie N.

    2010-09-14

    The UV photoirradiation of pyrimidine in pure H{sub 2}O ices has been explored using second-order Moeller-Plesset perturbation theory and density functional theory methods, and compared with experimental results. Mechanisms studied include those starting with neutral pyrimidine or cationic pyrimidine radicals, and reacting with OH radical. The ab initio calculations reveal that the formation of some key species, including the nucleobase uracil, is energetically favored over others. The presence of one or several water molecules is necessary in order to abstract a proton which leads to the final products. Formation of many of the photoproducts in UV-irradiated H{sub 2}O:pyrimidine=20:1 ice mixtures was established in a previous experimental study. Among all the products, uracil is predicted by quantum chemical calculations to be the most favored, and has been identified in experimental samples by two independent chromatography techniques. The results of the present study strongly support the scenario in which prebiotic molecules, such as the nucleobase uracil, can be formed under abiotic processes in astrophysically relevant environments, namely in condensed phase on the surface of icy, cold grains before being delivered to the telluric planets, like Earth.

  17. Probing the Watson-Crick, wobble, and sugar-edge hydrogen bond sites of uracil and thymine.

    PubMed

    Müller, Andreas; Frey, Jann A; Leutwyler, Samuel

    2005-06-16

    The nucleobases uracil (U) and thymine (T) offer three hydrogen-bonding sites for double H-bond formation via neighboring N-H and C=O groups, giving rise to the Watson-Crick, wobble and sugar-edge hydrogen bond isomers. We probe the hydrogen bond properties of all three sites by forming hydrogen bonded dimers of U, 1-methyluracil (1MU), 3-methyluracil (3MU), and T with 2-pyridone (2PY). The mass- and isomer-specific S1 <-- S0 vibronic spectra of 2PY.U, 2PY.3MU, 2PY.1MU, and 2PY.T were measured using UV laser resonant two-photon ionization (R2PI). The spectra of the Watson-Crick and wobble isomers of 2PY.1MU were separated using UV-UV spectral hole-burning. We identify the different isomers by combining three different diagnostic tools: (1) Selective methylation of the uracil N3-H group, which allows formation of the sugar-edge isomer only, and methylation of the N1-H group, which leads to formation of the Watson-Crick and wobble isomers. (2) The experimental S1 <-- S0 origins exhibit large spectral blue shifts relative to the 2PY monomer. Ab initio CIS calculations of the spectral shifts of the different hydrogen-bonded dimers show a linear correlation with experiment. This correlation allows us to identify the R2PI spectra of the weakly populated Watson-Crick and wobble isomers of both 2PY.U and 2PY.T. (3) PW91 density functional calculation of the ground-state binding and dissociation energies De and D0 are in agreement with the assignment of the dominant hydrogen bond isomers of 2PY.U, 2PY.3MU and 2PY.T as the sugar-edge form. For 2PY.U, 2PY.T and 2PY.1MU the measured wobble:Watson-Crick:sugar-edge isomer ratios are in good agreement with the calculated ratios, based on the ab initio dissociation energies and gas-phase statistical mechanics. The Watson-Crick and wobble isomers are thereby determined to be several kcal/mol less strongly bound than the sugar-edge isomers. The 36 observed intermolecular frequencies of the nine different H-bonded isomers give

  18. An extended version of Boyd's force field method applicable to heteroatomic molecules. Part 1. Adenine and uracil

    NASA Astrophysics Data System (ADS)

    Espinosa-Müller, A. W.; Bravo, A. N.

    The force field method developed by Boyd is extended to include molecules containing atoms other than C and H (e.g., N, O, P, S, Cl, Br,…). A new set of force field parameters is determined in order to redefine the potential energy functions that govern the dynamics of the internal (valence coordinates) degrees of freedom of a molecule. It is shown that the minimum of the partial potential energy surface is significantly affected by electrostatic intramolecular interactions. In this regard the non-bonded interactions appears to be less important than the dipole-dipole type interactions for a given interatomic distance when heteroatoms are present in the molecular framework. The reliability of the extended method as regards minimized structure, vibrational spectra and thermodynamic properties has been checked for more than 20 polyatomic molecules. From the correlation between calculated and experimental properties it is concluded that the method has good potential for further applications on polyatomic molecules with increasing size and topological compexities such as adenine and uracil.

  19. High-performance liquid chromatographic assay with UV detection for measurement of dihydrouracil/uracil ratio in plasma.

    PubMed

    Déporte, R; Amiand, M; Moreau, A; Charbonnel, C; Campion, L

    2006-04-13

    A rapid, robust and sensitive HPLC method for analysis of uracil (U) and dihydrouracil (UH2) in plasma was developed using solid phase extraction and ultraviolet detection. Separation was achieved with a SymmetryShield RP18 column and an Atlantis dC18 column using a 10 mM potassium phosphate buffer as mobile phase. Compounds were eluted within 15 min without interference. Recovery was 80.4 and 80.6% for U and UH2. Calibration curves were linear from 2.5 to 80 ng/mL for U and 6.75 to 200 ng/mL for UH2. The LLQ was, respectively, 2.5 ng/mL for U, and 6.75 ng/mL for UH2. Within-run and between-run precision were less than 5.94% and inaccuracy did not exceed 7.80%. The overall procedure has been applied to correlate UH2/U ratio with dihydropyrimidine dehydrogenase activity in 165 cancer patients.

  20. Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii.

    PubMed Central

    Donald, R G; Roos, D S

    1995-01-01

    Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7777580

  1. Infrared depletion spectra of 2-aminopyridineṡ2-pyridone, a Watson-Crick mimic of adenineṡuracil

    NASA Astrophysics Data System (ADS)

    Frey, Jann A.; Müller, Andreas; Frey, Hans-Martin; Leutwyler, Samuel

    2004-11-01

    The 2-aminopyridineṡ2-pyridone (2APṡ2PY) dimer is linked by N-H⋯O=C and N-H⋯N hydrogen bonds, providing a model for the Watson-Crick hydrogen bond configuration of the adenineṡthymine and adenineṡuracil nucleobase pairs. Mass-specific infrared spectra of 2APṡ2PY and its seven N-H deuterated isotopomers have been measured between 2550 and 3650 cm-1 by IR laser depletion combined with UV two-color resonant two-photon ionization. The 2PY amide N-H stretch is a very intense band spread over the range 2700-3000 cm-1 due to large anharmonic couplings. It is shifted to lower frequency by 710 cm-1 or ≈20% upon H bonding to 2AP. On the 2AP moiety, the "bound" amino N-H stretch gives rise to a sharp band at 3140 cm-1, which is downshifted by 354 cm-1 or ≈10% upon H bonding to 2PY. The amino group "free" N-H stretch and the H-N-H bend overtone are sharp bands at ≈3530 cm-1 and 3320 cm-1. Ab initio structures and harmonic vibrations were calculated at the Hartree-Fock level and with the PW91 and B3LYP density functionals. The PW91/6-311++G(d,p) method provides excellent predictions for the frequencies and IR intensities of all the isotopomers.

  2. C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells

    PubMed Central

    Brognara, Eleonora; Lampronti, Ilaria; Breveglieri, Giulia; Accetta, Alessandro; Corradini, Roberto; Manicardi, Alex; Borgatti, Monica; Canella, Alessandro; Multineddu, Chiara; Marchelli, Rosangela; Gambari, Roberto

    2011-01-01

    The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds acting by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antiproliferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect without activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge this compound should be considered among the most efficient inducers of erythroid differentiation of K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects. PMID:21958870

  3. Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach

    NASA Astrophysics Data System (ADS)

    Weiss, Jason T.; Dawson, John C.; MacLeod, Kenneth G.; Rybski, Witold; Fraser, Craig; Torres-Sánchez, Carmen; Patton, E. Elizabeth; Bradley, Mark; Carragher, Neil O.; Unciti-Broceta, Asier

    2014-02-01

    A bioorthogonal organometallic reaction is a biocompatible transformation undergone by a synthetic material exclusively through the mediation of a non-biotic metal source; a selective process used to label biomolecules and activate probes in biological environs. Here we report the in vitro bioorthogonal generation of 5-fluorouracil from a biologically inert precursor by heterogeneous Pd0 catalysis. Although independently harmless, combined treatment of 5-fluoro-1-propargyl-uracil and Pd0-functionalized resins exhibits comparable antiproliferative properties to the unmodified drug in colorectal and pancreatic cancer cells. Live-cell imaging and immunoassay studies demonstrate that the cytotoxic activity of the prodrug/Pd0-resin combination is due to the in situ generation of 5-fluorouracil. Pd0-resins can be carefully implanted in the yolk sac of zebrafish embryos and display excellent biocompatibility and local catalytic activity. The in vitro efficacy shown by this masking/activation strategy underlines its potential to develop a bioorthogonally activated prodrug approach and supports further in vivo investigations.

  4. Ion pairs and their role in modulating stability of cold- and warm-active uracil DNA glycosylase.

    PubMed

    Olufsen, Magne; Papaleo, Elena; Smalås, Arne Oskar; Brandsdal, Bjørn Olav

    2008-05-15

    MD simulations and continuum electrostatics calculations have been used to study the observed differences in thermostability of cold- and warm-active uracil DNA glycosylase (UDG). The present study focuses on the role of ion pairs and how they affect the thermal stability of the two enzymes. Analysis of the MD generated structural ensembles show that cod UDG (cUDG) and human UDG (hUDG) have 11 and 12 ion pairs which are present in at least 30% of the conformations. The electrostatic contribution of the ion pairs, computed using continuum electrostatics, is slightly more favorable in cUDG at 298 K. This is primarily attributed to more optimized interactions between the ion pairs and nearby dipoles/charges in cUDG. More global salt bridges are found in hUDG and are more stabilizing when compared to cUDG, possibly playing a role in maintaining overall stability and reducing conformational entropy. Both enzymes contain one three-member ionic network, but the one found in hUDG is far more stabilizing. Our results also suggest that care should be taken when performing statistical analysis of crystal structures with respect to ion pairs, and that crystallization conditions must be carefully examined when performing such analysis.

  5. Resonant photoelectron imaging of deprotonated uracil anion via vibrational levels of a dipole-bound excited state

    NASA Astrophysics Data System (ADS)

    Huang, Dao-Ling; Liu, Hong-Tao; Ning, Chuan-Gang; Dau, Phuong Diem; Wang, Lai-Sheng

    2017-01-01

    We report both non-resonant and resonant high-resolution photoelectron imaging of cryogenically-cooled deprotonated uracil anions, N1[U-H]-, via vibrational levels of a dipole-bound excited state. Photodetachment spectroscopy of N1[U-H]- was reported previously (Liu et al., 2014), in which forty-six vibrational autodetachment resonances due to the excited dipole-bound state were observed. By tuning the detachment laser to the vibrational levels of the dipole-bound state, we obtained high-resolution resonant photoelectron spectra, which are highly non-Franck-Condon. The resonant photoelectron spectra reveal many Franck-Condon inactive vibrational modes, significantly expanding the capability of photoelectron spectroscopy. A total of twenty one fundamental vibrational frequencies for the N1[U-H]rad radical are obtained, including all eight low-frequency out-of-plane modes, which are forbidden in non-resonant photoelectron spectroscopy. Furthermore, the breakdown of the Δv = -1 propensity rule is observed for autodetachment from many vibrational levels of the dipole-bound state, due to anharmonic effects. In particular, we have observed intramolecular electron rescattering in a number of resonant photoelectron spectra, leading to excitations of low-frequency vibrational modes. Further theoretical study may be warranted, in light of the extensive experimental data and new observations, to provide further insight into the autodetachment dynamics and vibronic coupling in dipole-bound states, as well as electron molecule interactions.

  6. Maternal nicotinamide supplementation causes global DNA hypomethylation, uracil hypo-incorporation and gene expression changes in fetal rats.

    PubMed

    Tian, Yan-Jie; Luo, Ning; Chen, Na-Na; Lun, Yong-Zhi; Gu, Xin-Yi; Li, Zhi; Ma, Qiang; Zhou, Shi-Sheng

    2014-05-01

    Recent evidence shows that excess nicotinamide can cause epigenetic changes in developing rats. The aim of the present study was to investigate the effects of maternal nicotinamide supplementation on the fetus. Female rats were randomised into four groups fed a standard chow diet (control group) or diets supplemented with 1 g/kg of nicotinamide (low-dose group), 4 g/kg of nicotinamide (high-dose group) or 4 g/kg of nicotinamide plus 2 g/kg of betaine (betaine group) for 14-16 d before mating and throughout the study. Fetal tissue samples were collected on the 20th day of pregnancy. Compared with the control group, the high-dose group had a higher fetal death rate, and the average fetal body weight was higher in the low-dose group but lower in the high-dose group. Nicotinamide supplementation led to a decrease in placental and fetal hepatic genomic DNA methylation and genomic uracil contents (a factor modifying DNA for diversity) in the placenta and fetal liver and brain, which could be completely or partially prevented by betaine. Moreover, nicotinamide supplementation induced tissue-specific alterations in the mRNA expression of the genes encoding nicotinamide N-methyltransferase, DNA methyltransferase 1, catalase and tumour protein p53 in the placenta and fetal liver. High-dose nicotinamide supplementation increased fetal hepatic α-fetoprotein mRNA level, which was prevented by betaine supplementation. It is concluded that maternal nicotinamide supplementation can induce changes in fetal epigenetic modification and DNA base composition. The present study raises the concern that maternal nicotinamide supplementation may play a role in the development of epigenetic-related diseases in the offspring.

  7. Theoretical study of the hydroxyl radical addition to uracil and photochemistry of the formed U6OH• adduct.

    PubMed

    Francés-Monerris, Antonio; Merchán, Manuela; Roca-Sanjuán, Daniel

    2014-03-20

    Hydroxyl radical ((•)OH) is produced in biological systems by external or endogenous agents. It can damage DNA/RNA by attacking pyrimidine nucleobases through the addition to the C5═C6 double bond. The adduct resulting from the attachment at the C5 position prevails in the experimental measurements, although the reasons for this preference remain unclear. The first aim of this work is therefore to shed light on the comprehension of this important process. Thus, the thermal (•)OH addition to the C5═C6 double bond of uracil has been studied theoretically by using DFT, MP2, and the multiconfigurational CASPT2//CASSCF methodologies. The in-vacuo results obtained with the latter protocol plus the analysis of solvent effects support the experimental observation. A significant lower barrier height is predicted for the C5 pathway with respect to that of the C6 route. In contrast to the C5 adduct, the C6 adduct is able to absorb visible light. Hence, the second aim of the work is to study the photochemistry of this species using the CASPT2//CASSCF methodology within the framework of the photochemical reaction path approach (PRPA). The nonradiative decay to the ground state of this compound has been characterized. A photoreactive character is predicted for the C6 adduct in the excited states according to the presence of excited-state minima along the main decay channel. Finally, a new mechanism of photodissociation has been explored, which implies the photoinduced regeneration of the canonical nucleobase by irradiating with visible light, being therefore relevant in RNA protection against damage by reactive oxygen species.

  8. Neoadjuvant therapy of rectal cancer using oral tegaful-uracil (UFT) plus concomitant radiotherapy--a case report.

    PubMed

    Yamaguchi, Yoshiyuki; Ohshita, Akiko; Hironaka, Katsuji; Emi, Manabu; Kawabuchi, Yoshiharu; Sakatani, Akio; Arihiro, Koji

    2005-09-01

    A 59-year-old male patient with rectal cancer 2 cm in diameter (T2) at the peritoneal reflection with suspicious left lateral node metastasis was treated with 400 mg of preoperative oral uracil and tegaful (UFT) for 5 weeks, 5 days a week in combination with concomitant radiotherapy of 45 Gy per 25 fractions for 5 weeks. After resting for another 5 weeks, colon fiberscopy, barium enema, and computed tomography revealed a trace of the primary tumor and a 40% shrinkage of the lateral metastasis. The serum CEA level decreased to the normal range during treatment. The adverse effects were nausea, bloody stool and elevation of transaminase, all at grade 1. Low anterior resection with a left hemi-lateral lymphadenectomy was performed through a suprapubic, one hand-size incision without laparoscopy. The preoperative treatment did not affect any operative procedures, and no postoperative complications occurred. The surgical specimen showed that the rectal tumor had been remarkably shrunk by the preoperative treatment, to the level of a superficial type tumor. Histological analysis indicated moderately differentiated adenocarcinoma cells that were present at only 2 mm in diameter in the mucosal layer, 6 mm in the submucosal layer, and 1 mm or less in the muscular layer with scar formation. No metastasis was detected in the 16 lymph nodes dissected, but an organizing tumor thrombus, which had preoperatively been diagnosed as lateral node metastasis, was detected. These results suggest that preoperative oral UFT plus concomitant radiotherapy may be a feasible, tolerable and effective treatment for patients with rectal cancer.

  9. Studies on molecular properties prediction and histamine H3 receptor affinities of novel ligands with uracil-based motifs.

    PubMed

    Lipani, Luca; Odadzic, Dalibor; Weizel, Lilia; Schwed, Johannes-Stephan; Sadek, Bassem; Stark, Holger

    2014-10-30

    The histamine H3 receptor (H3R) plays a role in cognitive and memory processes and is involved in different neurological disorders, including Alzheimer's disease, schizophrenia, and narcolepsy. Therefore, several hH3R antagonists/inverse agonists entered clinical phases for a broad spectrum of mainly centrally occurring diseases. However, many other promising candidates failed due to their pharmacokinetic profile, mostly because of their strong lipophilicity accompanied with low solubility. Analysis of previous potential H3R selective antagonists/inverse agonists, e.g. pitolisant, revealed promising results concerning physicochemical properties and drug-likeness. Herein, a series of new hH3R ligands 8-20 consisting of piperidin-1-yl or piperidin-1-yl-propoxyphenyl coupled to different uracil, thymine, and 5,6-dimethyluracil related moieties, were synthesized, evaluated on their binding properties at the hH3R and the estimation of different physicochemical and drug-likeness properties. Due to the coupling to various positions at pyrimidine-2,4-(1H,3H)-dione, affinity at hH3Rs and drug-likeness parameters have been improved. For instance, compound 9 showed in addition to high affinity at the hH3R (pKi (hH3R) = 8.14) clog S, clog P, LE, LipE, and drug-likeness score values of -4.36, 3.47, 0.34, 4.63, and 1.54, respectively. Also, the methyl substituted analog 17 (pKi (hH3R) = 8.15) revealed LE, LipE and drug-likeness score values of -3.29, 2.47, 0.49, 5.52, and 1.76, respectively.

  10. Tautomeric equilibrium of uracil and thymine in model protein-nucleic acid contacts. Spectroscopic and quantum chemical approach.

    PubMed

    Samijlenko, Svitlana P; Yurenko, Yevgen P; Stepanyugin, Andriy V; Hovorun, Dmytro M

    2010-01-28

    This work deals with tautomeric transformations of uracil (Ura) and thymine (Thy) in their model complexes with the deprotonated carboxylic group. Essential changes in the UV spectra of the bases upon their interaction with NaAc, vanishing signals of both imino protons in (1)H NMR spectra, and a perceptible decrease in intensity of both IR bands, related to the stretching vibrations nu(C=O) of the carbonyl groups, imply involvement of enolic tautomers. Results of quantum chemical calculations of the double complexes of the Ura(Thy) tautomers with CH(3)COO(-) at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory proved to be incompatible with the spectral features: despite the fact that the complexes of the enolic tautomers are much closer in energy to the diketo ones as compared to isolated tautomers, the energy gap between them is such that in tautomeric equilibrium dominate diketo forms. Calculations of triple complexes of the type CH(3)COO(-):Ura(Thy) tautomer:Na(+), taking into account the effect of the Na(+) coordination with tautomers, show that three triple complexes formed by enolic tautomers appeared more stable than those formed by diketo ones. This makes the UV and (1)H NMR data understandable, but the high residual intensity of the nu(C=O) bands in the IR spectra remains unclear. At that ion, Na(+) itself was not able to disturb the tautomeric equilibrium in the coordination complexes of the type Ura(Thy) tautomer:Na(+). To evaluate the DMSO effect, the CPCM solvation model was applied to triple complexes of the Ura tautomers. It appeared that in the solution there is coexistence between the diketo and enolic tautomers in a ratio of 53%:47%. This makes possible reconciliation of our experimental data. The biological significance of high-energy tautomers of nucleotide bases is discussed.

  11. Structures of protonated thymine and uracil and their monohydrated gas-phase ions from ultraviolet action spectroscopy and theory.

    PubMed

    Pedersen, Sara Øvad; Byskov, Camilla Skinnerup; Turecek, Frantisek; Brøndsted Nielsen, Steen

    2014-06-19

    The strong UV chromophores thymine (Thy) and uracil (Ura) have identical heteroaromatic rings that only differ by one methyl substituent. While their photophysics has been elucidated in detail, the effect on the excited states of base protonation and single water molecules is less explored. Here we report gas-phase absorption spectra of ThyH(+) and UraH(+) and monohydrated ions and demonstrate that the substituent is not only responsible for spectral shifts but also influences the tautomer distribution, being different for bare and monohydrated ions. Spectra interpretation is aided by calculations of geometrical structures and transition energies. The lowest free-energy tautomer (denoted 178, enol-enol form) accounts for 230-280 nm (ThyH(+)) and 225-270 nm (UraH(+)) bands. ThyH(+) hardly absorbs above 300 nm, whereas a discernible band is measured for UraH(+) (275-320 nm), ascribed to the second lowest free-energy tautomer (138, enol-keto form) comprising a few percent of the UraH(+) population at room temperature. Band widths are similar to those measured of cold ions in support of very short excited-state lifetimes. Attachment of a single water increases the abundance of 138 relative to 178, 138 now clearly present for ThyH(+). 138 resembles more the tautomer present in aqueous solution than 178 does, and 138 may indeed be a relevant transition structure. The band of ThyH(+)(178) is unchanged, that of UraH(+)(178) is nearly unchanged, and that of UraH(+)(138) blue-shifts by about 10 nm. In stark contrast to protonated adenine, more than one solvating water molecule is required to re-establish the absorption of ThyH(+) and UraH(+) in aqueous solution.

  12. Gamma-irradiated and nonirradiated Eimeria tenella sporozoites exhibit differential uracil uptake and expression of a 7- to 10-kDa metabolic antigen.

    PubMed

    Jenkins, M C; Chute, M B; Danforth, H D; Lillehoj, H S

    1995-06-01

    Eimeria tenella sporozoites were exposed in the oocyst form to either an optimum (15 kRad) or a high (25 kRad) dose of gamma irradiation and used to infect cultured chicken embryo fibroblasts (CEF). The sporozoite-infected CEF monolayer was pulsed at time of infection or 24 hr postinfection with [3H]uracil and harvested 24 hr later to measure sporozoite metabolic activity. Sporozoites exposed to either 0 or 15 kRad gamma irradiation incorporated similar (P > 0.05) amounts of [3H]uracil during the first and second 24-hr periods after infection. However, there was a significant decrease (P < 0.05) in [3H]uracil uptake by 25 kRad-exposed sporozoites compared to nonirradiated and 15 kRad-irradiated sporozoites. Indirect immunofluorescence (IFA) staining of E. tenella sporozoite-infected CEFs using monoclonal antibodies (MAb) specific for somatic or "metabolic" antigens showed that gamma irradiation also affected the release of intracellular metabolites. Regardless of irradiation dose, extracellular sporozoites exhibited similar intensity of immunofluorescence when stained with either somatic antigen- or metabolic antigen-reactive MAb. Also, somatic antigen expression was similar for intracellular parasites irrespective of radiation dose. However, metabolic 7- to 10-kDa antigen expression by 25 kRad-irradiated sporozoites was markedly reduced compared to nonirradiated or 15 kRad-irradiated intracellular sporozoites. These results were corroborated by immunostaining sporozoite/CEF protein-impregnated Immobilon membrane with somatic or metabolic 7- to 10-kDa antigen-reactive MAb. These findings may indicate that the metabolic 7- to 10-kDa antigen is involved in protective immunity elicited by nonirradiated and/or 15 kRad-irradiated E. tenella sporozoites.

  13. The methyl- and aza-substituent effects on nonradiative decay mechanisms of uracil in water: a transient absorption study in the UV region.

    PubMed

    Hua, XinZhong; Hua, LinQiang; Liu, XiaoJun

    2016-05-18

    The nonradiative decay dynamics of photo-excited uracil (Ura) and its derivatives, i.e., thymine (5-methyluracil, Thy), 6-methyluracil (6-MU) and 6-azauracil (6-AU) in water, has been studied using a femtosecond transient absorption method. The molecules are populated in the lowest (1)ππ* state by a pump pulse at 266 nm, and a broadband continuum in the deep UV region is then employed as the probe. The extension of the continuous UV probe down to 250 nm enables us to investigate comprehensively the population dynamics of the ground states for those molecules and to uncover the substituent effects on nonradiative decay dynamics of uracil. Vibrational cooling in the ground states of Ura, Thy and 6-MU has been directly observed for the first time, providing solid evidence of the ultrafast (1)ππ* → S0 decay. In combination with the ground state bleaching signals, it is consolidated that their lowest (1)ππ* state decays via two parallel pathways, i.e., (1)ππ* → S0 and (1)ππ* → (1)nπ*. Moreover, the contribution of the (1)ππ* → (1)nπ* channel is found to be much smaller for Thy or 6-MU than for Ura. Different from methyl-substitution, the initial (1)ππ* state of the aza-substituent 6-AU decays primarily to the (1)nπ* state, while the (1)ππ* → S0 channel can be negligible. Our study provides a comprehensive understanding of the substituent effects on the excited-state dynamics of uracil in water.

  14. Elucidating collision induced dissociation products and reaction mechanisms of protonated uracil by coupling chemical dynamics simulations with tandem mass spectrometry experiments.

    PubMed

    Molina, Estefanía Rossich; Ortiz, Daniel; Salpin, Jean-Yves; Spezia, Riccardo

    2015-12-01

    In this study we have coupled mixed quantum-classical (quantum mechanics/molecular mechanics) direct chemical dynamics simulations with electrospray ionization/tandem mass spectrometry experiments in order to achieve a deeper understanding of the fragmentation mechanisms occurring during the collision induced dissociation of gaseous protonated uracil. Using this approach, we were able to successfully characterize the fragmentation pathways corresponding to ammonia loss (m/z 96), water loss (m/z 95) and cyanic or isocyanic acid loss (m/z 70). Furthermore, we also performed experiments with isotopic labeling completing the fragmentation picture. Remarkably, fragmentation mechanisms obtained from chemical dynamics simulations are consistent with those deduced from isotopic labeling.

  15. Sulfolobus acidocaldarius UDG Can Remove dU from the RNA Backbone: Insight into the Specific Recognition of Uracil Linked with Deoxyribose

    PubMed Central

    Yi, Gang-Shun; Wang, Wei-Wei; Cao, Wei-Guo; Wang, Feng-Ping; Liu, Xi-Peng

    2017-01-01

    Sulfolobus acidocaldarius encodes family 4 and 5 uracil-DNA glycosylase (UDG). Two recombinant S. acidocaldarius UDGs (SacUDG) were prepared and biochemically characterized using oligonucleotides carrying a deaminated base. Both SacUDGs can remove deoxyuracil (dU) base from both double-stranded DNA and single-stranded DNA. Interestingly, they can remove U linked with deoxyribose from single-stranded RNA backbone, suggesting that the riboses on the backbone have less effect on the recognition of dU and hydrolysis of the C-N glycosidic bond. However, the removal of rU from DNA backbone is inefficient, suggesting strong steric hindrance comes from the 2′ hydroxyl of ribose linked to uracil. Both SacUDGs cannot remove 2,2′-anhydro uridine, hypoxanthine, and 7-deazaxanthine from single-stranded DNA and single-stranded DNA. Compared with the family 2 MUG, other family UDGs have an extra N-terminal structure consisting of about 50 residues. Removal of the 46 N-terminal residues of family 5 SacUDG resulted in only a 40% decrease in activity, indicating that the [4Fe-4S] cluster and truncated secondary structure are not the key elements in hydrolyzing the glycosidic bond. Combining our biochemical and structural results with those of other groups, we discussed the UDGs’ catalytic mechanism and the possible repair reactions of deaminated bases in prokaryotes. PMID:28106786

  16. RutR is the uracil/thymine-sensing master regulator of a set of genes for synthesis and degradation of pyrimidines.

    PubMed

    Shimada, Tomohiro; Hirao, Kiyo; Kori, Ayako; Yamamoto, Kaneyoshi; Ishihama, Akira

    2007-11-01

    Using the genomic SELEX, a total of six Escherichia coli DNA fragments have been identified, which formed complexes with transcription factor RutR. The RutR regulon was found to include a large number of genes encoding components for not only degradation of pyrimidines but also transport of glutamate, synthesis of glutamine, synthesis of pyrimidine nucleotides and arginine, and degradation of purines. DNase I footprinting indicated that RutR recognizes a palindromic sequence of TTGACCAnnTGGTCAA. The RutR box in P1 promoter of carAB encoding carbamoyl phosphate synthetase, a key enzyme of pyrimidine synthesis, overlaps with the PepA (CarP) repressor binding site, implying competition between RutR and PepA. Adding either uracil or thymine abolished RutR binding in vitro to the carAB P1 promoter. Accordingly, in the rutR-deletion mutant or in the presence of uracil, the activation in vivo of carAB P1 promoter was markedly reduced. Northern blot analysis of the RutR target genes indicated that RutR represses the Gad system genes involved in glutamate-dependent acid resistance and allantoin degradation. Altogether we propose that RutR is the pyrimidine sensor and the master regulator for a large set of the genes involved in the synthesis and degradation of pyrimidines.

  17. Intramolecular vibrational redistribution in the non-radiative excited state decay of uracil in the gas phase: an ab initio molecular dynamics study.

    PubMed

    Carbonniere, Philippe; Pouchan, Claude; Improta, Roberto

    2015-05-07

    We report a study of intramolecular vibrational distribution (IVR) occurring in the electronic ground state of uracil (S0) in the gas phase, following photoexcitation in the lowest energy bright excited state (Sπ) and decay through the ethylene-like Sπ/S0 Conical Intersection (CI-0π). To this aim we have performed 20 independent ab initio molecular dynamics simulations starting from CI-0π (ten of them with 1 eV kinetic energy randomly distributed over the different molecular degrees of freedom) and 10 starting from the ground state minimum (Franck-Condon, FC, point), with the excess kinetic energy equal to the energy gap between CI-0π and the FC point. The simulations, exploiting PBE0/6-31G(d) calculations, were performed over an overall period of 10 ps. A thorough statistical analysis of the variation of the geometrical parameters of uracil during the simulation time and of the distribution of the kinetic energy among the different vibrational degrees of freedom provides a consistent picture of the IVR process. In the first 0-200 fs the structural dynamics involve mainly the recovery of the average planarity. In the 200-600 fs time range, a substantial activation of CO and NH degrees of freedom is observed. After 500-600 fs most of the geometrical parameters reach average values similar to those found after 10 ps, though the system cannot be considered to be in equilibrium yet.

  18. Sulfolobus acidocaldarius UDG Can Remove dU from the RNA Backbone: Insight into the Specific Recognition of Uracil Linked with Deoxyribose.

    PubMed

    Yi, Gang-Shun; Wang, Wei-Wei; Cao, Wei-Guo; Wang, Feng-Ping; Liu, Xi-Peng

    2017-01-18

    Sulfolobus acidocaldarius encodes family 4 and 5 uracil-DNA glycosylase (UDG). Two recombinant S. acidocaldarius UDGs (SacUDG) were prepared and biochemically characterized using oligonucleotides carrying a deaminated base. Both SacUDGs can remove deoxyuracil (dU) base from both double-stranded DNA and single-stranded DNA. Interestingly, they can remove U linked with deoxyribose from single-stranded RNA backbone, suggesting that the riboses on the backbone have less effect on the recognition of dU and hydrolysis of the C-N glycosidic bond. However, the removal of rU from DNA backbone is inefficient, suggesting strong steric hindrance comes from the 2' hydroxyl of ribose linked to uracil. Both SacUDGs cannot remove 2,2'-anhydro uridine, hypoxanthine, and 7-deazaxanthine from single-stranded DNA and single-stranded DNA. Compared with the family 2 MUG, other family UDGs have an extra N-terminal structure consisting of about 50 residues. Removal of the 46 N-terminal residues of family 5 SacUDG resulted in only a 40% decrease in activity, indicating that the [4Fe-4S] cluster and truncated secondary structure are not the key elements in hydrolyzing the glycosidic bond. Combining our biochemical and structural results with those of other groups, we discussed the UDGs' catalytic mechanism and the possible repair reactions of deaminated bases in prokaryotes.

  19. Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil.

    PubMed

    Narayanan, Sharmila; Sanpui, Pallab; Sahoo, Lingaraj; Ghosh, Siddhartha Sankar

    2016-10-01

    In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively.

  20. Nucleic acid related compounds. 65. New syntheses of 1-(beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (IVAraU) from vinylsilane precursors. Radioiodine uptake as a marker for thymidine kinase positive herpes viral infections

    SciTech Connect

    Robins, M.J.; Manfredini, S.; Wood, S.G.; Wanklin, R.J.; Rennie, B.A.; Sacks, S.L. )

    1991-07-01

    (Trimethylsilyl)acetylene was coupled with 1-(2,3,5-tri-O-acetyl-beta-D- arabinofuranosyl)-5-iodouracil to give 1- (2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)-5-(2-(trimethylsilyl)eth yny l) uracil. Lindlar hydrogenation of 4 gave 1-(2,3,4-tri-O-acetyl-beta-D-arabinofuranosyl)-5(Z)-(2- (trimethylsilyl)vinyl)uracil. Treatment of 5 with iodine monochloride (or sodium iodide/phenyliodine(III) dichloride) in benzene gave 1-(2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (7), whereas polar solvents favored the (Z)-iodovinyl isomer 8. Deacetylation of 7 gave 1-(beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (IVAraU, 9). A microscale in situ synthesis with Na{asterisk}I gave ({asterisk}I)IVAraU. Treatment of HSV-infected cells with (125I)IVAraU resulted in virus-dependent uptake associated with nucleoside phosphorylation by wild type or acyclovir-resistant DNA polymerase mutants (but not with TK-HSV-1 mutants). Uptake was virus-inoculum dependent and was detectable within 4 h postinfection. The process was not completely reversible. Virus-specified uptake of (125I)IVAraU may allow automated in vitro detection of HSV isolates.

  1. Crystal structure of N-(7-di­bromo­methyl-5-methyl-1,8-naphthyridin-2-yl)benzamide–pyrrolidine-2,5-dione (1/1)

    PubMed Central

    Wang, Bang Zhong; Zhou, Jun Ping; Zhou, Yong; Luo, Jian Song; Yang, Jun Jie; Chi, Shao M.ing

    2017-01-01

    The title compound, C17H13Br2N3O·C4H5NO2, is a co-crystal of N-(7-di­bromo­methyl-5-methyl-1,8-naphthyridin-2-yl)benzamide and pyrrolidine-2,5-dione (succinimide). The benzamide mol­ecule exhibits pseudo-mirror symmetry, with an r.m.s. deviation of the non-H atoms of 0.09 Å (except for the two Br atoms). The angle between the least-squares planes of the two mol­ecules is 26.2 (2)°. In the crystal, the two mol­ecules are mutually linked by N—H⋯O and N—H⋯N hydrogen bonds. The packing is consolidated by C—H⋯(O,N) hydrogen bonds and π–π stacking inter­actions. PMID:28083121

  2. Alum Catalyzed Simple, Efficient, and Green Synthesis of 2-[3-Amino-5-methyl-5-(pyridin-3-yl)-1,5-dihydro-4H-1,2,4-triazol-4-yl]propanoic Acid Derivatives in Aqueous Media

    PubMed Central

    Sachdeva, Harshita; Dwivedi, Diksha; Saroj, Rekha

    2013-01-01

    Alum (KAl(SO4)2·12H2O) is an inexpensive, efficient, and nontoxic catalyst used for the synthesis of 2-[3-amino-5-methyl-5-(pyridin-3-yl)-1,5-dihydro-4H-1,2,4-triazol-4-yl]propanoic acid derivatives in aqueous media by the reaction of 3-acetyl pyridine (1), amino acids (2)/(6), and thiosemicarbazide (4) at 80°C. This methodology offers significant improvements for the synthesis of products with regards to the yield of products, simplicity in operation, and green aspects by avoiding toxic catalysts which uphold the motto of green chemistry. Synthesized compounds have been characterized by FT-IR, 13C NMR, and 1HNMR spectroscopy. PMID:24288503

  3. Insights into the mechanisms of ifosfamide encephalopathy: drug metabolites have agonistic effects on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and induce cellular acidification in mouse cortical neurons.

    PubMed

    Chatton, J Y; Idle, J R; Vågbø, C B; Magistretti, P J

    2001-12-01

    Therapeutic value of the alkylating agent ifosfamide has been limited by major side effects including encephalopathy. Although the underlying biochemical processes of the neurotoxic side effects are still unclear, they could be attributed to metabolites rather than to ifosfamide itself. In the present study, the effects of selected ifosfamide metabolites on indices of neuronal activity have been investigated, in particular for S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA). Because of structural similarities of SCMC with glutamate, the Ca(2+)(i) response of single mouse cortical neurons to SCMC and TDGA was investigated. SCMC, but not TDGA, evoked a robust increase in Ca(2+)(i) concentration that could be abolished by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only partly diminished by the N-methyl-D-aspartate receptor antagonist 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK=801). Cyclothiazide (CYZ), used to prevent AMPA/kainate receptor desensitization, potentiated the response to SCMC. Because activation of AMPA/kainate receptors is known to induce proton influx, the intracellular pH (pH(i)) response to SCMC was investigated. SCMC caused a concentration-dependent acidification that was amplified by CYZ. Since H(+)/monocarboxylate transporter (MCT) activity leads to similar cellular acidification, we tested its potential involvement in the pH(i) response. Application of the lactate transport inhibitor quercetin diminished the pH(i) response to SCMC and TDGA by 43 and 51%, respectively, indicating that these compounds may be substrates of MCTs. Taken together, this study indicates that hitherto apparently inert ifosfamide metabolites, in particular SCMC, activate AMPA/kainate receptors and induce cellular acidification. Both processes could provide the biochemical basis of the observed ifosfamide-associated encephalopathy.

  4. Aquatic toxicity of nine aircraft deicer and anti-icer formulations and relative toxicity of additive package ingredients alkylphenol ethoxylates and 4,5-methyl-1H-benzotriazoles

    USGS Publications Warehouse

    Corsi, S.R.; Geis, S.W.; Loyo-Rosales, J. E.; Rice, C.P.

    2006-01-01

    Characterization of the effects of aircraft deicer and anti-icer fluid (ADAF) runoff on aquatic organisms in receiving streams is a complex issue because the identities of numerous toxic additives are proprietary and not publicly available. Most potentially toxic and endocrine disrupting effects caused by ADAF are due to the numerous additive package ingredients which vary among manufacturers and types of ADAF formulation. Toxicity investigations of nine ADAF formulations indicate that endpoint concentrations for formulations of different manufacturers are widely variable. Type IV ADAF (anti-icers) are more toxic than Type I (deicers) for the four organisms tested (Vibrio fischeri, Pimephales promelas, Ceriodaphnia dubia, and Selenastrum capricornutum). Acute toxicity endpoint concentrations ranged from 347 to 7700 mg/L as ADAF for Type IV and from 1550 to 45 100 mg/L for Type I formulations. Chronic endpoint concentrations ranged from 70 to 1300 mg/L for Type IV and from 37 to 18 400 mg/L for Type I formulations. Alkylphenol ethoxylates and tolyltriazoles are two known classes of additives. Nonylphenol, nonylphenol ethoxylates, octylphenol, octylphenol ethoxylates, and 4,5-methyl-1H-benzotriazoles were quantified in the nine ADAF formulations, and toxicity tests were conducted with nonylphenol ethoxylates and 4,5-methyl-1H-benzotriazoles. Toxicity units computed for glycol and these additives, with respect to toxicity of the ADAF formulations, indicate that a portion of ADAF toxicity can be explained by the known additives and glycols, but much of the toxicity is due to unidentified additives. ?? 2006 American Chemical Society.

  5. Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

    PubMed Central

    Kim, Eun-Mi; Jeon, Hyo-Sung; Kim, Ji-Jung; Shin, Yeun-Kyung; Lee, Youn-Jeong; Yeo, Sang-Geon

    2016-01-01

    Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples. PMID:26726027

  6. The pH optimum of native uracil-DNA glycosylase of Archaeoglobus fulgidus compared to recombinant enzyme indicates adaption to cytosolic pH.

    PubMed

    Knævelsrud, Ingeborg; Kazazic, Sabina; Birkeland, Nils-Kåre; Bjelland, Svein

    2014-01-01

    Uracil-DNA glycosylase of Archaeoglobus fulgidus (Afung) in cell extracts exhibited maximal activity around pH 6.2 as compared to pH 4.8 for the purified recombinant enzyme expressed in Escherichia coli. Native Afung thus seems to be adapted to the intracellular pH of A. fulgidus, determined to be 7.0±0.1. Both recombinant and native Afung exhibited a broad temperature optimum for activity around 80°C, reflecting the A. fulgidus optimal growth temperature of 83°C. Adaption to the neutral conditions in the A. fulgidus cytoplasm might be due to covalent modifications or accessory factors, or due to a different folding when expressed in the native host.

  7. Biasing Simulations of DNA Base Pair Parameters with Application to Propellor Twisting in AT/AT, AA/TT, and AC/GT Steps and Their Uracil Analogs.

    PubMed

    Peguero-Tejada, Alfredo; van der Vaart, Arjan

    2017-01-23

    An accurate and efficient implementation of the six DNA base pair parameters as order parameters for enhanced sampling simulations is presented. The parameter definitions are defined by vector algebra operations on a reduced atomic set of the base pair, and correlate very well with standard definitions. Application of the model is illustrated by umbrella sampling simulations of propeller twisting within AT/AT, AA/TT, and AC/GT steps and their uracil analogs. Strong correlations are found between propeller twisting and a number of conformational parameters, including buckle, opening, BI/BII backbone configuration, and sugar puckering. The thymine methyl group is observed to notably alter the local conformational free energy landscape, with effects within and directly upstream of the thymine containing base pair.

  8. Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination.

    PubMed

    Kim, Eun-Mi; Jeon, Hyo-Sung; Kim, Ji-Jung; Shin, Yeun-Kyung; Lee, Youn-Jeong; Yeo, Sang-Geon; Park, Choi-Kyu

    2016-09-30

    Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

  9. Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi)

    SciTech Connect

    Assefa, Netsanet Gizaw; Niiranen, Laila; Johnson, Kenneth A.; Leiros, Hanna-Kirsti Schrøder; Smalås, Arne Oskar; Willassen, Nils Peder; Moe, Elin

    2014-08-01

    A structural and biophysical study of the interactions between cod and human uracil-DNA N-glycosylase (UNG) and their inhibitor Ugi is presented. The stronger interaction between cod UNG and Ugi can be explained by a greater positive electrostatic surface potential. Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme–substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG–Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG–Ugi complex with previously determined structures of UNG–Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (K{sub b}) which is one order of magnitude larger than that for hUNG–Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein–Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.

  10. Evaluation of the Role of the Vaccinia Virus Uracil DNA Glycosylase and A20 Proteins as Intrinsic Components of the DNA Polymerase Holoenzyme*

    PubMed Central

    Boyle, Kathleen A.; Stanitsa, Eleni S.; Greseth, Matthew D.; Lindgren, Jill K.; Traktman, Paula

    2011-01-01

    The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ∼1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N′ terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site. PMID:21572084

  11. Expression of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR2/3 receptors in the developing rat pineal gland.

    PubMed

    Kaur, C; Sivakumar, V; Ling, E A

    2005-10-01

    The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate (GluR2/3) receptors and N-methyl-D-aspartate receptor subtype 1 (NMDAR1) was carried out by immunohistochemistry, double immunofluorescence and real-time RT-PCR analysis in the pineal glands of 1-day to 6-wk-old rats in the present study. GluR2/3 immunopositive cells were distributed throughout the pineal gland and showed branching processes in all age groups. The NMDAR1 immunoreactivity, however, was observed in fewer branched cells. A constitutive mRNA expression of NMDAR1, GluR2 and GluR3 was detected in the pineal glands of various ages and showed no significant difference between the age groups studied. Immunohistochemical and double immunofluorescence results showed that the GluR2/3 were mainly expressed and co-localized with OX-42-positive microglia/macrophages and the glial fibrillary acidic protein (GFAP)-positive astrocytes. Co-localization of NMDAR1 with OX-42- and GFAP-positive cells was much less. The expression of these receptors on the glial cells suggests that they may be involved in the development and growth of the pineal gland in the early postnatal period (1 day to 3 wk) and subsequently in the regulation of melatonin synthesis.

  12. Design, synthesis, and biological evaluation of novel transrepression-selective liver X receptor (LXR) ligands with 5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-one skeleton.

    PubMed

    Aoyama, Atsushi; Endo-Umeda, Kaori; Kishida, Kenji; Ohgane, Kenji; Noguchi-Yachide, Tomomi; Aoyama, Hiroshi; Ishikawa, Minoru; Miyachi, Hiroyuki; Makishima, Makoto; Hashimoto, Yuichi

    2012-09-13

    To obtain novel transrepression-selective liver X receptor (LXR) ligands, we adopted a strategy of reducing the transactivational agonistic activity of the 5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-one derivative 10, which exhibits LXR-mediated transrepressional and transactivational activity. Structural modification of 10 based on the reported X-ray crystal structure of the LXR ligand-binding domain led to a series of compounds, of which almost all exhibited transrepressional activity at 1 or 10 μM but showed no transactivational activity even at 30 μM. Among the compounds obtained, 18 and 22 were confirmed to have LXR-dependent transrepressional activity by using peritoneal macrophages from wild-type and LXR-null mice. A newly developed fluorescence polarization assay indicated that they bind directly to LXRα. Next, further structural modification was performed with the guidance of docking simulations with LXRα, focusing on enhancing the binding of the ligands with LXRα through the introduction of substituents or heteroatom(s). Among the compounds synthesized, compound 48, bearing a hydroxyl group, showed potent, selective, and dose-dependent transrepressional activity.

  13. 2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells

    SciTech Connect

    Hsu, Mei-Hua; Liu, Chin-Yu; Lin, Chiao-Min; Chen, Yen-Jung; Chen, Chun-Jen; Lin, Yu-Fu; Huang, Li-Jiau; Lee, Kuo-Hsiung; Kuo, Sheng-Chu

    2012-03-01

    2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1 appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe. Highlights: ► HKL-1 is a potential antimitotic agent against HL-60 cells. ► HKL-1 induces spindle disruption and sustained resulted in mitotic catastrophe. ► CENP-E and aurora B protein expressions significantly reduced. ► Bcl-2 family protein expressions altered and caspase-9/-3 activation. ► HKL-1 is an attractive candidate for possible use as a novel antimitotic agent.

  14. In vivo antitumor effects of 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from the fruiting body of Antrodia camphorata through activation of the p53-mediated p27/Kip1 signaling pathway.

    PubMed

    Tu, Shih-Hsin; Wu, Chih-Hsiung; Chen, Li-Ching; Huang, Ching-Shui; Chang, Hui-Wen; Chang, Chien-Hsi; Lien, Hsiu-Man; Ho, Yuan-Soon

    2012-04-11

    In this study, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (SY-1) was isolated from three different sources of dried Antrodia camphorata (AC) fruiting bodies. AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), which is an endemic species that is used in Chinese medicine for its antitumor properties. We demonstrated that SY-1 [given as a 1-30 mg/kg body weight intraperitoneal (ip) injection three times per week] profoundly decreased the growth of COLO-205 human colon cancer cell tumor xenografts in an athymic nude mouse model. We further demonstrated that significant AC extract-mediated antitumor effects were observed at the highest concentration (5 g/kg body weight/day). No gross toxicity signs were observed (i.e., body weight changes, general appearance, or individual organ effects). Frozen COLO-205 xenograft tumors were pulverized in liquid N(2), and the expression of cell cycle regulatory proteins was detected by immunoblotting. We found that the p53-mediated p27/Kip1 protein was significantly induced in the low-dose (1 mg/kg body weight) SY-1-treated tumors, whereas the p21/Cip1 protein levels did not change. The G0/G1 phase cell cycle regulators induced by SY-1 were also associated with a significant decrease in cyclins D1, D3, and A. These results provide further evidence that SY-1 may have significance for cancer chemotherapy.

  15. 4,7-Dimethoxy-5-methyl-1,3-benzodioxole from Antrodia camphorata inhibits LPS-induced inflammation via suppression of NF-κB and induction HO-1 in RAW264.7 cells.

    PubMed

    Shie, Pei-Hsin; Wang, Sheng-Yang; Lay, Horng-Liang; Huang, Guan-Jhong

    2016-02-01

    Several benzenoid compounds have been isolated from Antrodia camphorata are known to have excellent anti-inflammatory activity. In this study, we investigated the anti-inflammatory potential of 4,7-dimethoxy-5-methyl-1,3-benzodioxole (DMB), one of the major benzenoid compounds isolated from the mycelia of A. camphorata. DMB significantly decreased the LPS-induced production of pro-inflammatory molecules, such as nitric oxide (NO), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in RAW264.7 cells. In addition, DMB suppressed the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose dependent manner. Moreover, DMB significantly suppressed LPS-induced nuclear translocation of nuclear factor-κB (NF-κB), and this inhibition was found to be associated with decreases in the phosphorylation and degradation of its inhibitor, inhibitory κB-α (IκB-α). Moreover, we found that DMB markedly inhibited the protein expression level of Toll-like receptor 4 (TLR4). Furthermore, treatment with DMB significantly increased hemoxygenase-1 (HO-1) expression in RAW264.7 cells, which is further confirmed by hemin, a HO-1 enhancer, significantly attenuated the LPS-induced pro-inflammatory molecules and iNOS and TLR4 protein levels. Taken together, the present study suggests that DMB may have therapeutic potential for the treatment of inflammatory diseases.

  16. Normal coordinate analysis and vibrational spectroscopy (FT-IR and FT-Raman) studies of 5-methyl-N-[4-(trifluoromethyl) phenyl]-isoxazole-4-carboxamide using density functional method.

    PubMed

    Shahidha, R; Muthu, S; Elamurugu Porchelvi, E; Govindarajan, M

    2014-11-11

    Vibrational spectral analysis of 5-methyl-N-[4-(trifluoromethyl) phenyl]-isoxazole-4-carboxamide is (5MN4TPI4C) molecule was carried out using FT-IR and FT-Raman spectroscopic techniques. The equilibrium geometry, harmonic vibrational wavenumbers, various bonding features have been computed using density functional B3LYP method with 6-311G(d,p) as basis set. The assignments of the vibrational spectra have been carried out with the aid of normal coordinate analysis (NCA) following the scaled quantum mechanical force field methodology (SQMFFM). Stability of the molecule arising from hyper conjugative interactions, charge delocalization has been analyzed using natural bond orbital (NBO) analysis. The non-linear optical (NLO) behavior of 5MN4TPI4C has been studied by determination of the electric dipole moment (μ) and hyperpolarizability (β) by using B3LYP/6-311G(d,p) method. The molecular orbital compositions and their contributions to the chemical bonding are studied by Total density of energy states (TDOS), sum of α and β electron (αβDOS) density of states. Thermodynamic properties (heat capacity, entropy and enthalpy) of the title compound at different temperatures are calculated.

  17. Synthesis and biological evaluation of the 1-arylpyrazole class of σ(1) receptor antagonists: identification of 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862).

    PubMed

    Díaz, José Luis; Cuberes, Rosa; Berrocal, Joana; Contijoch, Montserrat; Christmann, Ute; Fernández, Ariadna; Port, Adriana; Holenz, Jörg; Buschmann, Helmut; Laggner, Christian; Serafini, Maria Teresa; Burgueño, Javier; Zamanillo, Daniel; Merlos, Manuel; Vela, José Miguel; Almansa, Carmen

    2012-10-11

    The synthesis and pharmacological activity of a new series of 1-arylpyrazoles as potent σ(1) receptor (σ(1)R) antagonists are reported. The new compounds were evaluated in vitro in human σ(1)R and guinea pig σ(2) receptor (σ(2)R) binding assays. The nature of the pyrazole substituents was crucial for activity, and a basic amine was shown to be necessary, in accordance with known receptor pharmacophores. A wide variety of amines and spacer lengths between the amino and pyrazole groups were tolerated, but only the ethylenoxy spacer and small cyclic amines provided compounds with sufficient selectivity for σ(1)R vs σ(2)R. The most selective compounds were further profiled, and compound 28, 4-{2-[5-methyl-1-(naphthalen-2-yl)-1H-pyrazol-3-yloxy]ethyl}morpholine (S1RA, E-52862), which showed high activity in the mouse capsaicin model of neurogenic pain, emerged as the most interesting candidate. In addition, compound 28 exerted dose-dependent antinociceptive effects in several neuropathic pain models. This, together with its good physicochemical, safety, and ADME properties, led compound 28 to be selected as clinical candidate.

  18. 1-(1,3-Benzodioxol-5-yl-carbo-nyl) piperidine, a modulator of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, ameliorates exercise-induced fatigue in mice.

    PubMed

    Fan, Wutu; Wu, Xianglong; Pan, Yalei; Li, Chenrui; Niu, Yinbo; Zhai, Yuankun; Mei, Qibing

    2014-01-01

    The current study was designed to investigate the effects of 1-(1,3-benzodioxol-5-yl-carbonyl) piperidine (1-BCP) on swimming endurance capacity which as one indicator of fatigue in the weight-loaded forced swimming mice. Mice were given either vehicle or 1-BCP (0.1, or 0.2 mmol/kg body weight daily) by intraperitoneal injection once daily for 2 weeks. The 1-BCP groups showed a significant increase in swimming time to exhaustion compared with the control group. 1-BCP increased the liver glycogen (LG) and muscle glycogen (MG) contents significantly, while decreased the lactic acid (LA) and blood urea nitrogen (BUN) levels notably compared with control group. Besides, 1-BCP treatment also significantly improved the endogenous cellular antioxidant enzymes in mice by increasing the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). Therefore, this study demonstrated for the first time that the supplementation of 1-BCP, as a positive allosteric modulator of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, could enhance the endurance capacity of mice and facilitated them recovery from fatigue. Thus, we provide a new effective therapeutic strategy for fatigue.

  19. Effects of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline on glutamate transporter 1 and cysteine/glutamate exchanger as well as ethanol drinking behavior in male, alcohol-preferring rats.

    PubMed

    Aal-Aaboda, Munaf; Alhaddad, Hasan; Osowik, Francis; Nauli, Surya M; Sari, Youssef

    2015-06-01

    Alcohol consumption is largely associated with alterations in the extracellular glutamate concentrations in several brain reward regions. We recently showed that glutamate transporter 1 (GLT-1) is downregulated following chronic exposure to ethanol for 5 weeks in alcohol-preferring (P) rats and that upregulation of the GLT-1 levels in nucleus accumbens and prefrontal cortex results, in part, in attenuating ethanol consumption. Cystine glutamate antiporter (xCT) is also downregulated after chronic ethanol exposure in P rats, and its upregulation could be valuable in attenuating ethanol drinking. This study examines the effect of a synthetic compound, (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), on ethanol drinking and expressions of GLT-1 and xCT in the amygdala and the hippocampus of P rats. P rats were exposed to continuous free-choice access to water, 15% and 30% ethanol, and food for 5 weeks, after which they received treatments of MS-153 or vehicle for 5 days. The results show that MS-153 treatment significantly reduces ethanol consumption. It was revealed that GLT-1 and xCT expressions were downregulated in both the amygdala and the hippocampus of ethanol-vehicle-treated rats (ethanol-vehicle group) compared with water-control animals. MS-153 treatment upregulated GLT-1 and xCT expressions in these brain regions. These findings demonstrate an important role for MS-153 in these glutamate transporters for the attenuation of ethanol-drinking behavior.

  20. Lanthanide complexes containing 5-methyl-1,2,4-triazolo[1,5-a] pyrimidin-7(4H)-one and their therapeutic potential to fight leishmaniasis and Chagas disease.

    PubMed

    Caballero, Ana B; Rodríguez-Diéguez, Antonio; Salas, Juan M; Sánchez-Moreno, Manuel; Marín, Clotilde; Ramírez-Macías, Inmaculada; Santamaría-Díaz, Noelia; Gutiérrez-Sánchez, Ramón

    2014-09-01

    In the last years, numerous and significant advances in lanthanide coordination chemistry have been achieved. The unique chemical nature of these metal ions which is conferred by their f-electrons has led to a wide range of coordination compounds with interesting structural, physical and also biological properties. Consequently, lanthanide complexes have found applications mainly in catalysis, gas adsorption, photochemistry and as diagnostic tools. However, research on their therapeutic potential and the understanding of their mechanism of action is still taking its first steps, and there is a distinct lack of research in the parasitology field. In the present work, we describe the synthesis and physical properties of seven new lanthanide complexes with the anionic form of the bioactive ligand 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one (HmtpO), namely [Ln(mtpO)3(H2O)6]·9H2O (Ln=La(III), Nd(III), Eu(III), Gd(III), Tb(III), Dy(III) and Er(III)). In addition, results on the in vitro antiproliferative activity against Leishmania spp. and Trypanosoma cruzi are described. The high activity of the new compounds against parasite proliferation and their low cytotoxicity against reference host cell lines show a great potential of this type of compounds to become a new generation of highly effective and non-toxic antiparasitic agents to fight the so considered neglected diseases leishmaniasis and Chagas disease.

  1. Extraction studies of selected actinide ions from aqueous solutions with 4-benzoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione and tri-n-octylphosphine oxide

    SciTech Connect

    Hannink, N.J.; Hoffman, D.C. |; Smith, B.F.

    1991-11-01

    The first measurements of distribution coefficients (K{sub d}) for Cm(III), Bk(III), Cf(III), Es(III), and Fm(III) between aqueous perchlorate solutions and solutions of 4-benzoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione (BMPPT) and the synergist tri-n-octylphosphine oxide (TOPO) in toluene are reported. Curium-243, berkelium-250, californium-249, einsteinium-254, and fermium-253 were used in these studies. The K{sub d} for {sup 241}Am was also measured and is in agreement with previously published results. Our new results show that the K{sub d}`s decrease gradually with increasing atomic number for the actinides with a dip at Cf. In general, the K{sub d}`s for these actinides are about a factor of 5 to 10 greater than the K{sub d}`s for the homologous lanthanides at a pH of 2.9, a BMPPT concentration of 0.2 M, and a TOPO concentration of 0.04 M. The larger K{sub d}`s for the actinides are consistent with greater covalent bonding between the actinide metal ion and the sulfur bonding site in the ligand.

  2. Extraction studies of selected actinide ions from aqueous solutions with 4-benzoyl-2,4-Dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione and Tri-n-octylphosphine oxide

    SciTech Connect

    Hannink, N.J.; Hoffman, D.C.; Smith, B.F.

    1992-07-01

    The first measurements of distribution coefficients (k{sub d}) for Cm(III), Bk(III), Cf(III), Es(III), and Fm(III) between aqueous perchlorate solutions and solutions of 4-benzoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione (BMPPT) and the synergist tri-n-octylphosphine oxide (TOPO) in toluene are reported. Curium-243, berkelium-250, californium-249, einsteinium-254, and fermium-253 were used in these studies. The K{sub d} for {sup 241}Am was also measured and is in agreement with previously published results. Our new results show that the K{sub d}`s decrease gradually with increasing atomic number for the actinides with a dip at Cf. In general, the K{sub d}`s for these actinides are about about a factor of 10 greater than the K{sub d}`s for the homologous lanthanides at a pH of 2.9, a BMPPT concentration of 0.2 M, and a TOPO concentration of 0.04 M. The larger K{sub d}`s for the actinides are consistent with greater covalent bonding between the actinide metal ion and the sulfur bonding site in the ligand. 9 refs., 2 figs., 1 tab.

  3. Extraction studies of selected actinide ions from aqueous solutions with 4-benzoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione and tri-n-octylphosphine oxide

    SciTech Connect

    Hannink, N.J.; Hoffman, D.C. California Univ., Berkeley, CA . Dept. of Chemistry); Smith, B.F. )

    1991-11-01

    The first measurements of distribution coefficients (K{sub d}) for Cm(III), Bk(III), Cf(III), Es(III), and Fm(III) between aqueous perchlorate solutions and solutions of 4-benzoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-thione (BMPPT) and the synergist tri-n-octylphosphine oxide (TOPO) in toluene are reported. Curium-243, berkelium-250, californium-249, einsteinium-254, and fermium-253 were used in these studies. The K{sub d} for {sup 241}Am was also measured and is in agreement with previously published results. Our new results show that the K{sub d}'s decrease gradually with increasing atomic number for the actinides with a dip at Cf. In general, the K{sub d}'s for these actinides are about a factor of 5 to 10 greater than the K{sub d}'s for the homologous lanthanides at a pH of 2.9, a BMPPT concentration of 0.2 M, and a TOPO concentration of 0.04 M. The larger K{sub d}'s for the actinides are consistent with greater covalent bonding between the actinide metal ion and the sulfur bonding site in the ligand.

  4. Effects of tricyclazole (5-methyl-1,2,4-triazol[3,4] benzothiazole), a specific DHN-melanin inhibitor, on the morphology of Fonsecaea pedrosoi conidia and sclerotic cells.

    PubMed

    Franzen, Anderson J; Cunha, Marcel M L; Batista, Evander J O; Seabra, Sergio H; De Souza, Wanderley; Rozental, Sonia

    2006-09-01

    The influence of tricyclazole (5-methyl-1,2,4-triazol[3,4]benzothiazole), a specific DHN-melanin inhibitor, on the cell walls and intracellular structures of Fonsecaea pedrosoi conidia and sclerotic cells was analyzed by transmission electron microscopy (TEM), deep-etching, and field emission scanning electron microscopy. The treatment of the fungus with 16 microg mL(-1) of tricyclazole (TC) did not significantly affect fungal viability, but electron microscopy observations showed several important morphological differences between TC-treated and non-TC treated cells. Control sclerotic cells presented patched granules, with an average diameter of 47 nm, on the cell surface, which were absent in TC-treated cells. Also, TC-treated sclerotic cells showed an undulated relief. TC treatment leads to an accumulation of electron lucent vacuoles in the fungal cytoplasm of both conidia and sclerotic cells, and treated conidia observed by deep etching showed a relevant thickening of the fungal cell wall. Together, these observations support the previous data of our group that F. pedrosoi synthesizes melanin in intracellular organelles. In addition, we suggest that melanin is not only an extracellular constituent but could also be dispersing all over the cell walls and could have an effective role in cross-linking different cell wall compounds that help maintain the regular shape of the cell wall.

  5. Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

    PubMed

    Pierre, Karin; Chatton, Jean-Yves; Parent, Annabelle; Repond, Cendrine; Gardoni, Fabrizio; Di Luca, Monica; Pellerin, Luc

    2009-05-01

    MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

  6. A study of the oligomeric state of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptors in the synaptic junctions of porcine brain.

    PubMed Central

    Wu, T Y; Liu, C I; Chang, Y C

    1996-01-01

    The number of the subunits in an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring L-glutamate receptor in the synaptic junctions of porcine brain was investigated in this study. Upon incubation of the synaptic junctions with three cross-linking regents, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (SADP), AMPA receptor subunits in higher-molecular-mass aggregates were detected by immunoblotting. These aggregates migrated as proteins of approx. 200, 300 and 400 kDa. The number and identity of the subunits in a solubilized AMPA receptor were also investigated here. Two samples, W1 and W2, enriched in AMPA receptors were prepared from synaptic junctions by a combination of detergent-solubilization, anion-exchange chromatography and wheatgerm agglutinin affinity chromatography. Hydrodynamic behaviour analyses revealed that the majority of the AMPA receptors in either one of these samples were asymmetrical detergent-surrounded particles with a protein mass around 350 kDa. SDS/PAGE analysis revealed that the majority of AMPA receptors in the W1 sample were comprised of dimers of 106 kDa subunits which were covalently linked by disulphide bonds. Cross-linking these receptors with SADP yielded a new band of approx. 400 kDa. The results obtained here, either from the studies of AMPA receptors embedding in synaptic junctions or from those of detergent-solubilized and partially purified receptors, suggest that AMPA receptors contain a basic core structure comprising of four 106 kDa subunits. PMID:8920974

  7. Poly(ADP-Ribose)Polymerase 1 (PARP-1) Activation and Ca(2+) Permeable α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid (AMPA) Channels in Post-Ischemic Brain Damage: New Therapeutic Opportunities?

    PubMed

    Gerace, Elisabetta; Pellegrini-Giampietro, Domenico E; Moroni, Flavio; Mannaioni, Guido

    2015-01-01

    A significant number of laboratories observed that poly (ADP-ribose) polymerase (PARP) inhibitors, administered a few hours after ischemic or traumatic brain injury, may drastically reduce the subsequent neurological damage. It has also been shown that PARP inhibitors, administered for 24 hours to rats with permanent middle cerebral artery occlusion (MCAO), may reduce the number of dying neurons for a long period after surgery, thus suggesting that these agents could reduce the delayed brain damage and the neurological and cognitive impairment (dementia) frequently observed a few months after a stroke. In organotypic hippocampal slices exposed to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG), an alkylating agent able to activate PARP, a selective and delayed degeneration of the CA1 pyramidal cells which was anatomically similar to that observed after a short period of oxygen and glucose deprivation (OGD) has been described. Biochemical and electrophysiological approaches showed that MNNG exposure caused an increased expression and function of the calcium permeable α-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) channels in the CA1 but not in the CA3 hippocampal region. PARP inhibitors prevented this increase and reduced CA1 cell death. The AMPA receptor antagonist 2,3-dihydroxy-6- nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione or the selective Ca(2+) permeable AMPA channel blocker 1-Naphthyl acetyl spermine (NASPM), also reduced the MNNG-induced CA1 pyramidal cell death. Since activation of PARP-1 facilitate the expression of Ca(2+) permeable channels and the subsequent delayed cell death, PARP inhibitors administered a few hours after a stroke may not only reduce the early post-ischemic brain damage but also the late neuronal death frequently occurring after severe stroke.

  8. Roles of fragile X mental retardation protein in dopaminergic stimulation-induced synapse-associated protein synthesis and subsequent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) receptor internalization.

    PubMed

    Wang, Hansen; Kim, Susan S; Zhuo, Min

    2010-07-09

    Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.

  9. Probing the Conical Intersection Dynamics of the RNA Base Uracil by UV-Pump Stimulated-Raman-Probe Signals; Ab Initio Simulations

    PubMed Central

    2015-01-01

    Nonadiabatic electron and nuclear dynamics of photoexcited molecules involving conical intersections is of fundamental importance in many reactions such as the self-protection mechanism of DNA and RNA bases against UV irradiation. Nonlinear vibrational spectroscopy can provide an ultrafast sensitive probe for these processes. We employ a simulation protocol that combines nonadiabatic on-the-fly molecular dynamics with a mode-tracking algorithm for the simulation of femtosecond stimulated Raman spectroscopy (SRS) signals of the high frequency C–H- and N–H-stretch vibrations of the photoexcited RNA base uracil. The simulations rely on a microscopically derived expression that takes into account the path integral of the excited state evolution and the pulse shapes. Analysis of the joint time/frequency resolution of the technique reveals a matter chirp contribution that limits the inherent temporal resolution. Characteristic signatures of relaxation dynamics mediated in the vicinity of conical intersection are predicted. The C–H and N–H spectator modes provide high sensitivity to their local environment and act as local probes with submolecular and high temporal resolution. PMID:24803857

  10. Therapeutic usefulness of postoperative adjuvant chemotherapy with Tegafur-Uracil (UFT) in patients with breast cancer: focus on the results of clinical studies in Japan.

    PubMed

    Nakayama, Takahiro; Noguchi, Shinzaburo

    2010-01-01

    In Japan, the history of postoperative chemotherapy for breast cancer started with 5-fluorouracil (5-FU), launched in the 1980s. Currently, oral fluoropyrimidine-based regimens indicated for the treatment of breast cancer in Japan include tegafur plus uracil (UFT); tegafur, gimeracil, and oteracil (TS-1); doxifluridine; and capecitabine. In particular, UFT represents an important option for long-term treatment because of minimal adverse events and the potential for long-term maintenance of effective plasma concentrations of 5-FU to inhibit micrometastasis after surgery. Therefore, various clinical studies of postoperative adjuvant chemotherapy with UFT have been conducted in patients with completely resected tumors. Recent studies have shown that UFT prolongs survival after tumor resection in patients with gastric cancer, colorectal cancer, and lung cancer. In patients with breast cancer, large clinical trials of UFT-based postoperative chemotherapy conducted in Japan have shown that UFT is useful for the treatment of intermediate-risk patients with no lymph node metastasis. This paper reviews the results of clinical studies of UFT conducted in Japan to assess the therapeutic usefulness of this oral 5-FU. The types of patients most likely to benefit from UFT are discussed on the basis of currently available evidence and a global consensus of treatment recommendations. The optimal timing of endocrine therapy and strategies for postoperative adjuvant chemotherapy with UFT in patients with breast cancer are also discussed.

  11. WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae.

    PubMed

    Gajewska, B; Kamińska, J; Jesionowska, A; Martin, N C; Hopper, A K; Zoładek, T

    2001-01-01

    Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.

  12. 20-150-keV proton-impact-induced ionization of uracil: Fragmentation ratios and branching ratios for electron capture and direct ionization

    SciTech Connect

    Tabet, J.; Eden, S.; Feil, S.; Abdoul-Carime, H.; Farizon, B.; Farizon, M.; Ouaskit, S.; Maerk, T. D.

    2010-01-15

    Fragmentation ratios and branching ratios are measured for ionization and dissociative ionization for 20-150 keV (0.9-2.4v{sub 0}) proton collisions with gas-phase uracil molecules. Through event-by-event determination of the postcollision projectile charge, it is possible for such a key biomolecule to distinguish between electron capture (EC) by the incident proton and direct ionization (DI) without projectile neutralization. While the same fragment ion groups are observed in the mass spectra for both processes, EC induces dissociation with greater efficiency than DI in the impact energy range of 35-150 keV (1.2-2.4v{sub 0}). In this range EC is also less abundant than DI with a branching ratio for EC/total ionization of <50%. Moreover, whereas fragmentation ratios do not change with energy in the case of EC, DI mass spectra show a tendency for increased fragmentation at lower impact energies.

  13. Polymeric Cd(II), trinuclear and mononuclear Ni(II) complexes of 5-methyl-4-phenyl-1,2,4-triazole-3-thione: Synthesis, structural characterization, thermal behaviour, fluorescence properties and antibacterial activity

    NASA Astrophysics Data System (ADS)

    Bharty, M. K.; Paswan, S.; Dani, R. K.; Singh, N. K.; Sharma, V. K.; Kharwar, R. N.; Butcher, R. J.

    2017-02-01

    Syntheses of a polymeric Cd(II) complex, [Cd(mptt)2]n (1), a trinuclear Ni(II) complex, [Ni3(μ-mptt)4(μ-H2O)2(H2O)2(ttfa)2]·3H2O (2) and a mononuclear Ni(II) complex [Ni(mptt)2(en)2] (3) have been performed using the ligand 5-methyl-4-phenyl-1,2,4-triazole-3-thione (Hmptt) and nickel(II)/cadmium(II) salts {ttfa = thenoyltrifluroacetonate). The ligand and the complexes have been characterized by various physicochemical methods in addition to their single crystal X-ray structure. The Cd centre in complex 1 adopts a distorted tetrahedral geometry with one sulfur atom and two mptt ligands provide three nitrogen atoms from three triazole units. The sulfur atom of the ligand binds covalently and overall the ligand acts as uninigative N,S/N,N bidentate moiety. The polymeric structure of complex 1 results from the N atoms of the neighboring triazole units coordinating with the Cd(II) centre. The three Ni(II) centres in the trinuclear Ni(II) complex 2 form a linear arrangement and all have six coordinated arrangements. The middle Ni(II) binds with four deprotonated triazole ring nitrogens and two water molecules form two bridges. The terminal Ni(II) centres bind through two thenoyl oxygens, two triazole nitrogens and water molecules that formed bridges with the middle Ni centre. In complex 3, the nickel(II) centre is covalently bonded through two deprotonated triazole ring nitrogens from two ligand moieties and other four sites are occupied by four nitrogens from two bidentate en ligands. Thermogravimetric analyses (TGA) of the complexes indicated for NiO as the final residue. The bioefficacy of the ligand and complexes 2 and 3 have been examined against the growth of bacteria to evaluate their anti-microbial potential. Complex 2 showed high antibacterial activity as compared to the ligand and complex 3. Complexes 1, 2 and 3 are fluorescent materials with maximum emissions at 425, 421 and 396 nm at an excitation wavelength of 323, 348 and 322 nm, respectively.

  14. X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

    NASA Astrophysics Data System (ADS)

    Prokofev, I. I.; Lashkov, A. A.; Gabdulkhakov, A. G.; Balaev, V. V.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-11-01

    In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2'-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation

  15. Polymerization of non-complementary RNA: systematic symmetric nucleotide exchanges mainly involving uracil produce mitochondrial RNA transcripts coding for cryptic overlapping genes.

    PubMed

    Seligmann, Hervé

    2013-03-01

    Usual DNA→RNA transcription exchanges T→U. Assuming different systematic symmetric nucleotide exchanges during translation, some GenBank RNAs match exactly human mitochondrial sequences (exchange rules listed in decreasing transcript frequencies): C↔U, A↔U, A↔U+C↔G (two nucleotide pairs exchanged), G↔U, A↔G, C↔G, none for A↔C, A↔G+C↔U, and A↔C+G↔U. Most unusual transcripts involve exchanging uracil. Independent measures of rates of rare replicational enzymatic DNA nucleotide misinsertions predict frequencies of RNA transcripts systematically exchanging the corresponding misinserted nucleotides. Exchange transcripts self-hybridize less than other gene regions, self-hybridization increases with length, suggesting endoribonuclease-limited elongation. Blast detects stop codon depleted putative protein coding overlapping genes within exchange-transcribed mitochondrial genes. These align with existing GenBank proteins (mainly metazoan origins, prokaryotic and viral origins underrepresented). These GenBank proteins frequently interact with RNA/DNA, are membrane transporters, or are typical of mitochondrial metabolism. Nucleotide exchange transcript frequencies increase with overlapping gene densities and stop densities, indicating finely tuned counterbalancing regulation of expression of systematic symmetric nucleotide exchange-encrypted proteins. Such expression necessitates combined activities of suppressor tRNAs matching stops, and nucleotide exchange transcription. Two independent properties confirm predicted exchanged overlap coding genes: discrepancy of third codon nucleotide contents from replicational deamination gradients, and codon usage according to circular code predictions. Predictions from both properties converge, especially for frequent nucleotide exchange types. Nucleotide exchanging transcription apparently increases coding densities of protein coding genes without lengthening genomes, revealing unsuspected functional DNA

  16. Elevated levels of plasma homocysteine, deficiencies in dietary folic acid and uracil-DNA glycosylase impair learning in a mouse model of vascular cognitive impairment.

    PubMed

    Jadavji, Nafisa M; Farr, Tracy D; Lips, Janet; Khalil, Ahmed A; Boehm-Sturm, Philipp; Foddis, Marco; Harms, Christoph; Füchtemeier, Martina; Dirnagl, Ulrich

    2015-04-15

    Dietary deficiencies in folic acid result in elevated levels of plasma homocysteine, which has been associated with the development of dementia and other neurodegenerative disorders. Previously, we have shown that elevated levels of plasma homocysteine in mice deficient for a DNA repair enzyme, uracil-DNA glycosylase (UNG), result in neurodegeneration. The goal of this study was to evaluate how deficiencies in folic acid and UNG along with elevated levels of homocysteine affect vascular cognitive impairment, via chronic hypoperfursion in an animal model. Ung(+/+) and Ung(-/-) mice were placed on either control (CD) or folic acid deficient (FADD) diets. Six weeks later, the mice either underwent implantation of microcoils around both common carotid arteries. Post-operatively, behavioral tests began at 3-weeks, angiography was measured after 5-weeks using MRI to assess vasculature and at completion of study plasma and brain tissue was collected for analysis. Learning impairments in the Morris water maze (MWM) were observed only in hypoperfused Ung(-/-) FADD mice and these mice had significantly higher plasma homocysteine concentrations. Interestingly, Ung(+/+) FADD produced significant remodeling of the basilar artery and arterial vasculature. Increased expression of GFAP was observed in the dentate gyrus of Ung(-/-) hypoperfused and FADD sham mice. Chronic hypoperfusion resulted in increased cortical MMP-9 protein levels of FADD hypoperfused mice regardless of genotypes. These results suggest that elevated levels of homocysteine only, as a result of dietary folic acid deficiency, don't lead to memory impairments and neurobiochemical changes. Rather a combination of either chronic hypoperfusion or UNG deficiency is required.

  17. Differences in nitric oxide steady states between arginine, hypoxanthine, uracil auxotrophs (AHU) and non-AHU strains of Neisseria gonorrhoeae during anaerobic respiration in the presence of nitrite.

    PubMed

    Barth, Kenneth; Clark, Virginia L

    2008-08-01

    Neisseria gonorrhoeae can grow by anaerobic respiration using nitrite as an alternative electron acceptor. Under these growth conditions, N. gonorrhoeae produces and degrades nitric oxide (NO), an important host defense molecule. Laboratory strain F62 has been shown to establish and maintain a NO steady-state level that is a function of the nitrite reductase/NO reductase ratio and is independent of cell number. The nitrite reductase activities (122-197 nmol NO2 reduced x min(-1) x OD600(-1)) and NO reductase activities (88-155 nmol NO reduced x min(-1) x OD600(-1)) in a variety of gonococcal clinical isolates were similar to the specific activities seen in F62 (241 nmol NO2 reduced x min(-1) x OD600(-1) and 88 nmol NO reduced x min(-1) x OD600(-1), respectively). In seven gonococcal strains, the NO steady-state levels established in the presence of nitrite were similar to that of F62 (801-2121 nmol x L-1 NO), while six of the strains, identified as arginine, hypoxanthine, and uracil auxotrophs (AHU), that cause asymptomatic infection in men had either two- to threefold (373-579 nmol x L-1 NO) or about 100-fold (13-24 nmol x L-1 NO) lower NO steady-state concentrations. All tested strains in the presence of a NO donor, 2,2'-(hydroxynitrosohydrazono)bis-ethanimine/NO, quickly lowered and maintained NO levels in the noninflammatory range of NO (<300 nmol x L-1). The generation of a NO steady-state concentration was directly affected by alterations in respiratory control in both F62 and an AHU strain, although differences in membrane function are suspected to be responsible for NO steady-state level differences in AHU strains.

  18. Preoperative Chemoradiotherapy for Rectal Cancer: Randomized Trial Comparing Oral Uracil and Tegafur and Oral Leucovorin Vs. Intravenous 5-Fluorouracil and Leucovorin

    SciTech Connect

    Torre, Alejandro de la Garcia-Berrocal, Maria Isabel; Arias, Fernando; Marino, Alfonso; Valcarcel, Francisco; Magallon, Rosa; Regueiro, Carlos A.; Romero, Jesus; Zapata, Irma; Fuente, Cristina de la; Fernandez-Lizarbe, Eva; Vergara, Gloria; Belinchon, Belen; Veiras, Maria; Moleron, Rafael; Millan, Isabel

    2008-01-01

    Purpose: To compare, in a randomized trial, 5-fluorouracil (FU) plus leucovorin (LV) (FU+LV) vs. oral uracil and tegafur (UFT) plus LV (UFT+LV) given concomitantly with preoperative irradiation in patients with cT3-4 or N+ rectal cancer. Methods and Materials: A total of 155 patients were entered onto the trial. Patients received pelvic radiotherapy (4500-5,040 cGy in 5 to 6 weeks) and chemotherapy consisting of two 5-day courses of 20 mg/m{sup 2}/d LV and 350 mg/m{sup 2}/d FU in the first and fifth weeks of radiotherapy (77 patients) or one course of 25 mg/d oral LV and 300 mg/m{sup 2}/d UFT for 4 weeks beginning in the second week of radiotherapy (78 patients). The primary endpoints were pathologic complete response (pCR) and resectability rate. Secondary endpoints included downstaging rate, toxicity, and survival. Results: Grade 3-5 acute hematologic toxicity occurred only with FU+LV (leukopenia 9%; p = 0.02). There were no differences in resectability rates (92.1% vs. 93.4%; p = 0.82). The pCR rate was 13.2% in both arms. Tumor downstaging was more frequent with UFT+LV (59.2% vs. 43.3%; p = 0.04). Three-year overall survival was 87% with FU+LV and 74% with UFT+LV (p = 0.37). The 3-year cumulative incidences of local recurrence were 7.5% and 8.9%, respectively (p = 0.619; relative risk, 1.46; 95% confidence interval 0.32-6.55). Conclusion: Although this study lacked statistical power to exclude clinically significant differences between both groups, the outcome of patients treated with UFT+LV did not differ significantly from that of patients treated with FU+LV, and hematologic toxicity was significantly lower in the experimental arm.

  19. Electron configuration and hydrogen-bonding pattern in several thymine and uracil analogues studied by 1H-14N NQDR and DFT/QTAIM.

    PubMed

    Seliger, Janez; Žagar, Veselko; Latosińska, Magdalena; Latosińska, Jolanta Natalia

    2012-08-02

    Some thio- and aza-derivatives of natural nucleobases uracil and thymine: 2-thiouracil, 4-thiouracil, 6-methyl-2-thiouracil, 6-azauracil, and 6-aza-2-thiothymine have been studied experimentally in solid state by (1)H-(14)N NMR-NQR double resonance (NQDR) and theoretically by the Density Functional Theory (DFT)/Quantum Theory of Atoms in Molecules (QTAIM). The (14)N resonance frequencies have been measured at 173 and 295 K and assigned to particular nitrogen sites (-N═ and -NH-). The temperature factor has been found negligible. The changes in the molecular skeletons, electric charge distribution, intermolecular interactions pattern, and molecular aggregations caused by oxygen replacement with sulfur and carbon replacement with nitrogen are discussed in detail. Correlations between all the principal components of the (14)N quadrupole coupling tensor have been found helpful in the search for the experimental (14)N NQR frequencies, their assignment to a particular nitrogen positions and estimation of the strength of the inter- and intramolecular interactions. The variation in the NQR parameters have been mainly related to the variation in the population of π-electron orbital. For thiouracil derivatives a general trend is that the stronger the hydrogen bond is, the lower is the asymmetry parameter, while for thymine and 6-aza-2-thiotymine, the opposite relation holds. Differences in correlations of the principal components of the (14)N quadrupole coupling tensor at the amino and iminonitrogen positions in heterocyclic rings are discussed. The effect of C→H and C→N substitution at the amino nitrogen position and C→N substitution at the iminonitrogen position on the quadrupole coupling tensor is analyzed. This study also demonstrates the advantages of combining NQR and DFT/QTAIM to predict an unsolved crystalline structure of 4-thiouracil.

  20. Effect of Hydrogen Bonding on Barrier-Free Proton Transfer in Anionic Complexes of Uracil with Weak Acids: (U...HCN){sup -} versus (U...H{sub 2}S){sup -}

    SciTech Connect

    Haranczyk, Maciej; Rak, Janusz; Gutowski, Maciej S.; Radisic, Dunja; Stokes, Sarah T.; Bowen, Kit H.

    2004-10-26

    A photoelectron spectrum is reported for an anionic complex of uracil (U) with HCN. The effects of electron attachment to a complex of U with HA (A = CN, NC) have been studied at the density functional theory level with 6-31++G** basis sets and the B3LYP and MPW1K exchange correlation functionals. Critical anionic structures have been reexamined at the MP2/6-31++G** level. The B3LYP gas phase deprotonation enthalpies are equal to 14.56, 15.13, and 15.12 eV for HNC, HCN, and H{sub 2}S, respectively. The experimental deprotonation enthalpies are 15.100 {+-} 0.008 and 15.214 {+-}0.125 eV for HCN and H{sub 2}S, respectively. Hence, HCN and H{sub 2}S have very similar deprotonation enthalpies. The photoelectron spectra of anionic complexes of uracil with HCN and H{sub 2}S are, however, very different. The (UHCN){sup -} spectrum reveals a broad feature with a maximum between 1.2-1.4 eV, whereas the main feature of the (UH{sub 2}S){sup -} spectrum has a maximum between 1.7 and 2.1 eV. We suggest that barrier-free proton transfer (BFPT) [Eur. Phys. J. D 2002, 20, 431-439; J. Phys. Chem. B 2003, 107, 7889-7895] occurs in the (UH{sub 2}S){sup -} complex but not in (UHCN){sup -}. Critical factors for the occurrence of BFPT have been analyzed. The difference between the (UHCN){sup -} and (UH{sub 2}S){sup -} complexes is attributed to differences in hydrogen bonds formed by HCN and H{sub 2}S with uracil.

  1. Uracil/ftorafur/leucovorin combined with irinotecan (TEGAFIRI) or oxaliplatin (TEGAFOX) as first-line treatment for metastatic colorectal cancer patients: results of randomised phase II study

    PubMed Central

    Bajetta, E; Di Bartolomeo, M; Buzzoni, R; Mariani, L; Zilembo, N; Ferrario, E; Lo Vullo, S; Aitini, E; Isa, L; Barone, C; Jacobelli, S; Recaldin, E; Pinotti, G; Iop, A

    2007-01-01

    This randomised phase II study evaluates the safety and efficacy profile of uracil/tegafur/leucovorin combined with irinotecan (TEGAFIRI) or with oxaliplatin (TEGAFOX). One hundred and forty-three patients with measurable, non-resectable metastatic colorectal cancer were randomised in a multicentre study to receive TEGAFIRI (UFT 250 mg m−2 day days 1–14, LV 90 mg day days 1–14, irinotecan 240 mg m−2 day 1; q21) or TEGAFOX (UFT 250 mg m−2 day days 1–14, LV 90 mg day days 1–14, oxaliplatin 120 mg m−2 day 1; q21). Among 143 randomised patients, 141 were analysed (68 received TEGAFIRI and 73 TEGAFOX). The main characteristics of the two arms were well balanced. The most common grade 3–4 treatment-related adverse events were neutropenia (13% of cases with TEGAFIRI; 1% in the TEGAFOX group). Diarrhoea was prevalent in the TEGAFIRI arm (16%) vs TEGAFOX (4%). Six complete remission (CR) and 19 partial remission (PR) were recorded in the TEGAFIRI arm (odds ratio (OR): 41.7; 95% confidence limit (CL), 29.1–55.1%), and six CR and 22 PR were recorded in the TEGAFOX group, (OR: 38.9; 95% CL, 27.6–51.1). At a median time follow-up of 17 months (intequartile (IQ) range 12–23), a median survival probability of 20 and 19 months was obtained in the TEGAFIRI and TEGAFOX groups, respectively. Median time to progression was 8 months for both groups. TEGAFIRI and TEGAFOX are both effective and tolerable first-line therapies in MCRC patients. The employment of UFT/LV given in doublet combination is interesting and the presented data appear comparable to equivalent infusion regimens described in the literature. The safety profile of the two combinations also allows an evaluation with other biological agents such as monoclonal antibodies. PMID:17245343

  2. Toward feasible and comprehensive computational protocol for simulation of the spectroscopic properties of large molecular systems: the anharmonic infrared spectrum of uracil in the solid state by the reduced dimensionality/hybrid VPT2 approach.

    PubMed

    Fornaro, Teresa; Carnimeo, Ivan; Biczysko, Malgorzata

    2015-05-28

    Feasible and comprehensive computational protocols for simulating the spectroscopic properties of large and complex molecular systems are very sought after. Indeed, due to the great variety of intra- and intermolecular interactions that may take place, the interpretation of experimental data becomes more and more difficult as the system under study increases in size or is placed in a complex environment, such as condensed phases. In this framework, we are actively developing a comprehensive and robust computational protocol aimed at quantitative reproduction of the spectra of nucleic acid base complexes, with increasing complexity toward condensed phases and monolayers of biomolecules on solid supports. We have resorted to fully anharmonic quantum mechanical computations within the generalized second-order vibrational perturbation theory (GVPT2) approach, combined with the cost-effective B3LYP-D3 method, in conjunction with basis sets of double-ζ plus polarization quality. Such an approach has been validated in a previous work ( Phys. Chem. Chem. Phys. 2014 , 16 , 10112 - 10128 ) for simulating the IR spectra of the monomers of nucleobases and some of their dimers. In the present contribution we have extended such computational protocol to simulate spectroscopic properties of a molecular solid, namely polycrystalline uracil. First we have selected a realistic molecular model for representing the spectroscopic properties of uracil in the solid state, the uracil heptamer, and then we have computed the relative anharmonic frequencies combining less demanding approaches such as the hybrid B3LYP-D3/DFTBA one, in which the harmonic frequencies are computed at a higher level of theory (B3LYP-D3/N07D) whereas the anharmonic shifts are evaluated at a lower level of theory (DFTBA), and the reduced dimensionality VPT2 (RD-VPT2) approach, where only selected vibrational modes are computed anharmonically along with the couplings with other modes. The good agreement between the

  3. Eight new crystal structures of 5-(hydroxymethyl)uracil, 5-carboxyuracil and 5-carboxy-2-thiouracil: insights into the hydrogen-bonded networks and the predominant conformations of the C5-bound residues.

    PubMed

    Seiler, Vanessa Kristina; Hützler, Wilhelm Maximilian; Bolte, Michael

    2016-05-01

    In order to examine the preferred hydrogen-bonding pattern of various uracil derivatives, namely 5-(hydroxymethyl)uracil, 5-carboxyuracil and 5-carboxy-2-thiouracil, and for a conformational study, crystallization experiments yielded eight different structures: 5-(hydroxymethyl)uracil, C5H6N2O3, (I), 5-carboxyuracil-N,N-dimethylformamide (1/1), C5H4N2O4·C3H7NO, (II), 5-carboxyuracil-dimethyl sulfoxide (1/1), C5H4N2O4·C2H6OS, (III), 5-carboxyuracil-N,N-dimethylacetamide (1/1), C5H4N2O4·C4H9NO, (IV), 5-carboxy-2-thiouracil-N,N-dimethylformamide (1/1), C5H4N2O3S·C3H7NO, (V), 5-carboxy-2-thiouracil-dimethyl sulfoxide (1/1), C5H4N2O3S·C2H6OS, (VI), 5-carboxy-2-thiouracil-1,4-dioxane (2/3), 2C5H4N2O3S·3C6H12O3, (VII), and 5-carboxy-2-thiouracil, C10H8N4O6S2, (VIII). While the six solvated structures, i.e. (II)-(VII), contain intramolecular S(6) O-H...O hydrogen-bond motifs between the carboxy and carbonyl groups, the usually favoured R2(2)(8) pattern between two carboxy groups is formed in the solvent-free structure, i.e. (VIII). Further R2(2)(8) hydrogen-bond motifs involving either two N-H...O or two N-H...S hydrogen bonds were observed in three crystal structures, namely (I), (IV) and (VIII). In all eight structures, the residue at the ring 5-position shows a coplanar arrangement with respect to the pyrimidine ring which is in agreement with a search of the Cambridge Structural Database for six-membered cyclic compounds containing a carboxy group. The search confirmed that coplanarity between the carboxy group and the cyclic residue is strongly favoured.

  4. Synthesis, crystal structure, insecticidal activity and DFT study on the geometry and vibration of O-( E)-1-{1-[(6-chloropyridin-3-yl)methyl]-5-methyl-1 H-1,2,3-triazol-4-yl}ethyleneamino- O-ethyl- O-phenylphosphorothioate

    NASA Astrophysics Data System (ADS)

    Shi, De-Qing; Zhu, Xiao-Fei; Song, Yuan-Zhi

    2008-12-01

    The title compound, O-( E)-1-{1-[(6-chloropyridin-3-yl)methyl]-5-methyl-1 H-1,2,3-triazol-4-yl}ethyleneamino- O-ethyl- O-phenylphosphorothioate, has been synthesized via the condensation reaction of 1-{1-[(6-chloropyridin-3-yl)methyl]-5-methyl-1 H-1,2,3-triazol-4-yl}ethanone oxime and O-ethyl- O-phenylphosphorochloridothioate in the presence of NaOH powder in refluxing EtOH. Its structure was characterized by 1H NMR, FTIR, Raman, elemental analysis and X-ray single crystal diffraction. The results of preliminary bioassays indicated that the title compound displays good insecticidal activity. Density functional (DFT) calculations have been carried out for the title compound by using the Becke-Lee-Yang-Parr's three-parameter hybrid functional (B3LYP) method at 6-31G** and 6-31G* basis sets. The calculated results show that the predicted geometry can well reproduce the structural parameters. The vibrational wave numbers of the title compound were calculated at same level. Predicted vibrational frequencies have been assigned and compared with experimental IR and Raman spectra and they are supported each other.

  5. Crystal structure of 3-[({2-[bis­(2-hy­droxy­benz­yl)amino]­eth­yl}(2-hy­droxy­benz­yl)amino)­meth­yl]-2-hydroxy-5-methyl­benzaldehyde

    PubMed Central

    Fonseca, Alexandra S.; Bortoluzzi, Adailton J.

    2014-01-01

    The non-symmetric title mol­ecule, C32H34N2O5, is based on a tetra­substituted ethyl­enedi­amine backbone. The mol­ecular structure consists of three hy­droxy­benzyl groups and one 2-hy­droxy-5-methyl­benzaldehyde group bonded to the N atoms of the di­amine unit. The ethyl­enedi­amine skeleton shows a regular extended conformation, while the spatial orientation of the phenol arms is governed by hydrogen bonds. In the 2-hy­droxy-5-methyl­benzaldehyde group, an intra­molecular S(6) O—H⋯O hydrogen bond is observed between the alcohol and aldehyde functions, and the neighbouring phenol arm participates in an intra­molecular S(6) O—H⋯N hydrogen bond. The third phenol group is involved in a bifurcated intra­molecular hydrogen bond with graph-set notation S(6) for O—H⋯N and O—H⋯O intra­molecular hydrogen bonds between neighbouring amine and phenol arms, respectively. Finally, the fourth phenol group acts as an acceptor in a bifurcated intra­molecular hydrogen bond and also acts as donor in an inter­molecular hydrogen bond, which connects inversion-related mol­ecules into dimers with R 4 4(8) ring motifs. PMID:25552993

  6. The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: a possible molecular basis for the site-specific cancer risk modification.

    PubMed

    Sohn, Kyoung-Jin; Jang, Hyeran; Campan, Mihaela; Weisenberger, Daniel J; Dickhout, Jeffrey; Wang, Yi-Cheng; Cho, Robert C; Yates, Zoe; Lucock, Mark; Chiang, En-Pei; Austin, Richard C; Choi, Sang-Woon; Laird, Peter W; Kim, Young-In

    2009-05-01

    The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation.

  7. Comment on "Structural and vibrational studies on 1-(5-Methyl- [1,3,4] thiadiazol-2-yl)-pyrolidin-2-ol" [Spectrochimica Acta Part A, 152 (2016) 252-261]. The importance of intramolecular OH ⋯ N hydrogen bonding in the conformational properties of thiadiazol-pyrrolidin-2-ol bearing species

    NASA Astrophysics Data System (ADS)

    Laurella, Sergio L.; Erben, Mauricio F.

    2016-07-01

    The title paper [1] reports a study on the spectroscopic and physicochemical properties of 1-(5-methyl- [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol (MTPN) based on experimental and theoretical data. The latter ones are based on the computed molecular structure for a rather unusual conformer. Here, after a careful analysis of the conformational space of MTPN, the most stable conformation was determined for the molecule isolated in a vacuum, which results to be 21.9 kJ/mol more stable than the conformer reported previously. Our study also includes the closely related species 1-(5-trifluoromethyl- [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol (FMTPN). An intramolecular OH ⋯ N hydrogen bond determines the conformational behavior of the [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol group as demonstrated by Natural Bond Orbital population analysis.

  8. Comment on "Structural and vibrational studies on 1-(5-Methyl- [1,3,4] thiadiazol-2-yl)-pyrolidin-2-ol" [Spectrochimica Acta Part A, 152 (2016) 252-261]. The importance of intramolecular OH⋯N hydrogen bonding in the conformational properties of thiadiazol-pyrrolidin-2-ol bearing species.

    PubMed

    Laurella, Sergio L; Erben, Mauricio F

    2016-07-05

    The title paper [1] reports a study on the spectroscopic and physicochemical properties of 1-(5-methyl- [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol (MTPN) based on experimental and theoretical data. The latter ones are based on the computed molecular structure for a rather unusual conformer. Here, after a careful analysis of the conformational space of MTPN, the most stable conformation was determined for the molecule isolated in a vacuum, which results to be 21.9kJ/mol more stable than the conformer reported previously. Our study also includes the closely related species 1-(5-trifluoromethyl- [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol (FMTPN). An intramolecular OH⋯N hydrogen bond determines the conformational behavior of the [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol group as demonstrated by Natural Bond Orbital population analysis.

  9. Balance of Attraction and Repulsion in Nucleic-Acid Base Stacking: CCSD(T)/Complete-Basis-Set-Limit Calculations on Uracil Dimer and a Comparison with the Force-Field Description

    SciTech Connect

    Morgado, Claudio A.; Jurecka, Petr; Svozil, Daniel; Hobza, Pavel; Sponer, Jiri

    2009-06-09

    We have carried out reference quantum-chemical calculations for about 100 geometries of the uracil dimer in stacked conformations. The calculations have been specifically aimed at geometries with unoptimized distances between the monomers including geometries with mutually tilted monomers. Such geometries are characterized by a delicate balance between local steric clashes and local unstacking and had until now not been investigated using reference quantummechanics (QM) methods. Nonparallel stacking geometries often occur in nucleic acids and are of decisive importance, for example, for local conformational variations in B-DNA. Errors in the shortrange repulsion region would have a major impact on potential energy scans which were often used in the past to investigate local geometry variations in DNA. An incorrect description of such geometries may also partially affect molecular dynamics (MD) simulations in applications when quantitative accuracy is required. The reference QM calculations have been carried out using the MP2 method extrapolated to the complete basis-set limit and corrected for higher-order electron-correlation contributions using CCSD(T) calculations with a medium-sized basis set. These reference calculations have been used as benchmark data to test the performance of the DFT-D, SCS(MI)-MP2, and DFTSAPT QM methods and of the AMBER molecular-mechanics (MM) force field. The QM methods show close to quantitative agreement with the reference data, albeit the DFT-D method tends to modestly exaggerate the repulsion of steric clashes. The force field in general also provides a good description of base stacking for the systems studied here. However, for geometries with close interatomic contacts and clashes, the repulsion effects are rather severely exaggerated. The discrepancy reported here should not affect the overall stability of MD simulations and qualitative applications of the force field. However, it may affect the description of subtle

  10. Simultaneous determination of 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) uracil (FAU) and 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) 5-methyluracil (FMAU) in human plasma by liquid chromatography/tandem mass spectrometry.

    PubMed

    Wiegand, Richard; Wu, Jianmei; Shields, Anthony F; Lorusso, Patricia; Li, Jing

    2012-04-01

    A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) uracil (FAU) and its active metabolite 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl) 5-methyluracil (FMAU) in human plasma. FAU and FMAU were extracted from plasma samples using solid-phase extraction with Waters Sep-Pak® Vac C₁₈ cartridge. Chromatographic separation was achieved on a Waters Atlantis T3 C₁₈ column with a gradient mobile phase consisting of methanol and water with 0.45% formic acid (v/v) running at a flow rate of 0.2 ml/min. The analytes were monitored by triple quadrupole mass spectrometer under positive ionization mode. The lower limit of quantitation (LLOQ) was 10 and 2 ng/ml for FAU and FMAU in plasma, respectively. Calibration curves were linear over FAU and FMAU plasma concentration range of 10-2000 and 2-1000 ng/ml, respectively. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the plasma pharmacokinetics of FAU and FMAU in cancer patients receiving 1-h intravenous infusion of FAU 50 mg/m².

  11. The products of the reaction between 6-amine-1,3-dimethyl uracil and bis-chalcones induce cytotoxicity with massive vacuolation in HeLa cervical cancer cell line.

    PubMed

    Solano, José D; González-Sánchez, Ignacio; Cerbón, Marco A; Guzmán, Ángel; Martínez-Urbina, Miguel A; Vilchis-Reyes, Miguel A; Martínez-Zuñiga, Eduardo C; Alvarado, Cuauhtémoc; Quintero, Angelina; Díaz, Eduardo

    2013-02-01

    As a part of our research in the chemistry of chalcones we have prepared four pyrimidine monoadducts of bis-chalcones through the reaction with 6-amino-1,3-dimethyl uracil. These compounds displayed cytotoxicity with a massive vacuolation in different human cell lines in vitro. Compound 6 was the most cytotoxic inducer of vacuoles, this compound induced G1 phase cell cycle arrest, and their cytotoxicity went without morphological and biochemical evidence of apoptotic cell death in HeLa cells. In addition, our results showed that this vacuole formation does not require de novo protein synthesis and the content vacuolar is acidic. Compound 6 induce necrotic cell death with excessive vacuolation, similar to a process of autophagy. Spautin-1 an inhibitor of autophagy, decreased the transformation of microtubule-associated protein 1 light chain 3 (LC3B-I) to LC3B-II and the vacuolation induced by compound 6 in HeLa cells, both autophagy processes. These compounds could be of pivotal importance in the study of non-apoptotic cell death with vacuole formation and could be useful in research into new autophagy inhibitors agents.

  12. 2-Amino-5-methyl-pyridinium 2-amino-benzoate.

    PubMed

    Thanigaimani, Kaliyaperumal; Farhadikoutenaei, Abbas; Khalib, Nuridayanti Che; Arshad, Suhana; Razak, Ibrahim Abdul

    2012-11-01

    In the 2-amino-benzoate anion of the title salt, C(6)H(9)N(2) (+)·C(7)H(6)NO(2) (-), an intra-molecular N-H⋯O hydrogen bond is observed. The dihedral angle between the ring and the CO(2) group is 8.41 (13)°. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl-ate O atoms via a pair of N-H⋯O hydrogen bonds, forming an R(2) (2)(8) ring motif. The ion pairs are further connected via N-H⋯O hydrogen bonds, resulting in a donor-donor-acceptor-acceptor (DDAA) array of quadruple hydrogen bonds. The crystal structure also features a weak N-H⋯O hydrogen bond and a C-H⋯π inter-action, resulting in a three-dimensional network.

  13. Synthesis, biological evaluation, and automated docking of constrained analogues of the opioid peptide H-Dmt-D-Ala-Phe-Gly-NH₂ using the 4- or 5-methyl substituted 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one scaffold.

    PubMed

    De Wachter, Rien; de Graaf, Chris; Keresztes, Atilla; Vandormael, Bart; Ballet, Steven; Tóth, Géza; Rognan, Didier; Tourwé, Dirk

    2011-10-13

    The Phe(3) residue of the N-terminal tetrapeptide of dermorphin (H-Dmt-d-Ala-Phe-Gly-NH(2)) was conformationally constrained using 4- or 5-methyl-substituted 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) stereoisomeric scaffolds. Several of the synthesized peptides were determined to be high affinity agonists for the μ opioid receptor (OPRM) with selectivity over the δ opioid receptor (OPRD). Interesting effects of the Aba configuration on ligand binding affinity were observed. H-Dmt-d-Ala-erythro-(4S,5S)-5-Me-Aba-Gly-NH(2)9 and H-Dmt-threo-(4R,5S)-5-Me-Aba-Gly-NH(2)12 exhibited subnanomolar affinity for OPRM, while they possess an opposite absolute configuration at position 4 of the Aba ring. However, in the 4-methyl substituted analogues, H-Dmt-d-Ala-(4R)-Me-Aba-Gly-NH(2)14 was significantly more potent than the (4S)-derivative 13. These unexpected results were rationalized using the binding poses predicted by molecular docking simulations. Interestingly, H-Dmt-d-Ala-(4R)-Me-Aba-Gly-NH(2)14 is proposed to bind in a different mode compared with the other analogues. Moreover, in contrast to Ac-4-Me-Aba-NH-Me, which adopts a β-turn in solution and in the crystal structure, the binding mode of this analogue suggests an alternative receptor-bound conformation.

  14. Synthesis, DFT calculations, electronic structure, electronic absorption spectra, natural bond orbital (NBO) and nonlinear optical (NLO) analysis of the novel 5-methyl-8H-benzo[h]chromeno[2,3-b][1,6] naphthyridine-6(5H),8-dione (MBCND)

    NASA Astrophysics Data System (ADS)

    Halim, Shimaa Abdel; Ibrahim, Magdy A.

    2017-02-01

    New derivative of heteroannulated chromone identified as 5-methyl-8H-benzo[h]chromeno[2,3-b][1,6]naphthyridine-6(5H),8-dione (5, MBCND) was easily and efficiently synthesized from DBU catalyzed condensation reaction of 2-aminochromone-3-carboxaldehyde (1) with 4-hydroxy-1-methylquinolin-2(1H)-one (2). The same product 5 was isolated from condensation reaction of aldeyde 1 with 3-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-3-oxopropanoic acid (3) or ethyl 4-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinolin-3-yl)-2,4-dioxobutanoate (4). Structure of compound (5, MBCND) was deduced based on their elemental analyses and spectral data (IR, 1H NMR and mass spectra). Density Functional Theory (DFT) calculations at the B3LYP/6-311G (d,p) level of theory have been carried out to investigate the equilibrium geometry of the novel compound (5, MBCND). Moreover, total energy, energy of HOMO and LUMO and Mullikan atomic charges were calculated. In addition, the dipole moment, theoretical study of the electronic structure, nonlinear optical properties (NLO), and natural bonding orbital (NBO) analysis and orientation have been performed and discussed. Also the electronic absorption spectra were measured in polar (methanol) as well as non polar (dioxane) solvents and the assignment of the observed bands has been discussed by TD-DFT calculations. The correspondences between calculated and experimental transitions energies are satisfactory.

  15. Electron Detachment as a Probe of Intrinsic Nucleobase Dynamics in Dianion-Nucleobase Clusters: Photoelectron Spectroscopy of the Platinum II Cyanide Dianion Bound to Uracil, Thymine, Cytosine, and Adenine.

    PubMed

    Sen, Ananya; Hou, Gao-Lei; Wang, Xue-Bin; Dessent, Caroline E H

    2015-09-03

    We report the first low-temperature photoelectron spectra of isolated gas-phase complexes of the platinum II cyanide dianion bound to nucleobases. These systems are models for understanding platinum-complex photodynamic therapies, and a knowledge of the intrinsic photodetachment properties is crucial for characterizing their broader photophysical properties. Well-resolved, distinct peaks are observed in the spectra, consistent with complexes where the Pt(CN)4(2-) moiety is largely intact. Adiabatic electron detachment energies for the dianion-nucleobase complexes are measured to be 2.39-2.46 eV. The magnitudes of the repulsive Coulomb barriers of the complexes are estimated to be between 1.9 and 2.1 eV, values that are lower than for the bare Pt(CN)4(2-) dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four dianion-nucleobase complexes and also in the 266 nm spectra of the Pt(CN)4(2-)·thymine and Pt(CN)4(2-)·adenine complexes. The selective excitation of these features in the 266 nm spectra is attributed to one-photon excitation of [Pt(CN)4(2-)·thymine]* and [Pt(CN)4(2-)·adenine]* long-lived excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment signals. We attribute the delayed electron detachment bands observed here for Pt(CN)4(2-)·thymine and Pt(CN)4(2-)·adenine but not for Pt(CN)4(2-)·uracil and Pt(CN)4(2-)·cytosine to fundamental differences in the individual nucleobase photophysics following 266 nm excitation. This indicates that the Pt(CN)4(2-) dianion in the clusters can be viewed as a "dynamic tag" which has the propensity to emit electrons when the attached nucleobase displays a long-lived excited state.

  16. Magnetic behavior of MnPS{sub 3} phases intercalated by [Zn{sub 2}L]{sup 2+} (LH{sub 2}: macrocyclic ligand obtained by condensation of 2-hydroxy-5-methyl-1,3-benzenedicarbaldehyde and 1,2-diaminobenzene)

    SciTech Connect

    Spodine, E.; Valencia-Galvez, P.; Fuentealba, P.; Manzur, J.; Ruiz, D.; Venegas-Yazigi, D.; Paredes-Garcia, V.; Cardoso-Gil, R.; Schnelle, W.; Kniep, R.

    2011-05-15

    The intercalation of the cationic binuclear macrocyclic complex [Zn{sub 2}L]{sup 2+} (LH{sub 2}: macrocyclic ligand obtained by the template condensation of 2-hydroxy-5-methyl-1,3-benzenedicarbaldehyde and 1,2-diaminobenzene) was achieved by a cationic exchange process, using K{sub 0.4}Mn{sub 0.8}PS{sub 3} as a precursor. Three intercalated materials were obtained and characterized: (Zn{sub 2}L){sub 0.05}K{sub 0.3}Mn{sub 0.8}PS{sub 3}(1), (Zn{sub 2}L){sub 0.1}K{sub 0.2}Mn{sub 0.8}PS{sub 3}(2) and (Zn{sub 2}L){sub 0.05}K{sub 0.3}Mn{sub 0.8}PS{sub 3}(3), the latter phase being obtained by an assisted microwave radiation process. The magnetic data permit to estimate the Weiss temperature {theta} of {approx}-130 K for (1); {approx}-155 K for (2) and {approx}-130 K for (3). The spin canting present in the potassium precursor remains unperturbed in composite (3), and spontaneous magnetization is observed under 50 K in both materials. However composites (1) and (2) do not present this spontaneous magnetization at low temperatures. The electronic properties of the intercalates do not appear to be significantly altered. The reflectance spectra of the intercalated phases (1), (2) and (3) show a gap value between 1.90 and 1.80 eV, lower than the value observed for the K{sub 0.4}Mn{sub 0.8}PS{sub 3} precursor of 2.8 eV. -- Graphical Abstract: Microwave assisted synthesis was used to obtain an intercalated MnPS{sub 3} phase with a binuclear Zn(II) macrocyclic complex. A comparative magnetic study of the composites obtained by assisted microwave and traditional synthetic methods is reported. Display Omitted Highlights: {yields} A rapid and efficient preparation of intercalated MnPS{sub 3} composites by assisted microwave synthesis is described. {yields} The exchange of potassium ions of the precursor by the macrocyclic Zn(II) complex is partial. {yields} The composite obtained by assisted microwave synthesis retains the spontaneous magnetization, observed in the low temperature

  17. Spectroscopic Studies of Fluorescence Effects in Bioactive 4-(5-Heptyl-1,3,4-Thiadiazol-2-yl)Benzene-1,3-Diol and 4-(5-Methyl-1,3,4-Thiadiazol-2-yl)Benzene-1,3-Diol Molecules Induced by pH Changes in Aqueous Solutions.

    PubMed

    Matwijczuk, Arkadiusz; Kluczyk, Dariusz; Górecki, Andrzej; Niewiadomy, Andrzej; Gagoś, Mariusz

    2017-03-01

    This paper presents the results of stationary fluorescence spectroscopy and time-resolved spectroscopy analyses of two 1,3,4-thiadiazole analogues, i.e. 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C1) and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C7) in an aqueous medium containing different concentrations of hydrogen ions. An interesting dual florescence effect was observed when both compounds were dissolved in aqueous solutions at pH below 7 for C1 and 7.5 for C7. In turn, for C1 and C7 dissolved in water at pH higher than the physiological value (mentioned above), single fluorescence was only noted. Based on previous results of investigations of the selected 1,3,4-thiadiazole compounds, it was noted that the presented effects were associated with both conformational changes in the analysed molecules and charge transfer (CT) effects, which were influenced by the aggregation factor. However, in the case of C1 and C7, the dual fluorescence effects were visible in a higher energetic region (different than that observed in the 1,3,4-thiadiazoles studied previously). Measurements of the fluorescence lifetimes in a medium characterised by different concentrations of hydrogen ions revealed clear lengthening of the excited-state lifetime in a pH range at which dual fluorescence effects can be observed. An important finding of the investigations presented in this article is the fact that the spectroscopic effects observed not only are interesting from the cognitive point of view but also can help in development of an appropriate theoretical model of molecular interactions responsible for the dual fluorescence effects in the analysed 1,3,4-thiadiazoles. Furthermore, the study will clarify a broad range of biological and pharmaceutical applications of these compounds, which are more frequently used in clinical therapies. Graphical Abstract Upper left corner - C7 molecule at high pH, right upper corner - fluorescence emission spectrum for C7 dissolved in H2

  18. Communication: Electronic UV-Vis transient spectra of the ∙OH reaction products of uracil, thymine, cytosine, and 5,6-dihydrouracil by using the complete active space self-consistent field second-order perturbation (CASPT2//CASSCF) theory.

    PubMed

    Francés-Monerris, Antonio; Merchán, Manuela; Roca-Sanjuán, Daniel

    2013-08-21

    Addition of ∙OH radicals to pyrimidine nucleobases is a common reaction in DNA/RNA damage by reactive oxygen species. Among several experimental techniques, transient absorption spectroscopy has been during the last decades used to characterize such compounds. Discrepancies have however appeared in the assignment of the adduct or adducts responsible for the reported transient absorption UV-Vis spectra. In order to get an accurate assignment of the transient spectra and a unified description of the absorption properties of the ∙OH reaction products of pyrimidines, a systematic complete active space self-consistent field second-order perturbation (CASPT2//CASSCF) theory study has been carried out on the uracil, thymine, and cytosine ∙OH addition adducts, as well as on the 5,6-dihydrouracil hydrogen abstraction products. With the obtained findings, the C5OH contributions to the lowest-energy band can be finally discarded. Instead, a bright (2)(π2) state of the C6OH adducts is determined to be the main responsible in all compounds for the absorption band in the Vis range.

  19. Ca2+ sensitization in idiopathic dilated human myocardium. Differential in vitro effects of (+)-(5-methyl-6-phenyl)-1,3,5,6-tetrahydro-3,6-methano-1,5-benzodiazoci ne-2,4-dione, a novel purely Ca2+sensitizing agent, and (+)-5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-6-meth yl-3, 6-dihydro-2H-1,3,4-thiadiazin-2-one on skinned fibres and isolated ventricular strips.

    PubMed

    Herzig, J W; Chiesi, M; Depersin, H; Grüninger, S; Hasenfuss, G; Kubalek, R; Leutert, T; Pieske, B; Pioch, K; Wenk, P; Holubarsch, C

    1996-06-01

    (+)-(5-Methyl-6-phenyl)-1,3,5,6-tetrahydro-3,6-methano-1, 5-benzodiazocine-2,4-dione (CAS 165755-40-8, CGP 48506) is a novel Ca2+ sensitizing agent devoid of any other positive inotropic mechanism, particularly phosphodiesterase (PDE) III inhibition. 5-(1-(3,4-Dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-6-met hyl-3, 6-dihydro-2H-1,3,4-thiadiazin-2-one (CAS 120223-04-3, EMD 53998) is a PDE III inhibitor with a Ca2+ sensitizing activity residing in its (+)-enantiomer, EMD 57033 (CAS 147527-31-9). In skinned fibres and electrically stimulated left ventricular strips from idiopathic dilated human hearts, New York Heart Association (NYHA) class IV, the Ca2+ sensitizing and inotropic effects of the benzodiazocine CGP 48506 and the thiadiazinones EMD 53998 or EMD 57033 were compared. Both CGP 48506 and EMD 53998 induce a left shift of the Ca2+ activation curve of force towards lower Ca2+ concentrations in skinned fibres, which indicates Ca2+ sensitization. Only EMD 53998, but not CGP 48506, increases skinned fibre force at both minimum (resting) and maximally activating Ca2+ concentrations. This is taken as an argument for a principal difference in the mechanisms of the Ca2+ sensitizing actions of the two compounds. CGP 48506 is shown not to influence the amplitude of the Ca2+ transient in rat cardiomyocytes. On the other hand, both CGP 48506 and EMD 57033 show comparable, though quantitatively different, positive inotropic effects in electrically stimulated left ventricular strip preparations. It is unclear whether the PDE III inhibitory component of the profile of actions of EMD 57033 may play a role in preventing the increase in diastolic tension as expected from the skinned fibre experiments. It is noteworthy that both Ca2+ sensitizing agents act as positive inotropic compounds in the end-stage failing human heart where other inotropic agents like beta 1-adrenergic agonists or PDE inhibitors have been described to fail.

  20. Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression

    PubMed Central

    Ponnaluri, V. K. Chaithanya; Ehrlich, Kenneth C.; Zhang, Guoqiang; Lacey, Michelle; Johnston, Douglas; Pradhan, Sriharsa; Ehrlich, Melanie

    2017-01-01

    ABSTRACT Differentially methylated or hydroxymethylated regions (DMRs) in mammalian DNA are often associated with tissue-specific gene expression but the functional relationships are still being unraveled. To elucidate these relationships, we studied 16 human genes containing myogenic DMRs by analyzing profiles of their epigenetics and transcription and quantitatively assaying 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) at specific sites in these genes in skeletal muscle (SkM), myoblasts, heart, brain, and diverse other samples. Although most human promoters have little or no methylation regardless of expression, more than half of the genes that we chose to study—owing to their myogenic DMRs—overlapped tissue-specific alternative or cryptic promoters displaying corresponding tissue-specific differences in histone modifications. The 5mC levels in myoblast DMRs were significantly associated with 5hmC levels in SkM at the same site. Hypermethylated myogenic DMRs within CDH15, a muscle- and cerebellum-specific cell adhesion gene, and PITX3, a homeobox gene, were used for transfection in reporter gene constructs. These intragenic DMRs had bidirectional tissue-specific promoter activity that was silenced by in vivo-like methylation. The CDH15 DMR, which was previously associated with an imprinted maternal germline DMR in mice, had especially strong promoter activity in myogenic host cells. These findings are consistent with the controversial hypothesis that intragenic DNA methylation can facilitate transcription and is not just a passive consequence of it. Our results support varied roles for tissue-specific 5mC- or 5hmC-enrichment in suppressing inappropriate gene expression from cryptic or alternative promoters and in increasing the plasticity of gene expression required for development and rapid responses to tissue stress or damage. PMID:27911668

  1. Crystal structure of N-(2-hy-droxy-5-methyl-phen-yl)benzamide.

    PubMed

    Moreno-Fuquen, Rodolfo; Mariño, Nory J; Kennedy, Alan R

    2015-12-01

    In the title compound, C14H13NO2, the mean plane of the non-H atoms of the central amide fragment C-N-C(=O)-C (r.m.s. deviation = 0.029 Å) forms dihedral angles of 5.63 (6) and 10.20 (5)° with the phenyl and hy-droxy-phenyl rings, respectively. A short intra-molecular N-H⋯O contact is present. In the crystal, the mol-ecules are linked by O-H⋯O hydrogen bonds to generate C(7) chains along [100]. The chains are reinforced by weak C-H⋯O contacts, which together with the O-H⋯O bonds lead to R 2 (2)(7) loops. Very weak N-H⋯O inter-actions link the mol-ecules into inversion dimers.

  2. 2-Amino-5-methyl­pyridinium 3-chloro­benzoate

    PubMed Central

    Thanigaimani, Kaliyaperumal; Farhadikoutenaei, Abbas; Khalib, Nuridayanti Che; Arshad, Suhana; Razak, Ibrahim Abdul

    2012-01-01

    The 3-chloro­benzoate anion of the title salt, C6H9N2 +·C7H4ClO2 −, is nearly planar with a dihedral angle of 2.44 (13)° between the benzene ring and the carboxyl­ate group. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl­ate O atoms of the anion via a pair of N—H⋯O hydrogen bonds with an R 2 2(8) ring motif, forming an approximately planar ion pair with a dihedral angle of 7.92 (5)° between the pyridinium and benzene rings. The ion pairs are further connected via N—H⋯O and C—H⋯O hydrogen bonds, forming a two-dimensional network parallel to the bc plane. PMID:23284506

  3. 2-Amino-5-methyl­pyridinium 4-chloro­benzoate

    PubMed Central

    Thanigaimani, Kaliyaperumal; Farhadikoutenaei, Abbas; Arshad, Suhana; Razak, Ibrahim Abdul

    2013-01-01

    The 4-chloro­benzoate anion of the title salt, C6H9N2 +·C7H4ClO2 −, is nearly planar with a dihedral angle of 5.14 (16)° between the benzene ring and the carboxyl­ate group. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl­ate O atoms of the anion via a pair of N—H⋯O hydrogen bonds with an R 2 2(8) ring motif. The ion pairs are further connected via N—H⋯O and weak C—H⋯O hydrogen bonds, forming a two-dimensional network parallel to the bc plane. The crystal structure also features a π–π stacking inter­action between the pyridinium and benzene rings with a centroid–centroid distance of 3.7948 (9) Å. PMID:23476392

  4. 2-Amino-5-methyl­pyridinium 4-methyl­benzoate

    PubMed Central

    Thanigaimani, Kaliyaperumal; Farhadikoutenaei, Abbas; Arshad, Suhana; Razak, Ibrahim Abdul

    2013-01-01

    The 4-methyl­benzoate anion of the title salt, C6H9N2 +·C8H7O2 −, is nearly planar, with a dihedral angle of 6.26 (10)° between the benzene ring and the carboxyl­ate group. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl­ate O atoms of the anion via a pair of N—H⋯O hydrogen bonds with an R 2 2(8) ring motif, forming an approximately planar ion pair with a dihedral angle of 9.63 (4)° between the pyridinium and benzene rings. The ion pairs are further connected via N—H⋯O and weak C—H⋯O hydrogen bonds, forming a two-dimensional network parallel to the bc plane. PMID:23476474

  5. 2-Amino-5-methyl­pyridinium trifluoro­acetate

    PubMed Central

    Thanigaimani, Kaliyaperumal; Farhadikoutenaei, Abbas; Khalib, Nuridayanti Che; Arshad, Suhana; Razak, Ibrahim Abdul

    2012-01-01

    In the title salt, C6H9N2 +·C2F3O2 −, the F atoms of the anion are disordered over two sets of sites, with refined occupancies in a ratio of 0.505 (17):0.495 (17). In the crystal, cations and anions are linked via N—H⋯O hydrogen bonds, forming R 2 2(8) ring motifs. The ionic units are linked into a two-dimensional network parallel to (100) by N—H⋯O and weak C—H⋯O hydrogen bonds. The crystal structure is further stabilized by weak C—H⋯F hydrogen bonds, resulting in a three-dimensional network. PMID:23476163

  6. Bromidotetra-kis-(1H-2-ethyl-5-methyl-imidazole-κN)copper(II) bromide.

    PubMed

    Godlewska, Sylwia; Baranowska, Katarzyna; Socha, Joanna; Dołęga, Anna

    2011-12-01

    The Cu(II) ion in the title compound, [CuBr(C(6)H(10)N(2))(4)]Br, is coordinated in a square-based-pyramidal geometry by the N atoms of four imidazole ligands and a bromide anion in the apical site. Both the Cu(II) and Br(-) atoms lie on a crystallographic fourfold axis. In the crystal, the [CuBr(C(6)H(10)N(2))(4)](+) complex cations are linked to the uncoordinated Br(-) anions (site symmetry [Formula: see text]) by N-H⋯Br hydrogen bonds, generating a three-dimensional network. The ethyl group of the imidazole ligand was modelled as disordered over two orientations with occupancies of 0.620 (8) and 0.380 (8).

  7. 2-Isopropyl-4-meth­oxy-5-methyl­phenyl benzoate

    PubMed Central

    Moumou, Mohamed; Akssira, Mohamed; Elhakmaoui, Ahmed; El Ammari, Lahcen; Benharref, Ahmed; Berraho, Moha

    2010-01-01

    The title compound, C18H20O3, a hemisynthetic product, was obtained by the reaction of benzoyl chloride and p-methoxy­thymol. The structure comprises two benzene rings bridged by a carboxyl group; the dihedral angle between the rings is 73.54 (8)°. PMID:21580674

  8. 2-Amino-5-methyl­pyridinium 2-amino­benzoate

    PubMed Central

    Thanigaimani, Kaliyaperumal; Farhadikoutenaei, Abbas; Khalib, Nuridayanti Che; Arshad, Suhana; Razak, Ibrahim Abdul

    2012-01-01

    In the 2-amino­benzoate anion of the title salt, C6H9N2 +·C7H6NO2 −, an intra­molecular N—H⋯O hydrogen bond is observed. The dihedral angle between the ring and the CO2 group is 8.41 (13)°. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl­ate O atoms via a pair of N—H⋯O hydrogen bonds, forming an R 2 2(8) ring motif. The ion pairs are further connected via N—H⋯O hydrogen bonds, resulting in a donor–donor–acceptor–acceptor (DDAA) array of quadruple hydrogen bonds. The crystal structure also features a weak N—H⋯O hydrogen bond and a C—H⋯π inter­action, resulting in a three-dimensional network. PMID:23284507

  9. 8-Bromo-3-(cyclo­propanylcarbon­yl)-5-methyl­indolizine-1-carbonitrile

    PubMed Central

    Fan, Dahe; Tang, Fan; Wang, Wei

    2012-01-01

    The asymmetric unit of the title compound, C14H11BrN2O, contains three independent mol­ecules with very similar geometries. The dihedral angles between the side chain of the cyclo­propyl plane and the five-membered ring to which it is attached are 55.0 (2), 58.1 (2) and 60.2 (3)° for the three mol­ecules. Each mol­ecule forms an intra­molecular C—H⋯O hydrogen bond. PMID:22719653

  10. New Boron-Nitrogen Analogues of Uracil Derivatives

    DTIC Science & Technology

    1989-07-01

    obtained on treatment of the betaine 1,3,5-trimehyl-2-(N, N’, N"- trimetLhlbiuret-l-vi)-1,3,5-triaza-2-boracyclohexa-4,6-dione 6a [5] with anhydrous ...quantitative yield on treatment of the betaine 6a [5] with anhydrous dimethylamine in chloroform solution. After evaporation of excess amine and solvent...of the salts 4a--4c under vacuum, the betaine 5a was obtained. ,F R\\ N- I 11 R’ R II 0 0 5 a: R = RI = CH3 b: R = CH3, RI = C2 H5 c: R = CH 3, RI

  11. Increase in the photoreactivity of uracil derivatives by doubling thionation.

    PubMed

    Pollum, M; Jockusch, S; Crespo-Hernández, C E

    2015-11-07

    The ability of 4-thiouracil to strongly absorb UVA radiation and to populate a reactive triplet state in high yield has enabled its use as a versatile photocrosslinker for nearly 50 years. In this contribution, we present a detailed spectroscopic and photochemical investigation of the 2-thiouracil, 4-thiouracil, and 2,4-dithiouracil series in an effort to further advance this chemistry and to scrutinize the photoreactivity of 2,4-dithiouracil. Our results reveal that excitation of 2,4-dithiouracil leads to intersystem crossing to the triplet manifold in 220 ± 40 fs, which enables the population of the reactive triplet state with near unity yield (ΦT = 0.90 ± 0.15) and ultimately leads to a ca. 50% singlet oxygen generation (ΦΔ = 0.49 ± 0.02)-one of the highest singlet oxygen yields reported to date for a photoexcited thiobase. In addition, the long-lived triplet state of 2,4-dithiouracil reacts efficiently with the nucleic acid base adenine 5'-monophosphate through a direct, oxygen-independent photocycloaddition mechanism and at a rate that is at least 3-fold faster than that of 4-thiouracil under equal conditions. The new physico-chemical insights reported for these RNA-thiobase derivatives are compared to those of the DNA and RNA bases and the DNA-thiobase derivatives. Furthermore, the strong near-visible absorption and increased photoreactivity measured for 2,4-dithiouracil lays a solid foundation for developing RNA-targeted photocrosslinking and phototherapeutic agents that are more effective than those currently available.

  12. Methotrexate-induced misincorporation of uracil into DNA

    PubMed Central

    Goulian, M.; Bleile, B.; Tseng, B. Y.

    1980-01-01

    A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [3H]dUrd contained dUMP in DNA in readily detectable amounts (≈0.8 pmol of dUMP per μmol of total DNA nucleotide), and this was increased ≈3-fold if the cells were also treated with Ura at the same time. No dUMP (<1 fmol/μmol of DNA) could be detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompany inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. The results suggest an active lesion of DNA in cells in which thymidylate synthetase is inhibited. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general. Images PMID:6929529

  13. Aquadi-n-but­yl(5-methyl­pyrazine-2-carboxyl­ato)tin(IV) methanol solvate

    PubMed Central

    Gao, Zhongjun

    2008-01-01

    In the monomeric title compound, [Sn(C4H9)2(C6H5N2O2)2(H2O)]·CH3OH, the Sn atom is seven-coordinate, displaying a distorted penta­gonal bipyramidal SnC2N2O3 geometry with the two C atoms in the axial sites. In the crystal structure, inter­molecular O—H⋯O hydrogen bonds link the complex and solvent mol­ecules into infinite chains. PMID:21202739

  14. 1,2-Bis(2-hy­droxy-5-methyl­benzyl­idene)hydrazine

    PubMed Central

    Saravanan, B.; Jayamani, A.; Sengottuvelan, N.; Chakkaravarthi, G.; Manivannan, V.

    2013-01-01

    The mol­ecular structure of the title compound, C16H16N2O2, is stabilized by intra­molecular O—H⋯N hydrogen bonds with S(6) graph-set motifs, so that the mol­ecule is almost planar, with a C=N—N=C torsion angle of −179.7 (2)° and a dihedral angle of 1.82 (12)° between the aromatic rings. In the crystal, weak C—H⋯π inter­actions lead to the formation of a three-dimensional network. PMID:24427035

  15. 2-Hy­droxy-3-meth­oxy­methyl-5-methyl­benzaldehyde

    PubMed Central

    Gunasekaran, B.; Jayamani, A.; Sengottuvelan, N.; Chakkaravarthi, G.

    2013-01-01

    In the title mol­ecule, C10H12O3, all non-H atoms lie in a common plane (r.m.s deviation = 0.010 Å). The mol­ecular conformation is stabilized by an intra­molecular O—H⋯O hydrogen bond. PMID:23424581

  16. 2-Amino-5-methyl­pyridinium 2-hy­droxy-5-chloro­benzoate

    PubMed Central

    Thanigaimani, Kaliyaperumal; Farhadikoutenaei, Abbas; Arshad, Suhana; Razak, Ibrahim Abdul

    2013-01-01

    In the 5-chloro­salicylate anion of the title salt, C6H9N2 +·C7H4ClO3 −, an intra­molecular O—H⋯O hydrogen bond with an S(6) graph-set motif is observed and the dihedral angle between the benzene ring and the –CO2 group is 1.6 (6)°. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl­ate O atoms via a pair of N—H⋯O hydrogen bonds, forming an R 2 2(8) ring motif. The crystal structure also features N—H⋯O and weak C—H⋯O inter­actions, resulting in a layer parallel to (10-1). PMID:23476391

  17. 2-Amino-5-methyl­pyridinium 3-hy­droxy­pyridine-2-carboxyl­ate

    PubMed Central

    Farhadikoutenaei, Abbas; Thanigaimani, Kaliyaperumal; Arshad, Suhana; Razak, Ibrahim Abdul

    2013-01-01

    In the 3-hy­droxy­picolinate anion of the title salt, C6H9N2 +·C6H4NO3 −, an intra­molecular O—H⋯O hydrogen bond with an S(6) graph-set motif is formed, so that the anion is essentially planar, with a dihedral angle of 9.55 (9)° between the pyridine ring and the carboxyl­ate group. In the crystal, the cations and anions are linked via N—H⋯O hydrogen bonds, forming a centrosymmetric 2 + 2 aggregate with R 2 2(8) and R 4 2(8) ring motifs. The crystal structure also features N—H⋯N and weak C—H⋯π inter­actions. PMID:24046674

  18. Synthesis and crystal structure studies of ethyl 5-methyl-1, 3-diphenyl-1H-pyrazole-4-carboxylate

    SciTech Connect

    Chandra,; Babu, E. A. Jithesh; Mahendra, M.; Srikantamurthy, N.; Umesha, K. B.

    2014-04-24

    The title compound, C{sub 19}H{sub 18}N{sub 2}O{sub 2}, was investigated by single crystal X-ray diffraction method. It crystallizes in monoclinic class under the space group P2{sub 1}/c with cell parameters a= 8.4593(4) Å, b=15.6284(6) Å, c=12.4579(5) Å, α=90°, β=98.241(3)°, γ=90° and Z=2. The ethoxycarbonyl group is slightly twisted from the pyrazole ring, and adopts syn-periplanar conformation. The crystal structure is stabilized by intermolecular C-H….O hydrogen bonds, which help in stabilizing the crystal structure.

  19. Synthesis and spectroscopy studies of the inclusion complex of 3-amino-5-methyl pyrazole with beta-cyclodextrin.

    PubMed

    Louiz, S; Labiadh, H; Abderrahim, R

    2015-01-05

    Amino pyrazole belongs to anti-inflammatory class, and is characterized by a low solubility in water. (In order to increase its solubility in water, inclusion complex of amino pyrazole with β-CD was obtained.) The inclusion complex obtained between AMP and β-cyclodextrin, was characterized by FT-IR, (1)H NMR, (1)H-(1)H NOESY, (13)C NMR, DEPT, XHCOR, spectra, through TG analysis, DTA, DSC and Scanning Electron Microscopy (SEM). The stoichiometry of inclusion complex is 1:1 (guest-host) and K stability is 1.1 × 10(4)M(-1).

  20. Synthesis and crystal structure studies of ethyl 5-methyl-1, 3-diphenyl-1H-pyrazole-4-carboxylate

    NASA Astrophysics Data System (ADS)

    Chandra, Srikantamurthy, N.; Babu, E. A. Jithesh; Umesha, K. B.; Mahendra, M.

    2014-04-01

    The title compound, C19H18N2O2, was investigated by single crystal X-ray diffraction method. It crystallizes in monoclinic class under the space group P21/c with cell parameters a= 8.4593(4) Å, b=15.6284(6) Å, c=12.4579(5) Å, α=90°, β=98.241(3)°, γ=90° and Z=2. The ethoxycarbonyl group is slightly twisted from the pyrazole ring, and adopts syn-periplanar conformation. The crystal structure is stabilized by intermolecular C-H….O hydrogen bonds, which help in stabilizing the crystal structure.

  1. Synthesis and spectroscopy studies of the inclusion complex of 3-amino-5-methyl pyrazole with beta-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Louiz, S.; Labiadh, H.; Abderrahim, R.

    2015-01-01

    Amino pyrazole belongs to anti-inflammatory class, and is characterized by a low solubility in water. (In order to increase its solubility in water, inclusion complex of amino pyrazole with β-CD was obtained.) The inclusion complex obtained between AMP and β-cyclodextrin, was characterized by FT-IR, 1H NMR, 1H-1H NOESY, 13C NMR, DEPT, XHCOR, spectra, through TG analysis, DTA, DSC and Scanning Electron Microscopy (SEM). The stoichiometry of inclusion complex is 1:1 (guest-host) and K stability is 1.1 × 104 M-1.

  2. QM/MM lineshape simulation of the hydrogen-bonded uracil NH stretching vibration of the adenine:uracil base pair in CDCl3

    NASA Astrophysics Data System (ADS)

    Yan, Yun-an; Krishnan, Gireesh M.; Kühn, Oliver

    2008-10-01

    A hybrid Car-Parrinello QM/MM molecular dynamics simulation has been carried out for the Watson-Crick base pair of 9-ethyl-8-phenyladenine and 1-cyclohexyluracil in deuterochloroform solution at room temperature. The resulting trajectory is analyzed putting emphasis on the N-H⋯N hydrogen bond geometry. Using an empirical correlation between the N⋯N-distance and the fundamental NH-stretching frequency, the time-dependence of this energy gap along the trajectory is obtained. From the gap-correlation function we determine the infrared absorption spectrum using lineshape theory in combination with a multimode oscillator model. The obtained average transition frequency and the width of the spectrum are in reasonable agreement with recent experimental data.

  3. Partial uracil-DNA-glycosylase treatment for screening of ancient DNA.

    PubMed

    Rohland, Nadin; Harney, Eadaoin; Mallick, Swapan; Nordenfelt, Susanne; Reich, David

    2015-01-19

    The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.

  4. Uracil moiety is required for toxicity of the cyanobacterial hepatotoxin cylindrospermopsin.

    PubMed

    Banker, R; Carmeli, S; Werman, M; Teltsch, B; Porat, R; Sukenik, A

    2001-02-23

    A new natural derivative of the sulfated guanidinium zwitterionic toxin cylindrospermopsin, 7-epi-cylindrospermopsin, was recently isolated from the cyanobacterium Aphanizomenon ovalisporum (Forti). The toxicity of the molecule (LD50 ip 5 d), estimated by mouse bioassay, was 200 microg/kg mouse, a value similar to that of cylindrospermopsin. Treatment of cylindrospermopsin with chlorine solution or chlorine-related oxidants produced two new derivatives. The chemical structure of these products was elucidated by nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques and toxicity was determined. In the first derivative, the vinylic proton at position 5 of the pyrimidine ring was substituted by chlorine to yield 5-chlorocylindrospermopsin. The other product is a truncated one, where C-6 of the pyrimidine ring was oxidized to a carboxylic acid. A trivial name, cylindrospermic acid, was given to this compound. Both products showed no toxic effects even at doses 50 times higher than the LD50 of cylindrospermopsin (10 mg/kg mouse ip). Based on these results, the pyrimidine ring is postulated as the molecule component essential for the toxicity of cylindrospermopsin.

  5. Contrasting reactions of hydrated electron and formate radical with 2-thio analogues of cytosine and uracil.

    PubMed

    Prasanthkumar, Kavanal P; Alvarez-Idaboy, Juan R; Kumar, Pavitra V; Singh, Beena G; Priyadarsini, K Indira

    2016-10-19

    2-Thiocytosine (TC) and 2-thiouracil (TU) were subjected to hydrated electron (eaq(-)), formate radical (CO2˙(-)) and 2-hydroxypropan-2-yl radical ((CH3)2˙COH) reactions in aqueous medium. Transients were characterized by absorption spectroscopy and the experimental findings were rationalized by DFT calculations at LC-ωPBE and M06-2X levels using a 6-311+G(d,p) basis set and SMD solvation. In eaq(-) reactions, a ring N-atom protonated radical of TC and an exocyclic O-atom protonated radical of TU were observed via addition of eaq(-) and subsequent protonation by solvent molecules. However, two competing but simultaneous mechanisms are operative in CO2˙(-) reactions with TC and TU. The first one corresponds to formations of N(O)-atom protonated radicals (similar to eaq(-) reactions); the second mechanism led to 2 center-3 electron, sulfur-sulfur bonded neutral dimer radicals, TCdim˙ and TUdim˙. DFT calculations demonstrated that H-abstraction by CO2˙(-) from TC(TU) results in S-centered radical which upon combination with TC(TU) provide the dimer radical. In some cases, DFT energy profiles were further validated by CBS-QB3//M06-2X calculations. This is the first time report for a contradictory behavior in the mechanisms of eaq(-) and CO2˙(-) reactions with any pyrimidines or their thio analogues.

  6. 40 CFR 721.5590 - Oxirane, [[[(1R,2S,5R)-5-methyl-2- (1-methylethyl)cyclohexyl] oxy]methyl]-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... activities. Requirements as specified in § 721.80(g). (ii) Release to water. Requirements as specified § 721.90 (a)(1), (b)(1), and (c)(1). (b) Specific requirements. The provisions of subpart A of this part... requirements as specified in § 721.125 (a), (b), (c), (i), and (k) are applicable to manufacturers,...

  7. 3-{[Bis(pyridin-2-ylmeth­yl)amino]­meth­yl}-2-hy­droxy-5-methyl­benz­aldehyde

    PubMed Central

    Wang, Ruo-Xu; Gao, Da-Zhi; Ye, Fan; Wu, Yan-Fei; Zhu, Dun-Ru

    2012-01-01

    In the title compound, C21H21N3O2, the pyridine rings and the benzene ring lie in a propeller arrangement around the central tertiary amine N atom. The dihedral angles formed by the benzene ring with the pyridine rings are 61.0 (3) and 49.6 (3)°, while the dihedral angle between the pyridine rings is 69.7 (3)°. The mol­ecular conformation is stabilized by intramolecular bifurcated O—H⋯N hydrogen bonds. In the crystal, inversion dimers are formed via pairs of C—H⋯N hydrogen bonds. PMID:22719467

  8. 3-tert-Butyl 5-methyl (2R,4S,5R)-2-(4-methoxyphenyl)-4-(3-nitrophenyl)-1,3-oxazolidine-3,5-dicarboxylate

    PubMed Central

    Montiel-Smith, Sara; Bernès, Sylvain; Sandoval-Ramírez, Jesús; Meza-Reyes, Socorro; Dubois, Joëlle

    2012-01-01

    The title mol­ecule, C23H26N2O8, was synthesized in three steps starting from m-nitro­cinnamic acid. The central oxazolidine ring adopts an almost perfect envelope conformation with the O atom as the flap [puckering parameter ϕ = 0.3 (6)°]. The dihedral angle formed by the benzene rings is 61.81 (9)°. In the crystal, mol­ecules are connected into double chains parallel to [010] by C—H⋯O hydrogen bonds. The absolute configuration was assigned from the synthetic procedure. PMID:23284466

  9. Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

    PubMed Central

    Ahmed, Ahmed H.; Oswald, Robert E.

    2010-01-01

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to both GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators. PMID:20163115

  10. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    PubMed

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  11. Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8.

    PubMed

    Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon K; Ornstein, Paul L; Spinazze, Patrick; Stevens, F Craig; Hahn, Patric; Hollinshead, Sean P; Mayhugh, Daniel; Schkeryantz, Jeff; Khilevich, Albert; De Frutos, Oscar; Gleason, Scott D; Kato, Akihiko S; Luffer-Atlas, Debra; Desai, Prashant V; Swanson, Steven; Burris, Kevin D; Ding, Chunjin; Heinz, Beverly A; Need, Anne B; Barth, Vanessa N; Stephenson, Gregory A; Diseroad, Benjamin A; Woods, Tim A; Yu, Hong; Bredt, David; Witkin, Jeffrey M

    2016-05-26

    Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

  12. 3-Methyl-5-methyl­sulfanyl-1,3,4-thia­diazole-2(3H)-thione

    PubMed Central

    Suarez, Sebastian A.; Hazari, Saroj K. S.; Ganguly, Biplab; Doctorovich, Fabio; G. Roy, Tapashi; Baggio, Ricardo

    2012-01-01

    The title compound, C4H6N2S3, has two very similar mol­ecules per asymmetric unit. The nine non-H atoms in each mol­ecule are coplanar, both having comparable r.m.s. deviations of 0.002 Å. The main inter­est in the rather simple structure resides in a survey of very weak (in some cases, borderline) non-bonding inter­actions of various kinds, viz. S⋯S, C—H⋯π, π–π [centroid–centroid distance = 3.8958 (13) Å] and C—S⋯π [3.7271 (11) Å], which act as the major driving force for the arrangement of mol­ecules in the structure. The role of long, though highly directional, S⋯S contacts (d > 3.60 Å), and their relevance to the stability of the structure is discussed. PMID:23125808

  13. 2-Bromo-1-[1-(4-bromo­phen­yl)-5-methyl-1H-1,2,3-triazol-4-yl]ethanone

    PubMed Central

    Bunev, Alexander S.; Troshina, Marina A.; Ostapenko, Gennady I.; Pavlova, Andzhela P.; Khrustalev, Victor N.

    2014-01-01

    The asymmetric unit of the title compound, C11H9Br2N3O, contains two crystallographically independent mol­ecules with similar geometries; the Br—C—C=O torsion angles are 1.2 (4) and −2.8 (4)°, and the benzene and triazole rings are inclined o one another by 51.90 (16) and 51.88 (16)°. The two molecules are related by a pseudo-screw 21 axis directed along [100]. In the crystal, mol­ecules are linked into a three-dimensional network by weak C—H⋯O and C—H⋯N hydrogen bonds and secondary Br⋯Br [3.5991 (8) and 3.6503 (9) Å] inter­actions. PMID:25161595

  14. Bromidotetra­kis­(1H-2-ethyl-5-methyl­imidazole-κN 3)copper(II) bromide

    PubMed Central

    Godlewska, Sylwia; Baranowska, Katarzyna; Socha, Joanna; Dołęga, Anna

    2011-01-01

    The CuII ion in the title compound, [CuBr(C6H10N2)4]Br, is coordinated in a square-based-pyramidal geometry by the N atoms of four imidazole ligands and a bromide anion in the apical site. Both the CuII and Br− atoms lie on a crystallographic fourfold axis. In the crystal, the [CuBr(C6H10N2)4]+ complex cations are linked to the uncoordinated Br− anions (site symmetry ) by N—H⋯Br hydrogen bonds, generating a three-dimensional network. The ethyl group of the imidazole ligand was modelled as disordered over two orientations with occupancies of 0.620 (8) and 0.380 (8). PMID:22199662

  15. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... in paragraph (a) of this section may be used as a color additive in contact lenses in amounts not to...) of the Federal Food, Drug, and Cosmetic Act with respect to the contact lens in which the...

  16. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... in paragraph (a) of this section may be used as a color additive in contact lenses in amounts not to...) of the Federal Food, Drug, and Cosmetic Act with respect to the contact lens in which the...

  17. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... in paragraph (a) of this section may be used as a color additive in contact lenses in amounts not to...) of the Federal Food, Drug, and Cosmetic Act with respect to the contact lens in which the...

  18. 2-((Z)-{3-[(Z)-(2-Hy-droxy-5-methyl-benzyl-idene)amino]-2,2-dimethyl-prop-yl}imino-meth-yl)-4-methyl-phenol.

    PubMed

    Kia, Reza; Kargar, Hadi; Mirkhani, Valiollah; Ganji, Fatemeh; Tahir, Muhammad Nawaz

    2010-12-15

    In the title compound, C(21)H(26)N(2)O(2), the dihedral angle between the two benzene rings is 73.47 (16)°. Strong intra-molecular O-H⋯N hydrogen bonds generate S(6) ring motifs. The substituted benzene rings are twisted around the central quaternary C atom in opposite directions, making a vault geometry.

  19. WHY DOES 5-METHYL CHRYSENE INTERACT WITH DNA LIKE BOTH A PLANAR AND A NON-PLANAR POLYCYCLIC AROMATIC HYDROCARBON? QUANTUM MECHANICAL STUDIES

    EPA Science Inventory

    Polycyclic aromatic hydrocarbons are a large class of anthropogenic chemicals found in the environment. Some class members are potent animal carcinogens while other similar class members show little carcinogenic activity. When considering a series of in vitro studies of the int...

  20. Studies on an (S)-2-Amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic Acid (AMPA) Receptor Antagonist IKM-159: Asymmetric Synthesis, Neuroactivity, and Structural Characterization

    PubMed Central

    Juknaitė, Lina; Sugamata, Yutaro; Tokiwa, Kazuya; Ishikawa, Yuichi; Takamizawa, Satoshi; Eng, Andrew; Sakai, Ryuichi; Pickering, Darryl S.; Frydenvang, Karla; Swanson, Geoffrey T.; Kastrup, Jette S.; Oikawa, Masato

    2015-01-01

    IKM-159 was developed and identified as a member of a new class of heterotricyclic glutamate analogues that act as AMPA receptor-selective antagonists. However, it was not known which enantiomer of IKM-159 was responsible for its pharmacological activities. Here, we report in vivo and in vitro neuronal activities of both enantiomers of IKM-159 prepared by enantioselective asymmetric synthesis. By employment of (R)-2-amino-2-(4-methoxyphenyl)ethanol as a chiral auxiliary, (2R)-IKM-159 and the (2S)-counterpart were successfully synthesized in 0.70% and 1.5% yields, respectively, over a total of 18 steps. Both behavioral and electrophysiological assays showed that the biological activity observed for the racemic mixture was reproduced only with (2R)-IKM-159, whereas the (2S)-counterpart was inactive in both assays. Racemic IKM-159 was crystallized with the ligand-binding domain of GluA2, and the structure revealed a complex containing (2R)-IKM-159 at the glutamate binding site. (2R)-IKM-159 locks the GluA2 in an open form, consistent with a pharmacological action as competitive antagonist of AMPA receptors. PMID:23432124

  1. Synthesis of 2'-deoxy-2'-[.sup.18F]fluoro-5-methyl-1-B-D-arabinofuranosyluracil (.sup.18F-FMAU)

    DOEpatents

    Li, Zibo; Cai, Hancheng; Conti, Peter S

    2014-12-16

    The present invention relates to methods of synthesizing .sup.18F-FMAU. In particular, .sup.18F-FMAU is synthesized using one-pot reaction conditions in the presence of Friedel-Crafts catalysts. The one-pot reaction conditions are incorporated into a fully automated cGMP-compliant radiosynthesis module, which results in a reduction in synthesis time and simplifies reaction conditions. The one-pot reaction conditions are also suitable for the production of 5-substituted thymidine or cytidine analogs. The products from the one-pot reaction (e.g. the labeled thymidine or cytidine analogs) can be used as probes for imaging tumor proliferative activity. More specifically, these [.sup.18F]-labeled thymidine or cytidine analogs can be used as a PET tracer for certain medical conditions, including, but not limited to, cancer disease, autoimmunity inflammation, and bone marrow transplant.

  2. 1-(2-Hydr­oxy-5-methyl­phen­yl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one

    PubMed Central

    Thippeswamy, G. B.; Vijay Kumar, D.; Jayashree, B. S.; Sridhar, M. A.; Shashidhara Prasad, J.

    2010-01-01

    In the structure of the title compound, C15H14O2S, the benzene ring is nearly coplanar with the thio­phene ring. The hydroxy group substituted at C2 position is in an antiperi­planar conformation with respect to the phenyl ring. The crystal structure exhibits weak intramolecular O—H⋯O hydrogen bonding. PMID:21579466

  3. Effect of C5-methylation of cytosine on the photoreactivity of DNA: a joint experimental and computational study of TCG trinucleotides.

    PubMed

    Esposito, Luciana; Banyasz, Akos; Douki, Thierry; Perron, Marion; Markovitsi, Dimitra; Improta, Roberto

    2014-08-06

    DNA methylation, occurring at the 5 position of cytosine, is a natural process associated with mutational hotspots in skin tumors. By combining experimental techniques (optical spectroscopy, HPLC coupled to mass spectrometry) with theoretical methods (molecular dynamics, DFT/TD-DFT calculations in solution), we study trinucleotides with key sequences (TCG/T5mCG) in the UV-induced DNA damage. We show how the extra methyl, affecting the conformational equilibria and, hence, the electronic excited states, increases the quantum yield for the formation of cyclobutane dimers while reducing that of (6-4) adducts.

  4. Crystal structure of 1,3-bis-(3-tert-butyl-2-hy-droxy-5-methyl-benz-yl)-1,3-diazinan-5-ol monohydrate.

    PubMed

    Rivera, Augusto; Miranda-Carvajal, Ingrid; Ríos-Motta, Jaime; Bolte, Michael

    2016-09-01

    In the title hydrate, C28H42N2O3·H2O, the central 1,3-diazinan-5-ol ring adopts a chair conformation with the two benzyl substituents equatorial and the lone pairs of the N atoms axial. The dihedral angle between the aromatic rings is 19.68 (38)°. There are two intra-molecular O-H⋯N hydrogen bonds, each generating an S(6) ring motif. In the crystal, classical O-H⋯O hydrogen bonds connect the 1,3-diazinane and water mol-ecules into columns extending along the b axis. The crystal structure was refined as a two-component twin with a fractional contribution to the minor domain of 0.0922 (18).

  5. The contribution of the methyl groups on thymine bases to binding specificity and affinity by alanine-rich mutants of the bZIP motif.

    PubMed

    Kise, K J; Shin, J A

    2001-09-01

    We have used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) to short DNA duplexes, in which thymines were replaced with uracils, in order to quantify the contributions of the C5 methyl group on thymines with alanine methyl side chains. We simplified the alpha-helical GCN4 bZIP by alanine substitution: 4A, 11A, and 18A contain four, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. Titration of fluorescein-labeled duplexes with increasing amounts of protein yielded dissociation constants in the low-to-mid nanomolar range for all bZIP mutants in complex with the AP-1 target site (5'-TGACTCA-3'); binding to the nonspecific control duplex was >1000-fold weaker. Small changes of <1 kcal/mol in binding free energies were observed for wild-type bZIP and 4A mutant to uracil-containing AP-1, whereas 11A and 18A bound almost equally well to native AP-1 and uracil-containing AP-1. These modest changes in binding affinities may reflect the multivalent nature of protein-DNA interactions, as our highly mutated proteins still exhibit native-like behavior. These protein mutations may compensate for changes in enthalpic and entropic contributions toward DNA-binding in order to maintain binding free energies similar to that of the native protein-DNA complex.

  6. Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells.

    PubMed

    Paulin, R P; Ho, T; Balzer, H J; Holliday, R

    1998-06-01

    Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT- gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates "dual inheritance" at the APRT locus in CHO cells.

  7. Experimental and theoretical studies on the coordination chemistry of the N1-hexyl substituted pyrimidines (uracil, 5-fluorouracil and cytosine).

    PubMed

    Barceló-Oliver, Miquel; Baquero, Beatriz Adriana; Bauzá, Antonio; García-Raso, Angel; Vich, Roberto; Mata, Ignasi; Molins, Elies; Terrón, Angel; Frontera, Antonio

    2013-06-07

    N(1)-Hexyl substituted pyrimidines were shown to present solubility properties closer to the real bases than the commonly used methyl and ethyl derivatives, yielding bi-layered structures in the solid state. The study of their coordination capabilities, mainly with Ag(I) and Hg(II), is presented in order to prove their reactivity. A series of coordination complexes, namely, [Hg(N(1)-hexyl-5-fluorouracilate)2]4·6H2O (1), (Ag(+))·[Ag(N(1)-hexyl-5-fluorouracilate)2](-) (2), [Ag(NO3)(N(1)-hexyluracil-κO(4))4] (3), [ZnBr2(N(1)-hexylcytosine)2] (4), [CdBr2(N(1)-hexylcytosine)2] (5), [HgBr2(N(1)-hexylcytosine)2] (6) and [CoBr2(N(1)-hexylcytosine)2] (7), have been synthesized in good yields and X-ray characterized. The presence of the hexyl chains and the fluorine atoms causes the formation of interesting 3D architectures in the solid state. Their structures have been further characterized by infrared spectra (IR) and elemental analyses. In addition, DFT-D3 calculations are used to study interesting noncovalent interactions observed in the solid state, like fluorine-fluorine, fluorine-π and hydrophobic interactions.

  8. Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine and DNA uracil concentrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Folate is an essential nutrient which supports nucleotide synthesis and biological methylation reactions. Diminished folate status results in chromosome breakage and is associated with several diseases including colorectal cancer. Folate status is also inversely related to plasma homocys...

  9. UV-MALDI mass spectrometric quantitation of uracil based pesticides in fruit soft drinks along with matrix effects evaluation.

    PubMed

    Ivanova, Bojidarka; Spiteller, Michael

    2014-02-01

    This study focused on the development of the accurate and precise quantitative method for the determination of pesticides bromacil (1), terbacil (2), lenacil (3), butafenacil (4) and flupropacil (5) in fruit based soft drinks. Three different types of drinks are bought from market; huddled orange fruit drink (100%) (I), red-oranges (II) and multivitamin drink containing strawberry, orange, banana and maracuja (III). Samples were analyzed "with" and "without" pulp utilizing LC-ESI (or APCI) MS/MS, HPLC-ESI-(or APCI)-MS/MS and UV-MALDI-Orbitrap-MS methods. The effect of high complexity of the food matrix on the analysis was discussed. Study focuses on the advantages of the UV-MALDI-Orbitrap-MS method compared to the traditionally involved GC alone or hybrid methods such as GC-MS and LC-MS/MS for quantification of pesticides in water and soft drinks. The developed method included the techniques performed for validation, calibration and standardization. The target pesticides are widely used for the treatment of citrus fruits and pineapples, but for soft drink products, there are still no clear regulations on pesticide residues limits. The matrix effects in the analysis of fruit drinks required implementation of the exact standard reference material corresponds to the variety of food matrices. This paper contributed to the broad analytical implementation of the UV-MALDI-Orbitrap-MS method in the quality control and assessment programs for monitoring of pesticide contamination in fruit based sodas.

  10. Unlocked nucleic acids with a pyrene-modified uracil: synthesis, hybridization studies, fluorescent properties and i-motif stability.

    PubMed

    Perlíková, Pavla; Karlsen, Kasper K; Pedersen, Erik B; Wengel, Jesper

    2014-01-03

    The synthesis of two new phosphoramidite building blocks for the incorporation of 5-(pyren-1-yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene-modified nucleobase component were found to destabilize an i-motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene-modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene-modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady-state fluorescence emission studies of oligonucleotides containing pyrene-modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene-modified UNA monomers was observed after formation of i-motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene-modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.

  11. Electron transport through 5-substituted pyrimidines in DNA: electron affinities of uracil and cytosine derivatives differently affect the apparent efficiencies.

    PubMed

    Ito, Takeo; Kurihara, Ryohsuke; Utsumi, Nihiro; Hamaguchi, Yuta; Tanabe, Kazuhito; Nishimoto, Sei-ichi

    2013-11-11

    We investigated excess electron transport (EET) in DNA containing cytosine derivatives. By arranging the derivatives according to their electron affinities, the apparent EET efficiency was successfully regulated. Unexpectedly, however, providing gradients of electron affinity by inserting 5-fluorocytosine did not always enhance EET.

  12. Discovery of 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a Clinical p38[alpha] MAP Kinase Inhibitor for the Treatment of Inflammatory Diseases

    SciTech Connect

    Liu, Chunjian; Lin, James; Wrobleski, Stephen T.; Lin, Shuqun; Hynes, Jr., John; Wu, Hong; Dyckman, Alaric J.; Li, Tianle; Wityak, John; Gillooly, Kathleen M.; Pitt, Sidney; Shen, Ding Ren; Zhang, Rosemary F.; McIntyre, Kim W.; Salter-Cid, Luisa; Shuster, David J.; Zhang, Hongjian; Marathe, Punit H.; Doweyko, Arthur M.; Sack, John S.; Kiefer, Susan E.; Kish, Kevin F.; Newitt, John A.; McKinnon, Murray; Dodd, John H.; Barrish, Joel C.; Schieven, Gary L.; Leftheris, Katerina

    2013-11-20

    The discovery and characterization of 7k (BMS-582949), a highly selective p38{alpha} MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38{alpha} inhibitor. Unlike alkyl and other cycloalkyls, the sp{sup 2} character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38{alpha} enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38{alpha} was confirmed by X-ray crystallographic analysis.

  13. Novel Regulation of the Synthesis of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Subunit GluA1 by Carnitine Palmitoyltransferase 1C (CPT1C) in the Hippocampus*

    PubMed Central

    Fadó, Rut; Soto, David; Miñano-Molina, Alfredo J.; Pozo, Macarena; Carrasco, Patricia; Yefimenko, Natalia; Rodríguez-Álvarez, José; Casals, Núria

    2015-01-01

    The regulation of AMPA-type receptor (AMPAR) abundance in the postsynaptic membrane is an important mechanism involved in learning and memory formation. Recent data suggest that one of the constituents of the AMPAR complex is carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform located in the endoplasmic reticulum of neurons. Previous results had demonstrated that CPT1C deficiency disrupted spine maturation in hippocampal neurons and impaired spatial learning, but the role of CPT1C in AMPAR physiology had remained mostly unknown. In the present study, we show that CPT1C binds GluA1 and GluA2 and that the three proteins have the same expression profile during neuronal maturation. Moreover, in hippocampal neurons of CPT1C KO mice, AMPAR-mediated miniature excitatory postsynaptic currents and synaptic levels of AMPAR subunits GluA1 and GluA2 are significantly reduced. We show that AMPAR expression is dependent on CPT1C levels because total protein levels of GluA1 and GluA2 are decreased in CPT1C KO neurons and are increased in CPT1C-overexpressing neurons, whereas other synaptic proteins remain unaltered. Notably, mRNA levels of AMPARs remained unchanged in those cultures, indicating that CPT1C is post-transcriptionally involved. We demonstrate that CPT1C is directly involved in the de novo synthesis of GluA1 and not in protein degradation. Moreover, in CPT1C KO cultured neurons, GluA1 synthesis after chemical long term depression was clearly diminished, and brain-derived neurotrophic factor treatment was unable to phosphorylate the mammalian target of rapamycin (mTOR) and stimulate GluA1 protein synthesis. These data newly identify CPT1C as a regulator of AMPAR translation efficiency and therefore also synaptic function in the hippocampus. PMID:26338711

  14. Crystal structure of (E)-2-[(2S,5R)-2-isopropyl-5-methyl­cyclo­hexyl­idene]hydrazine-1-carbo­thio­amide

    PubMed Central

    de Oliveira, Adriano Bof; Beck, Johannes; Daniels, Jörg; de Farias, Renan Lira; de Godoy Netto, Adelino Vieira

    2014-01-01

    The title compound, C11H21N3S, consists of a menthone moiety attached to an extended thio­semicarbazone group with the N—N—C—N torsion angle being 11.92 (16)°. The cyclo­hexane ring has a chair conformation and the conformation about the C=N bond is E. In the crystal, mol­ecules are linked via pairs of N—H⋯S hydrogen bonds, forming chains along the a axis. The absolute structure could be assigned with reference to the starting material, i.e. enanti­opure (−)-menthone [Flack parameter = 0.05 (5)]. PMID:25309244

  15. Early Growth Response 1 (Egr-1) Regulates N-Methyl-d-aspartate Receptor (NMDAR)-dependent Transcription of PSD-95 and α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor (AMPAR) Trafficking in Hippocampal Primary Neurons*

    PubMed Central

    Qin, Xike; Jiang, Yongjun; Tse, Yiu Chung; Wang, Yunling; Wong, Tak Pan; Paudel, Hemant K.

    2015-01-01

    The N-methyl-d-aspartate receptor (NMDAR) controls synaptic plasticity and memory function and is one of the major inducers of transcription factor Egr-1 in the hippocampus. However, how Egr-1 mediates the NMDAR signal in neurons has remained unclear. Here, we show that the hippocampus of mice lacking Egr-1 displays electrophysiology properties and ultrastructure that are similar to mice overexpressing PSD-95, a major scaffolding protein of postsynaptic density involved in synapse formation, synaptic plasticity, and synaptic targeting of AMPA receptors (AMPARs), which mediate the vast majority of excitatory transmission in the CNS. We demonstrate that Egr-1 is a transcription repressor of the PSD-95 gene and is recruited to the PSD-95 promoter in response to NMDAR activation. Knockdown of Egr-1 in rat hippocampal primary neurons blocks NMDAR-induced PSD-95 down-regulation and AMPAR endocytosis. Likewise, overexpression of Egr-1 in rat hippocampal primary neurons causes reduction in PSD-95 protein level and promotes AMPAR endocytosis. Our data indicate that Egr-1 is involved in NMDAR-mediated PSD-95 down-regulation and AMPAR endocytosis, a process important in the expression of long term depression. PMID:26475861

  16. Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol: a novel, potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor.

    PubMed

    Watanabe, Kazushi; Kakefuda, Akio; Yasuda, Minoru; Enjo, Kentaro; Kikuchi, Aya; Furutani, Takashi; Naritomi, Yoichi; Otsuka, Yukio; Okada, Minoru; Ohta, Mitsuaki

    2013-09-01

    Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the human prostate. One of the main functions of 17β-HSD5 is to catalyze the conversion of the weak androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic 5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to remain as high as 39% of that of healthy men, with 17β-HSD5 shown to be involved in this androgen synthesis. Inhibition of 17β-HSD5 therefore represents a promising target for the treatment of castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening (HTS) and identified compound 2, which displayed a structure distinct from known 17β-HSD5 inhibitors. To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity, improved metabolic stability, and led to the identification of compound 17. Oral administration of compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced pharmacokinetic and physicochemical properties.

  17. Crystal structure of 1,3-bis­(3-tert-butyl-2-hy­droxy-5-methyl­benz­yl)-1,3-diazinan-5-ol monohydrate

    PubMed Central

    Rivera, Augusto; Miranda-Carvajal, Ingrid; Ríos-Motta, Jaime; Bolte, Michael

    2016-01-01

    In the title hydrate, C28H42N2O3·H2O, the central 1,3-diazinan-5-ol ring adopts a chair conformation with the two benzyl substituents equatorial and the lone pairs of the N atoms axial. The dihedral angle between the aromatic rings is 19.68 (38)°. There are two intra­molecular O—H⋯N hydrogen bonds, each generating an S(6) ring motif. In the crystal, classical O—H⋯O hydrogen bonds connect the 1,3-diazinane and water mol­ecules into columns extending along the b axis. The crystal structure was refined as a two-component twin with a fractional contribution to the minor domain of 0.0922 (18). PMID:27920933

  18. Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors*

    PubMed Central

    Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

    2015-01-01

    Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca2+, resulting from Ca2+ influxes through calcium-permeable AMPA receptors, voltage-gated Ca2+ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca2+ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca2+ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. PMID:26134564

  19. (E)-1-(1-Benzyl-5-methyl-1H-1,2,3-triazol-4-yl)-3-(4-fluoro­phen­yl)prop-2-en-1-one

    PubMed Central

    Fun, Hoong-Kun; Hemamalini, Madhukar; Shanmugavelan, Poovan; Ponnuswamy, Alagusundaram; Jagatheesan, Rathinavel

    2011-01-01

    The asymmetric unit of the title compound, C19H16FN3O, contains two crystallographically independent mol­ecules. The triazole rings in both mol­ecules are essentially planar with maximum deviations of 0.002 (1) and 0.001 (1) Å. The dihedral angles between the benzene and fluorophenyl rings are 79.36 (9) and 89.40 (10)° in the two molecules. In the crystal, the two independent mol­ecules are linked by C—H⋯N hydrogen bonds, forming dimers. Furthermore, the crystal structure is stabilized by C—H⋯π inter­actions. PMID:22065516

  20. A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators*

    PubMed Central

    Harms, Jonathan E.; Benveniste, Morris; Kessler, Markus; Stone, Leslie M.; Arai, Amy C.; Partin, Kathryn M.

    2014-01-01

    AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators. PMID:24550387

  1. A charge-inverting mutation in the "linker" region of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors alters agonist binding and gating kinetics independently of allosteric modulators.

    PubMed

    Harms, Jonathan E; Benveniste, Morris; Kessler, Markus; Stone, Leslie M; Arai, Amy C; Partin, Kathryn M

    2014-04-11

    AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.

  2. Persistent inflammation-induced up-regulation of brain-derived neurotrophic factor (BDNF) promotes synaptic delivery of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA1 subunits in descending pain modulatory circuits.

    PubMed

    Tao, Wenjuan; Chen, Quan; Zhou, Wenjie; Wang, Yunping; Wang, Lu; Zhang, Zhi

    2014-08-08

    The enhanced AMPA receptor phosphorylation at GluA1 serine 831 sites in the central pain-modulating system plays a pivotal role in descending pain facilitation after inflammation, but the underlying mechanisms remain unclear. We show here that, in the rat brain stem, in the nucleus raphe magnus, which is a critical relay in the descending pain-modulating system of the brain, persistent inflammatory pain induced by complete Freund adjuvant (CFA) can enhance AMPA receptor-mediated excitatory postsynaptic currents and the GluA2-lacking AMPA receptor-mediated rectification index. Western blot analysis showed an increase in GluA1 phosphorylation at Ser-831 but not at Ser-845. This was accompanied by an increase in distribution of the synaptic GluA1 subunit. In parallel, the level of histone H3 acetylation at bdnf gene promoter regions was reduced significantly 3 days after CFA injection, as indicated by ChIP assays. This was correlated with an increase in BDNF mRNA levels and BDNF protein levels. Sequestering endogenous extracellular BDNF with TrkB-IgG in the nucleus raphe magnus decreased AMPA receptor-mediated synaptic transmission and GluA1 phosphorylation at Ser-831 3 days after CFA injection. Under the same conditions, blockade of TrkB receptor functions, phospholipase C, or PKC impaired GluA1 phosphorylation at Ser-831 and decreased excitatory postsynaptic currents mediated by GluA2-lacking AMPA receptors. Taken together, these results suggest that epigenetic up-regulation of BDNF by peripheral inflammation induces GluR1 phosphorylation at Ser-831 sites through activation of the phospholipase C-PKC signaling cascade, leading to the trafficking of GluA1 to pain-modulating neuronal synapses.

  3. Positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors reverses sub-chronic PCP-induced deficits in the novel object recognition task in rats.

    PubMed

    Damgaard, Trine; Larsen, Dorrit Bjerg; Hansen, Suzanne L; Grayson, Ben; Neill, Jo C; Plath, Niels

    2010-02-11

    Cognitive deficits are a major clinical unmet need in schizophrenia. The psychotomimetic drug phencyclidine (PCP) is widely applied in rodents to mimic symptoms of schizophrenia, including cognitive deficits. Previous studies have shown that sub-chronic PCP induces an enduring episodic memory deficit in female Lister Hooded rats in the novel object recognition (NOR) task. Here we show that positive modulation of AMPA receptor (AMPAR) mediated glutamate transmission alleviates cognitive deficits induced by sub-chronic PCP treatment. Female Lister hooded rats were treated sub-chronically with either vehicle (0.9% saline) or PCP (2mg/kg two doses per day for 7 days), followed by a 7 days washout period. 30 min prior to the acquisition trial of the NOR task animals were dosed with either vehicle, CX546 (10, 40 or 80 mg/kg) or CX516 (0.5, 2.5, 10, 40 or 80 mg/kg). Our results show that sub-chronic PCP treatment induced a significant decrease in the discrimination index (DI) and both ampakines CX546 and CX516 were able to reverse this disruption of object memory in rats in the novel object recognition task. These data suggest that positive AMPAR modulation may represent a mechanism for treatment of cognitive deficits in schizophrenia.

  4. Synthesis and 1H and 13C NMR spectral study of some r(2),c(4)-bis(isopropylcarbonyl)-c(5)-hydroxy-t(5)-methyl-t(3)-substituted phenyl, cyclohexanones and their oximes

    NASA Astrophysics Data System (ADS)

    Balachander, R.; Sameera, S. A.; Mohan, R. T. Sabapathy

    2016-07-01

    All the synthesized compounds have been characterized by 1H, 13C, 2D NMR and mass spectral studies. The spectral data suggest that compounds 2, 3, 5 and 6 exist in chair conformation with axial orientation of the hydroxyl group and equatorial orientations of all the other substituent. Long-range coupling is observed between OH proton to H-6a proton should be in a W arrangement. Compounds 1 and 4 diamagnetic anisotropic effect of the furyl group is not pronounced and absence of long-rang coupling between OH proton to H-6a proton. The oximation effects were discussed to all synthesized compounds using 1H and 13C chemical shifts.

  5. Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

    2010-02-15

    Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing.

  6. The carcinostatic effects of 1-(2-tetrahydrofuryl)-5-fluorouracil and uracil (UFT) on tongue carcinoma induced by 4-nitroquinoline 1-oxide (4NQO) in rats.

    PubMed

    Katakura, A; Shiozaki, Y; Kouda, H; Hatada, K; Tonogi, M; Takaki, T; Yamane, G; Noma, H

    1991-11-01

    UFT is a carcinostatic agent used in adjuvant chemotherapy for head and neck cancer. In the present study. UFT was given orally to treat tongue carcinoma in rats induced by 4-nitroquinoline 1-oxide. The antitumor effects of UFT were studied macroscopically and histologically. In addition, the antitumor effects of UFT were evaluated in relationship to lesions of the clinical and, invasive types, and to there vascular structure. In clinical lesions, the antitumor effect of UFT was higher in extrovert tumor-mass lesions and lower in ulcerous lesions. With regard to vascular structure, the effect was higher in cases demonstrating irregular net-like patterns and branch-like patterns and lower in cases in which the pattern had been destroyed. There was a correlation between antitumor effect and invasive type. As invasive tendency the 3H-thymidine labeling index, and mitotic index increased, antitumor effect and degree of tumor cell degeneration decreased.

  7. Synthesis and DNA cleavage activities of mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes which linked with uracil.

    PubMed

    Wang, Xiao-Yan; Zhang, Ji; Li, Kun; Jiang, Ning; Chen, Shan-Yong; Lin, Hong-Hui; Huang, Yu; Ma, Li-Jian; Yu, Xiao-Qi

    2006-10-01

    Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid (PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and (1)H NMR spectroscopy. The catalytic activities on DNA cleavage of these mononuclear complexes with different central metals were subsequently studied, which showed that copper complex was better catalyst in the DNA cleavage process than zinc and cobalt complexes. The effects of reaction time, concentration of complexes were also investigated. The results indicated that the copper(II) complexes could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II, no Form III produced) with high yields. The mechanism of the cleavage process was also studied.

  8. Synthesis, structure and study of azo-hydrazone tautomeric equilibrium of 1,3-dimethyl-5-(arylazo)-6-amino-uracil derivatives

    NASA Astrophysics Data System (ADS)

    Debnath, Diptanu; Roy, Subhadip; Li, Bing-Han; Lin, Chia-Her; Misra, Tarun Kumar

    2015-04-01

    Azo dyes, 1,3-dimethyl-5-(arylazo)-6-aminouracil (aryl = -C6H5 (1), -p-CH3C6H4 (2), -p-ClC6H4 (3), -p-NO2C6H4 (4)) were prepared and characterized by UV-vis, FT-IR, 1H NMR, 13C NMR spectroscopic techniques and single crystal X-ray crystallographic analysis. In the light of spectroscopic analysis it evidences that of the tautomeric forms, the azo-enamine-keto (A) form is the predominant form in the solid state whereas in different solvents it is the hydrazone-imine-keto (B) form. The study also reveals that the hydrazone-imine-keto (B) form exists in an equilibrium mixture with its anionic form in various organic solvents. The solvatochromic and photophysical properties of the dyes in various solvents with different hydrogen bonding parameter were investigated. The dyes exhibit positive solvatochromic property on moving from polar protic to polar aprotic solvents. They are fluorescent active molecules and exhibit high intense fluorescent peak in some solvents like DMSO and DMF. It has been demonstrated that the anionic form of the hydrazone-imine form is responsible for the high intense fluorescent peak. In addition, the acid-base equilibrium in between neutral and anionic form of hydrazone-imine form in buffer solution of varying pH was investigated and evaluated the pKa values of the dyes by making the use of UV-vis spectroscopic methods. The determined acid dissociation constant (pKa) values increase according to the sequence of 2 > 1 > 3 > 4.

  9. Studies on the preferred uracil-adenine base pair at the cleavage site of 10-23 DNAzyme by functional group modifications on adenine.

    PubMed

    Zhu, Junfei; Li, Zhiwen; Yang, Zhenjun; He, Junlin

    2015-08-01

    10-23 DNAzyme is capable of catalytically cleaving RNA substrates with the preferred cleavage sites rAU and rGU, in which the common base pair U-dA0 forms between the substrate and the DNAzyme in the cleavage reaction. Here its conservation was studied with base modifications on dA and extra functional groups introduced. The nitrogen atom at 7- or 8-position of adenine was demonstrated to be equally important for the cleavage reaction, although it is not related to the thermal stability of the base pair. Deletion of 6-amino group led to decreased stability of the base pair and a slight slower reaction rate. Extra functional groups through 6-amino group were not favorably accommodated in the cleavage site. From these modifications at the level of functional groups, it demonstrated that the base pair U-dA0 not only contributes to the recognition and binding stability, but also it is involved in the active catalytic center by its functional groups and base stacking. This kind of chemical modifications with 7-substituted 8-aza-7-deaza-2'-deoxyadenosine at dA0 is favorable for the introduction of signal molecules for mechanistic studies and biological applications, without significant loss of the catalytic function and structural destruction.

  10. Clay catalysis of oligonucleotide formation: kinetics of the reaction of the 5'-phosphorimidazolides of nucleotides with the non-basic heterocycles uracil and hypoxanthine

    NASA Technical Reports Server (NTRS)

    Kawamura, K.; Ferris, J. P.

    1999-01-01

    The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine of substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.

  11. 7-Amino-5-methyl-2-phenyl-6-(phenyldiazenyl)pyrazolo[1,5-a]pyrimidine crystallizes with Z' = 2: pseudosymmetry and the formation of complex sheets built from N-H...N and C-H...pi(arene) hydrogen bonds.

    PubMed

    Portilla, Jaime; Estupiñan, Diego; Cobo, Justo; Glidewell, Christopher

    2010-03-01

    The title compound, C(19)H(16)N(6), crystallizes with Z' = 2 in the space group P2(1)/n. The two molecules in the selected asymmetric unit are approximate mirror images of one another; most corresponding pairs of atoms are related by an approximate half-cell translation along [100]. Each molecule contains an intramolecular N-H...N hydrogen bond and the molecules are linked into complex sheets by a combination of two intermolecular N-H...N and four C-H...pi(arene) hydrogen bonds. Comparisons are made with some other 7-aminopyrazolo[1,5-a]pyrimidines.

  12. 7-Amino-5-methyl-2-phenyl-6-(phenyl­diazenyl)pyrazolo[1,5-a]pyrimidine crystallizes with Z′ = 2: pseudosymmetry and the formation of complex sheets built from N—H⋯N and C—H⋯π(arene) hydrogen bonds

    PubMed Central

    Portilla, Jaime; Estupiñan, Diego; Cobo, Justo; Glidewell, Christopher

    2010-01-01

    The title compound, C19H16N6, crystallizes with Z′ = 2 in the space group P21/n. The two mol­ecules in the selected asym­metric unit are approximate mirror images of one another; most corresponding pairs of atoms are related by an approximate half-cell translation along [100]. Each mol­ecule contains an intra­molecular N—H⋯N hydrogen bond and the mol­ecules are linked into complex sheets by a combination of two inter­molecular N—H⋯N and four C—H⋯π(arene) hydrogen bonds. Comparisons are made with some other 7-amino­pyrazolo[1,5-a]pyrimidines. PMID:20203410

  13. [2-({Benz­yl[2-(benz­yl{5-methyl-2-oxido-3-[(pyridin-2-ylmeth­yl)imino­meth­yl]benz­yl}amino)­eth­yl]aza­nium­yl}meth­yl)-4-methyl-6-[(pyridin-2-ylmeth­yl)imino­meth­yl]phenolato]nickel(II) perchlorate methanol disolvate

    PubMed Central

    Liu, Ming; Mao, Jia-Wei; Pan, Zhi-Quan

    2012-01-01

    In the solvated title complex, [Ni(C46H47N6O2)]ClO4·2CH4O, the coordination sphere around the NiII ion can be described as distorted cis-NiO2N4 octa­hedral defined by two phenolate O atoms and four N atoms from the hexa­dentate ligand. An intra­molecular bifurcated N—H⋯(N,O) hydrogen bond helps to establish the conformation of the complex mol­ecule. In the crystal, the components are connected by O—H⋯O and C—H⋯O hydrogen bonds. PMID:22719393

  14. Crystal structure of 4-({(1E,2E)-3-[3-(4-fluoro-phen-yl)-1-isopropyl-1H-indol-2-yl]allyl-idene}amino)-5-methyl-1H-1,2,4-triazole-5(4H)-thione.

    PubMed

    Kulkarni, Ajaykumar D; Rahman, Md Lutfor; Mohd Yusoff, Mashitah; Kwong, Huey Chong; Quah, Ching Kheng

    2015-11-01

    The title compound, C23H22FN5S, exists in a trans conformation with respect to the methene C=C and the acyclic N=C bonds. The 1,2,4-triazole-5(4H)-thione ring makes dihedral angles of 88.66 (9) and 84.51 (10)°, respectively, with the indole and benzene rings. In the crystal, mol-ecules are linked by pairs of N-H⋯S hydrogen bonds, forming inversion dimers with an R 2 (2)(8) ring motif. The dimers are linked via C-H⋯π inter-actions, forming chains along [1-10]. The chains are linked via π-π inter-actions involving inversion-related triazole rings [centroid-centroid distance = 3.4340 (13) Å], forming layers parallel to the ab plane.

  15. [Di­aqua­sesqui(nitrato-κO)hemi(perchlorato-κO)copper(II)]-μ-{bis­[5-methyl-3-(pyridin-2-yl)-1H-pyrazol-4-yl] selenide}-[tri­aqua­(perchlorato-κO)copper(II)] nitrate monohydrate

    PubMed Central

    Seredyuk, Maksym; Pavlenko, Vadim A.; Znovjyak, Kateryna O.; Gumienna-Kontecka, Elzbieta; Iskenderov, Turganbay S.

    2013-01-01

    In the binuclear title complex, [Cu2(ClO4)1.5(NO3)1.5(C18H16N6Se)(H2O)5]NO3·H2O, both CuII ions are hexa­coordinated by O and N atoms, thus forming axially elongated CuO4N2 octa­hedra. The equatorial plane of each octa­hedron is formed by one chelating pyrazole–pyridine fragment of the organic ligand and two water mol­ecules. The axial positions in one octa­hedron are occupied by a water mol­ecule and a monodentately coordinated perchlorate anion, while those in the other are occupied by a nitrate anion and a disordered perchlorate/nitrate anion with equal site occupancy. The pyrazole–pyridine units of the organic selenide are trans-oriented to each other with a C—Se—C angle of 96.01 (14)°. In the crystal, uncoordinated nitrate anions and the coordinating water mol­ecules are involved in O—H⋯O and N—H⋯O hydrogen bonds, forming a bridge between the pyrazole group and the coordinating water mol­ecules. Further O—H⋯O hydrogen bonds between the complex mol­ecules and a π–π stacking inter­action with a centroid–centroid distance of 3.834 (4) Å are also observed. PMID:23794981

  16. Palladium(II) and platinum(II) organometallic complexes with the model nucleobase anions of thymine, uracil, and cytosine: antitumor activity and interactions with DNA of the platinum compounds.

    PubMed

    Ruiz, José; Lorenzo, Julia; Sanglas, Laura; Cutillas, Natalia; Vicente, Consuelo; Villa, María Dolores; Avilés, Francesc X; López, Gregorio; Moreno, Virtudes; Pérez, José; Bautista, Delia

    2006-08-07

    Pd(II) and Pt(II) complexes with the anions of the model nucleobases 1-methylthymine (1-MethyH), 1-methyluracil (1-MeuraH), and 1-methylcytosine (1-MecytH) of the types [Pd(dmba)(mu-L)]2 [dmba = N,C-chelating 2-((dimethylamino)methyl)phenyl; L = 1-Methy, 1-Meura or 1-Mecyt] and [M(dmba)(L)(L')] [L = 1-Methy or 1-Meura; L' = PPh(3) (M = Pd or Pt), DMSO (M = Pt)] have been obtained. Palladium complexes of the types [Pd(C6F5)(N-N)(L)] [L = 1-Methy or 1-Meura; N-N = N,N,N',N'-tetramethylethylenediamine (tmeda), 2,2'-bipyridine (bpy), or 4,4'-dimethyl-2,2'-bipyridine (Me2bpy)] and [NBu4][Pd(C6F5)(1-Methy)2(H2O)] have also been prepared. The crystal structures of [Pd(dmba)(mu-1-Methy)]2, [Pd(dmba)(mu-1-Mecyt)]2.2CHCl3, [Pd(dmba)(1-Methy)(PPh3)].3CHCl3, [Pt(dmba)(1-Methy)(PPh3)], [Pd(tmeda)(C6F5)(1-Methy)], and [NBu4][Pd(C6F5)(1-Methy)2(H2O)].H2O have been established by X-ray diffraction. The DNA adduct formation of the new platinum complexes synthesized was followed by circular dichroism and electrophoretic mobility. Atomic force microscopy images of the modifications caused by the platinum complexes on plasmid DNA pBR322 were also obtained. Values of IC50 were also calculated for the new platinum complexes against the tumor cell line HL-60. All the new platinum complexes were more active than cisplatin (up to 20-fold in some cases).

  17. Electron Detachment as a Probe of Intrinsic Nucleobase Dynamics in Dianion-Nucleobase Clusters: Photoelectron Spectroscopy of the Platinum II Cyanide Dianion Bound to Uracil, Thymine, Cytosine and Adenine

    SciTech Connect

    Sen, Ananya; Hou, Gao-Lei; Wang, Xue B.; Dessent, Caroline

    2015-08-05

    We report the first low-temperature photodetachment photoelectron spectra of isolated gas-phase complexes of the platinum II cyanide dianion bound to nucleobases. These systems are model systems for understanding platinum-complex photodynamic therapies, and knowledge of the intrinsic photodetachment properties is crucial for understanding their broader photophysical properties. Well-resolved, distinct peaks are observed in the spectra consistent with the complexes where the Pt(CN)42- moiety is largely intact. The adiabatic electron detachment energies for the dianion-nucleobase complexes are measured to be between 2.39-2.46 eV. The magnitudes of the repulsive Coulomb barriers of the complexes are estimated to be between 1.9 and 2.1 eV, values that are lower than for the bare Pt(CN)42- dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photodetachment spectra of the four nucleobase-dianion complexes, and also in the 266 nm spectra of the Pt(CN)42-∙thymine and Pt(CN)42-∙adenine complexes. The selective excitation of these features in the 266 nm spectra is attributed to one-photon excitation of [Pt(CN)42-∙T]* and [Pt(CN)42-∙A]* long-lived excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment signals. We attribute the resonant electron detachment bands observed here for Pt(CN)42-∙T and Pt(CN)42-∙A but not for Pt(CN)42-∙U and Pt(CN)42-∙C to fundamental differences in the individual nucleobase photophysics following 266 nm excitation. This indicates that the Pt(CN)42- dianion in the Pt(CN)42-∙M clusters can be viewed as a “dynamic tag” which has the propensity to emit electrons when the attached nucleobase disaplys a long-lived excited state.

  18. Accurate, Direct, and High-Throughput Analyses of a Broad Spectrum of Endogenously Generated DNA Base Modifications with Isotope-Dilution Two-Dimensional Ultraperformance Liquid Chromatography with Tandem Mass Spectrometry: Possible Clinical Implication.

    PubMed

    Gackowski, Daniel; Starczak, Marta; Zarakowska, Ewelina; Modrzejewska, Martyna; Szpila, Anna; Banaszkiewicz, Zbigniew; Olinski, Ryszard

    2016-12-20

    Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2'-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor's margin; also 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material

  19. Augmentation of a Novel Enzyme/Pro-Drug Gene Therapy "Distant Bystander Effect" to Target Prostate Cancer Metastasis

    DTIC Science & Technology

    2005-09-01

    enzyme/prodrug therapy(GDEPT)_ mIL-12; mIL-18; cytosine deaminase and uracil phopho-ribosyl transferase (CDUPRT) 16. SECURITY CLASSIFICATION OF: 17...therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase (CD/UPRT)) is delivered to a cancer cell so that harmless bacterial...reduction both at the treatment site and at remote locations. In this therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase

  20. THE STUDY OF THE MECHANISM OF TRANSFORMATION OF PHYSICAL ENERGY INTO NERVE ACTIVITY IN THE INTERNAL EAR

    DTIC Science & Technology

    HEARING, ANATOMICAL MODELS, ANATOMY, BATS, BIOCHEMISTRY, DEAFNESS, DRUGS, EAR, ECHO RANGING, ETHANOLS, HISTOLOGY, LEPIDOPTERA, MUCOPOLYSACCHARIDES , PATHOLOGY, PHYSIOLOGY, SCOPOLAMINE, URACILS, THYROID GLAND, VERTIGO.

  1. 40 CFR 180.490 - Imazapic-ammonium; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic acid and its metabolite... established for the combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic...

  2. 40 CFR 180.490 - Imazapic-ammonium; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic acid and its metabolite... established for the combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic...

  3. 40 CFR 180.490 - Imazapic-ammonium; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic acid and its metabolite... established for the combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic...

  4. 40 CFR 180.490 - Imazapic-ammonium; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic acid and its metabolite... established for the combined residues of the herbicide imazapic, (±)-2- -5-methyl-3-pyridinecarboxylic...

  5. Using quantum mechanics to investigate the photophysical properties of the DNA and RNA bases and their fluorescent analogs

    NASA Astrophysics Data System (ADS)

    Kistler, Kurt A.

    The ability of the nucleic acids to absorb ultraviolet light and remain relatively photostable is a property upon which life depends. The nucleobases, which are the primary chromophores, when irradiated display rapid radiationless decay back to the ground state, in general faster than is needed for photoreaction. Fluorescent analogs of these bases have structures similar to the nucleic acid bases, but display much longer excited state lifetimes. Theoretical investigations using quantum mechanical methods can provide insight into the precise mechanisms of these decay processes, and to the molecular specifics that contribute to them. The results of multi-reference configuration interaction (MRCI) ab initio investigations into these mechanisms are presented, with emphasis on cytosine and its fluorescent analog 5-methyl-2-pyrimidinone (5M2P). A comprehensive picture of the potential energy surfaces of these two bases is given, including stationary points and conical intersections, where radiationless transisitons are promoted, between up to three state surfaces, as well as pathways connecting these points for each base. Cytosine is shown to have two different energetically accessible radiationless decay channels. The fluorescence of 5M2P is also demonstrated theoretically, with mechanism proposed. The potential energy surfaces of the two bases have many close similarities, with the different photophysical properties being attributed to subtle energetic differences between the two bases. Nonadiabatic coupling and the geometric phase effect are analyzed in detail near conical intersections in cytosine, including in a region close to a three-state conical intersection. A substituent effect study on the 2-pyrimidinone ring system shows that the presence, position and orientation of the amino group in cytosine is central to its photophysical properties, particulary its high absorption energy, and can be explained with a simple Frontier Molecular Orbital model. The effects

  6. Tetra­kis(μ2-5-methyl­pyrazine-2-carboxyl­ato)-1:2κ3 N 1,O:O;2:3κ3 O:N 1,O;1:2κ2 O:O′;3:4κ2 O:O′-octa­octyl-1κ2 C,2κ2 C,3κ2 C,4κ2 C-di-μ3-oxido-1:2:3κ3 O;1:3:4κ3 O-tetra­tin(IV)

    PubMed Central

    Gao, Zhongjun; Li, Fahui

    2009-01-01

    The title compound, [Sn4(C8H17)8O2(C6H5N2O2)4], is a tetra­nuclear SnIV complex, built up by inversion symmetry around the central Sn2O2 ring. The SnIV coordination geometries are distorted SnO3C2 trigonal-bipyramidal and distorted SnO4C2 octa­hedral. The three-coordinate μ3-oxido bridging O atom in the Sn2O2 ring is attached to three Sn atoms. All non-H atoms, with the exception of the Sn-bonded octyl groups, lie approximately on a non-crystallographic mirror plane. PMID:21578611

  7. 5-Arylaminouracil Derivatives: New Inhibitors of Mycobacterium tuberculosis.

    PubMed

    Matyugina, Elena; Novikov, Mikhail; Babkov, Denis; Ozerov, Alexander; Chernousova, Larisa; Andreevskaya, Sofia; Smirnova, Tatiana; Karpenko, Inna; Chizhov, Alexander; Murthu, Pravin; Lutz, Stefan; Kochetkov, Sergei; Seley-Radtke, Katherine L; Khandazhinskaya, Anastasia L

    2015-12-01

    Three series of 5-arylaminouracil derivatives, including 5-(phenylamino)uracils, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, and 1,3-di-(4'-hydroxy-2'-cyclopenten-1'-yl)-5-(phenylamino)uracils, were synthesized and screened for potential antimicrobial activity. Most of compounds had a negative effect on the growth of the Mycobacterium tuberculosis H37Rv strain, with 100% inhibition observed at concentrations between 5 and 40 μg/mL. Of those, 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-3-(4‴-hydroxy-2‴-cyclopenten-1‴-yl)-5-(4″-butyloxyphenylamino)uracil proved to be the most active among tested compounds against the M. tuberculosis multidrug-resistant strain MS-115 (MIC90 5 μg/mL). In addition, the thymidylate kinase of M. tuberculosis was evaluated as a possible enzymatic target.

  8. Secondary tritium and solvent deuterium isotope effects as a probe of the reaction catalyzed by porcine recombinant dihydropyrimidine dehydrogenase.

    PubMed

    Rosenbaum, K; Jahnke, K; Schnackerz, K D; Cook, P F

    1998-06-23

    Dihydropyrimidine dehydrogenase catalyzes the rate-limiting step in the degradation of pyrimidines in mammals, the reduction of uracil or thymine to their 5,6-dihydro derivatives. The reduction of uracil by enzyme-bound reduced flavin involves both proton and hydride transfer. In order to determine whether hydride and proton transfer occur in a concerted or stepwise fashion, and to determine the nature of the transition state for the reduction, secondary tritium kinetic isotope effects were measured in H2O and D2O. The tritium isotope effect using 5-3H-uracil is 0.90 +/- 0.03 in H2O and becomes more inverse, 0.85 +/- 0.04, in D2O. Data are interpreted in terms of a stepwise reduction at C-6 followed by protonation at C-5. A late transition state is proposed for the proton transfer at C-5 of uracil.

  9. Formulated Delivery of Enzyme/Pro-Drug and Cytokine Gene Therapy to Promote Immune Reduction of Treated and Remote Tumors in Mouse Models of Prostate Cancer

    DTIC Science & Technology

    2005-01-01

    reduction both at the treatment site and at remote locations. In this therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase...In this therapy, a gene (a fusion of cytosine deaminase and uracil phosphoribosyltransferase (CDUPRT)) is delivered to a cancer cell so that...represents a total of 18 months of work. Task 1: GDEPT alone. Assess the ability of lentivirus expressing GDEPT (based on the fusion gene, cytosine deaminase

  10. Genetic code correlations - Amino acids and their anticodon nucleotides

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Lacey, J. C., Jr.

    1978-01-01

    The data here show direct correlations between both the hydrophobicity and the hydrophilicity of the homocodonic amino acids and their anticodon nucleotides. While the differences between properties of uracil and cytosine derivatives are small, further data show that uracil has an affinity for charged species. Although these data suggest that molecular relationships between amino acids and anticodons were responsible for the origin of the code, it is not clear what the mechanism of the origin might have been.

  11. Mapping the UV Photophysics of Platinum Metal Complexes Bound to Nucleobases

    NASA Astrophysics Data System (ADS)

    Sen, Ananya; Dessent, Caroline

    2015-03-01

    We report the first UV laser spectroscopic study of isolated gas-phase complexes of Platinum metal complex anions bound to a nucleobase as model systems for exploring at the molecular level the key photophysical processes involved in photodynamic therapy. Spectra of the PtIV CN 6 2 - • Uracil and PtII CN 4 2 - • Uracil complexes were acquired across the 220 -320 nm range using mass-selective photodepletion and photofragment action spectroscopy. The spectra of both complexes reveal prominent UV absorption bands that we assign primarily to excitation of the Uracil π - π * localized chromophore. Distinctive UV photofragments are observed for the complexes, with PtIV CN 6 2 - • Uracil photoexcitation resulting in complex fission, while PtII CN 4 2 - • Uracil photoexcitation initiates a nucleobase proton-transfer reaction across 4.4 -5.2 eV and electron detachment above 5.2 eV. The observed photofragments are consistent with ultrafast decay of a Uracil localized excited state back to the electronic ground state followed by intramolecular vibrational relaxation and ergodic complex fragmentation. In addition, we present recent results to explore how the photophysics of the Platinum complex-nucleobase clusters evolves as a function of nucleobase. Results are presented for PtII CN 4 2 - • Uracil complexed to Cytosine, Thymine and Adenine, reveal distinctive decay dynamics which we attribute to the intrinsic decay dynamics of the nucleobase. JPC. Lett. 2014, 5, 3281 to 3285 and PCCP 2014, 16, 15490 to 15500.

  12. 46 CFR Appendix I to Part 150 - Exceptions to the Chart

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... hydrosulfide solution (5) Methyl alcohol (20) Iso-Propyl alcohol (20) Sulfuric acid (2) Coconut oil (34) Coconut oil acid (34) Palm oil (34) Tallow (34) Sulfuric acid, 98% or less (2) Choice white grease tallow... solution (5) Methyl alcohol (20). Iso-Propyl alcohol (20). Sulfuric acid (2) Coconut oil (34). Coconut...

  13. Thymine and other prebiotic molecules produced from the ultraviolet photo-irradiation of pyrimidine in simple astrophysical ice analogs.

    PubMed

    Materese, Christopher K; Nuevo, Michel; Bera, Partha P; Lee, Timothy J; Sandford, Scott A

    2013-10-01

    The informational subunits of RNA or DNA consist of substituted N-heterocyclic compounds that fall into two groups: those based on purine (C₅H₄N₄) (adenine and guanine) and those based on pyrimidine (C₄H₄N₂) (uracil, cytosine, and thymine). Although not yet detected in the interstellar medium, N-heterocycles, including the nucleobase uracil, have been reported in carbonaceous chondrites. Recent laboratory experiments and ab initio calculations have shown that the irradiation of pyrimidine in ices containing H₂O, NH₃, or both leads to the abiotic production of substituted pyrimidines, including the nucleobases uracil and cytosine. In this work, we studied the methylation and oxidation of pyrimidine in CH₃OH:pyrimidine, H₂O:CH₃OH:pyrimidine, CH₄:pyrimidine, and H₂O:CH₄:pyrimidine ices irradiated with UV photons under astrophysically relevant conditions. The nucleobase thymine was detected in the residues from some of the mixtures. Our results suggest that the abundance of abiotic thymine produced by ice photolysis and delivered to the early Earth may have been significantly lower than that of uracil. Insofar as the delivery of extraterrestrial molecules was important for early biological chemistry on early Earth, these results suggest that there was more uracil than thymine available for emergent life, a scenario consistent with the RNA world hypothesis.

  14. Spectrofluorimetric determination of 5-fluorouracil by fluorescence quenching of 9-anthracenecarboxylic acid

    NASA Astrophysics Data System (ADS)

    Khot, M. S.; Bhattar, S. L.; Kolekar, G. B.; Patil, S. R.

    2010-09-01

    Photo-induced intermolecular electron transfer (PET) interaction between excited singlet (S 1) state of 9-anthracene carboxylic acid (9-ANCA) and DNA bases of pyrimidines as uracil and 5-fluorouracil (5-FU) has been studied in water and ethanol solutions using steady-state fluorescence spectroscopy. The intensity of all emission bands of 9-ANCA was quenched in presence of uracil and 5-FU by electron transfer reaction without formation of an exciplex. It was found that uracil and 5-fluorouracil acts as effective electron donors and simultaneously quench the fluorescence of electron-accepting sensitizer 9-ANCA. The quenching by diffusion-controlled rate coincides well with the dynamic Stern-Volmer correlation. The bimolecular quenching rate constant (kqss) and electron transfer rate constant ( ket) observed are seen to be much higher for 5-fluorouracil than those for uracil. The thermodynamic parameters estimated by using the Rehm-Weller equation were used to propose a suitable mechanism for PET occurring between uracils and 9-ANCA. The proposed method was used to determine 5-fluorouracil from pharmaceutical samples with satisfactory results. The technique is more selective, sensitive and relatively free from coexisting substances.

  15. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    PubMed

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

  16. Multiconfigurational Effects in Theoretical Resonance Raman Spectra

    PubMed Central

    Ma, Yingjin

    2017-01-01

    Abstract We analyze resonance Raman spectra of the nucleobase uracil in the short‐time approximation calculated with multiconfigurational methods. We discuss the importance of static electron correlation by means of density‐matrix renormalization group self‐consistent field (DMRG‐SCF) calculations. Our DMRG‐SCF results reveal that a minimal active orbital space that leads to a qualitatively correct description of the resonance Raman spectrum of uracil should encompass parts of the σ/σ* bonding/anti‐bonding orbitals of the pyrimidine ring. We trace these findings back to the considerable entanglement between the σ/σ* bonding/anti‐bonding as well as valence π/π* orbitals in the excited‐state electronic structure of uracil, which indicates non‐negligible non‐dynamical correlation effects that are less pronounced in the electronic ground state. PMID:27933695

  17. Homeostasis between gut-associated microorganisms and the immune system in Drosophila.

    PubMed

    You, Hyejin; Lee, Won Jun; Lee, Won-Jae

    2014-10-01

    The metabolic activities of a given gut bacterium or gut commensal community fluctuate in a manner largely depending on the physicochemical parameters within the gut niche. Recognition of the bacterial metabolic status in situ, by a sensing of the gut metabolites as a signature of a specific bacterial metabolic activity, has been suggested to be a highly beneficial means for the host to maintain gut-microbe homeostasis. Recently, analysis of Drosophila gut immunity revealed that bacterial-derived uracil and uracil-modulated intestinal reactive oxygen species (ROS) generation play a pivotal role in diverse aspects of host-microbe interactions, such as pathogen clearance, commensal protection, intestinal cell regeneration, colitogenesis, and possibly also interorgan immunological communication. A deeper understanding of the role of uracil in Drosophila immunity will provide additional insight into the molecular mechanisms underlying host-microbe symbiosis and dysbiosis.

  18. An Insight into the Environmental Effects of the Pocket of the Active Site of the Enzyme. Ab initio ONIOM-Molecular Dynamics (MD) Study on Cytosine Deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2008-02-01

    We applied the ONIOM-molecular dynamics (MD) method to cytosine deaminase to examine the environmental effects of the amino acid residues in the pocket of the active site on the substrate taking account of their thermal motion. The ab initio ONIOM-MD simulations show that the substrate uracil is strongly perturbed by the amino acid residue Ile33, which sandwiches the uracil with His62, through the steric contact due to the thermal motion. As a result, the magnitude of the thermal oscillation of the potential energy and structure of the substrate uracil significantly increases. TM and MA were partly supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

  19. Cleavage of DNA containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases.

    PubMed

    Olszewska, Agata; Dadová, Jitka; Mačková, Michaela; Hocek, Michal

    2015-11-01

    A systematic study of the cleavage of DNA sequences containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases (REs) was performed and the results compared with the same sequences containing natural pyrimidine bases, uracil or 5-methylcytosine. The results show that some REs recognize fluorine as a hydrogen on cytosine and cleave the corresponding sequences where the presence of m5dC leads to blocking of the cleavage. However, on uracil, the same REs recognize the F as a methyl surrogate and cleave the sequences which are not cleaved if uracil is incorporated instead of thymine. These results are interesting for understanding the recognition of DNA sequences by REs and for manipulation of the specific DNA cutting.

  20. Photoelectron spectroscopic study of the negative ions of 4-thiouracil and 2,4-dithiouracil

    NASA Astrophysics Data System (ADS)

    Li, Xiang; Chen, Jing; Bowen, Kit H.

    2011-02-01

    We report the photoelectron spectra of the negative ions of 4-thiouracil (4-TU)- and 2,4-dithiouracil (2,4-DTU)-. Both of these spectra are indicative of valence anions, and they are each dominated by a single broad band with vertical detachment energies of 1.05 and 1.4 eV, respectively. Complementary calculations by Dolgounitcheva, Zakrzewski, and Ortiz (see companion paper) are in accord with our experimental results and conclude that the (4-TU)- and (2,4-DTU)- anions, reported herein, are valence anions of canonical 4-thiouracil and canonical dithiouracil. Comparisons among the anions and corresponding neutrals of 4-thiouracil, 2,4-dithiouracil, 5-chlorouracil, 5-fluorouracil, and uracil itself show that both sulfur and halogen modifications of uracil give rise to significant changes in the electronic structure. The electron affinities of the first four are all substantially larger than that of the canonical uracil.

  1. Metabolism of dihydrouracil in Rhodosporidium toruloides.

    PubMed Central

    Davis, C H; Putnam, M D; Thwaites, W M

    1984-01-01

    Previous studies, including those done with a similar species, have indicated that dihydrouracil is formed by the breakdown of uracil and is degraded into N-carbamyl-beta-alanine. (Fink et al., J. Biol. Chem. 201:349-355, 1953; S. R. Vilks and M. Y. Vitols, Mikrobiologiya 42:567-583, 1973; O. A. Milstein and M. L. Bekker, J. Bacteriol. 127:1-6, 1976). In the present work the conversion of dihydrouracil to uracil is studied in Rhodosporidium toruloides, and the growth characteristics of mutants that have lost the ability to use dihydrouracil as a source of nitrogen are examined. It is concluded that dihydrouracil must be converted to uracil before catabolism of the pyrimidine ring can take place. PMID:6425266

  2. TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway.

    PubMed

    Du, Eun Jo; Ahn, Tae Jung; Kwon, Ilmin; Lee, Ji Hye; Park, Jeong-Ho; Park, Sun Hwa; Kang, Tong Mook; Cho, Hana; Kim, Tae Jin; Kim, Hyung-Wook; Jun, Youngsoo; Lee, Hee Jae; Lee, Young Sik; Kwon, Jae Young; Kang, KyeongJin

    2016-01-01

    Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1's role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

  3. Hydroxyl radical oxidation of cylindrospermopsin (cyanobacterial toxin) and its role in the photochemical transformation.

    PubMed

    Song, Weihua; Yan, Shuwen; Cooper, William J; Dionysiou, Dionysios D; O'Shea, Kevin E

    2012-11-20

    Cylindrospermopsin (CYN), an alkaloid guanidinium sulfated toxin, is produced by a number of cyanobacteria regularly found in lakes, rivers, and reservoirs. Steady-state and time-resolved radiolysis methods were used to determine reaction pathways and kinetic parameters for the reactions of hydroxyl radical with CYN. The absolute bimolecular reaction rate constant for the reaction of hydroxyl radical with CYN is (5.08 ± 0.16) × 10(9) M(-1) s(-1). Comparison of the overall reaction rate of CYN with hydroxyl radical with the individual reaction rate for addition to the uracil ring in CYN indicate the majority of the hydroxyl radicals (84%) react at the uracil functionality of CYN. Product analyses using liquid chromatography-mass spectrometry indicate the major products from the reaction of hydroxyl radical with CYN involve attack of hydroxyl radical at the uracil ring and hydrogen abstraction from the hydroxy-methine bridge linking the uracil ring to the tricyclic guanidine functionality. The role of hydroxyl radical initiated pathways in the natural organic matter (NOM) photosensitized transformation of CYN were evaluated. Scavenger and trapping experiments indicate that hydroxyl radical mediated transformations account for approximately ~70% of CYN destruction in surface waters under solar irradiation in the presence of NOM. The absence of solvent isotope effect indicates singlet oxygen does not play a significant role in the NOM sensitized transformation of CYN. The primary degradation pathways for HO• mediated and NOM photosensitized destruction of CYN involve destruction of the uracil ring. The fundamental kinetic parameters determined from these studies are critical for the accurate evaluation of hydroxyl-radical based technologies for the remediation of this problematic cyanotoxin in drinking water and important in the assessment of the environmental oxidative transformation of uracil based compounds.

  4. TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway

    PubMed Central

    Park, Jeong-Ho; Park, Sun Hwa; Kang, Tong Mook; Cho, Hana; Kim, Tae Jin; Kim, Hyung-Wook; Jun, Youngsoo; Lee, Hee Jae; Lee, Young Sik; Kwon, Jae Young; Kang, KyeongJin

    2016-01-01

    Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1’s role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems. PMID:26726767

  5. Spontaneous Oligomerization of Nucleotide Alternatives in Aqueous Solutions.

    PubMed

    Smith, Karen E; House, Christopher H; Dworkin, Jason P; Callahan, Michael P

    2017-03-01

    On early Earth, a primitive polymer that could spontaneously form from likely available precursors may have preceded both RNA and DNA as the first genetic material. Here, we report that heated aqueous solutions containing 5-hydroxymethyluracil (HMU) result in oligomers of uracil, heated solutions containing 5-hydroxymethylcytosine (HMC) result in oligomers of cytosine, and heated solutions containing both HMU and HMC result in mixed oligomers of uracil and cytosine. Oligomerization of hydroxymethylated pyrimidines, which may have been abundant on the primitive Earth, might have been important in the development of simple informational polymers.

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

    PubMed

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

    2016-05-04

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

  7. Life without dUTPase

    PubMed Central

    Kerepesi, Csaba; Szabó, Judit E.; Papp-Kádár, Veronika; Dobay, Orsolya; Szabó, Dóra; Grolmusz, Vince; Vértessy, Beáta G.

    2016-01-01

    Fine-tuned regulation of the cellular nucleotide pools is indispensable for faithful replication of Deoxyribonucleic Acid (DNA). The genetic information is also safeguarded by DNA damage recognition and repair processes. Uracil is one of the most frequently occurring erroneous bases in DNA; it can arise from cytosine deamination or thymine-replacing incorporation. Two enzyme activities are primarily involved in keeping DNA uracil-free: dUTPase (dUTP pyrophosphatase) activity that prevent thymine-replacing incorporation and uracil-DNA glycosylase activity that excise uracil from DNA and initiate uracil-excision repair. Both dUTPase and the most efficient uracil-DNA glycosylase (UNG) is thought to be ubiquitous in free-living organisms. In the present work, we have systematically investigated the genotype of deposited fully sequenced bacterial and Archaeal genomes. We have performed bioinformatic searches in these genomes using the already well described dUTPase and UNG gene sequences. For dUTPases, we have included the trimeric all-beta and the dimeric all-alpha families and also, the bifunctional dCTP (deoxycytidine triphosphate) deaminase-dUTPase sequences. Surprisingly, we have found that in contrast to the generally held opinion, a wide number of bacterial and Archaeal species lack all of the previously described dUTPase gene(s). The dut– genotype is present in diverse bacterial phyla indicating that loss of this (or these) gene(s) has occurred multiple times during evolution. We discuss potential survival strategies in lack of dUTPases, such as simultaneous lack or inhibition of UNG and possession of exogenous or alternate metabolic enzymes involved in uracil-DNA metabolism. The potential that genes previously not associated with dUTPase activity may still encode enzymes capable of hydrolyzing dUTP is also discussed. Our data indicate that several unicellular microorganisms may efficiently cope with a dut– genotype lacking all of the previously described d

  8. Spontaneous Oligomerization of Nucleotide Alternatives in Aqueous Solutions

    NASA Astrophysics Data System (ADS)

    Smith, Karen E.; House, Christopher H.; Dworkin, Jason P.; Callahan, Michael P.

    2017-03-01

    On early Earth, a primitive polymer that could spontaneously form from likely available precursors may have preceded both RNA and DNA as the first genetic material. Here, we report that heated aqueous solutions containing 5-hydroxymethyluracil (HMU) result in oligomers of uracil, heated solutions containing 5-hydroxymethylcytosine (HMC) result in oligomers of cytosine, and heated solutions containing both HMU and HMC result in mixed oligomers of uracil and cytosine. Oligomerization of hydroxymethylated pyrimidines, which may have been abundant on the primitive Earth, might have been important in the development of simple informational polymers.

  9. Anticoccidial activity of 1-phenyluracils.

    PubMed

    Miller, M W; Chappel, L R

    1983-07-01

    The minimum effective concentration (MEC) of 6-azauracil against Eimeria tenella when incorporated in feed is about 1000 ppm. Attachment of suitably substituted phenyl side chains at the 1-position reduces the MEC to less than 1 ppm. Uracil itself is devoid of anticoccidial activity, but side chains attached at the 1-position potentiate uracil and result in activity against E. tenella. Although effective at levels as low as 60 ppm, the 1-phenyluracils are less active than the corresponding 1-phenyl-6-azauracils. They also are less acidic.

  10. Identification of glucuronide metabolites of d- and 1-methadone in bile from the isolated perfused rat liver.

    PubMed

    Lynn, R K; Leger, R M; Gerber, N

    1976-09-01

    Three hydroxylated metabolites of methadone, 2-ethyl-3-(dihydroxyphenyl)-5-methyl-3-phenylpyrroline (di-HO-EMDP), 2-ethyl-3-(hydroxy-methoxy-phenyl)-5-methyl-3-phenylpyrroline (HO-MeO-EMDP) and 2-ethyl-3-(hydroxy-phenyl)-5-methyl-3-phenylpyrroline (HO-EMDP) were excreted as O-glucuronide conjugates in bile from the isolated perfused rat liver following the addition of either d- or 1-methadone to the perfusate. These glucuronide metabolites were identified as intact molecules by combined gas chromatographic-mass spectrometric (GC-MS) analysis of their permethylated derivatives.

  11. Structure-Based Evaluation of C5 Derivatives in the Catechol Diether Series Targeting HIV-1 Reverse Transcriptase

    PubMed Central

    Frey, Kathleen M.; Gray, William T.; Spasov, Krasimir A.; Bollini, Mariela; Gallardo-Macias, Ricardo; Jorgensen, William L.; Anderson, Karen S.

    2014-01-01

    Using a computationally driven approach, a class of inhibitors with picomolar potency known as the catechol diethers were developed targeting the non-nucleoside binding pocket (NNBP) of HIV-1 RT. Computational studies suggested that halogen bonding interactions between the C5 substituent of the inhibitor and backbone carbonyl of conserved residue Pro95 might be important. While the recently reported crystal structures of the RT complexes confirmed the interactions with the NNBP, they revealed the lack of a halogen bonding interaction with Pro95. In order to understand the effects of substituents at the C5 position, we determined additional crystal structures with 5-Br and 5-H derivatives. Using comparative structural analysis, we identified several conformations of the ethoxy uracil dependent on the strength of a van der Waals interaction with the Cγ of Pro95 and the C5 substitution. The 5-Cl and 5-F derivatives position the ethoxy uracil to make more hydrogen bonds, while the larger 5-Br and smaller 5-H position the ethoxy uracil to make fewer hydrogen bonds. EC50 values correlate with the trends observed in the crystal structures. The influence of C5 substitutions on the ethoxy uracil conformation may have strategic value, as future derivatives can possibly be modulated in order to gain additional hydrogen bonding interactions with resistant variants of RT. PMID:24289305

  12. Preparation of next-generation sequencing libraries from damaged DNA.

    PubMed

    Briggs, Adrian W; Heyn, Patricia

    2012-01-01

    Next-generation sequencing (NGS) has revolutionized ancient DNA research, especially when combined with high-throughput target enrichment methods. However, attaining high sequencing depth and accuracy from samples often remains problematic due to the damaged state of ancient DNA, in particular the extremely low copy number of ancient DNA and the abundance of uracil residues derived from cytosine deamination that lead to miscoding errors. It is therefore critical to use a highly efficient procedure for conversion of a raw DNA extract into an adaptor-ligated sequencing library, and equally important to reduce errors from uracil residues. We present a protocol for NGS library preparation that allows highly efficient conversion of DNA fragments into an adaptor-ligated form. The protocol incorporates an option to remove the vast majority of uracil miscoding lesions as part of the library preparation process. The procedure requires only two spin column purification steps and no gel purification or bead handling. Starting from an aliquot of DNA extract, a finished, highly amplified library can be generated in 5 h, or under 3 h if uracil removal is not required.

  13. Alternative bases in the RNA world: the prebiotic synthesis of urazole and its ribosides

    NASA Technical Reports Server (NTRS)

    Kolb, V. M.; Dworkin, J. P.; Miller, S. L.

    1994-01-01

    Urazole is a five-membered heterocyclic compound which is isosteric with uracil's hydrogen-bonding segment. Urazole reacts spontaneoulsy with ribose (and other aldoses) to give a mixture of four ribosides: alpha and beta pyranosides and furanosides. This reaction occurs in aqueous solution at mild temperatures. Thermodynamic and kinetic parameters for the reaction of urazole with ribose were determined. In contrast, uracil is completely unreactive with ribose under these conditions. Urazole's unusual reactivity is ascribed to the hydrazine portion of the molecule. Urazole can be synthesized from biuret and hydrazine under prebiotic conditions. The prebiotic synthesis of guanazole, which is isosteric in part to diaminopyrimidine and cytosine, is accomplished from dicyandiamide and hydrazine. Kinetic parameters for both prebiotic reactions were measured. Urazole and guanazole are transparent in the UV, which would be a favorable property in the absence of an ozone layer on the early Earth. Urazole makes hydrogen bonds with adenine in DMSO similar to those of uracil, as established by H NMR. All of these properties make urazole an attractive potential precursor to uracil and guanazole a potential precursor to cytosine in the RNA or pre-RNA world.

  14. Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus.

    PubMed Central

    Woloshuk, C P; Seip, E R; Payne, G A; Adkins, C R

    1989-01-01

    A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin. Images PMID:2495764

  15. Lone-pair-π interactions: analysis of the physical origin and biological implications.

    PubMed

    Novotný, Jan; Bazzi, Sophia; Marek, Radek; Kozelka, Jiří

    2016-07-28

    Lone-pair-π (lp-π) interactions have been suggested to stabilize DNA and protein structures, and to participate in the formation of DNA-protein complexes. To elucidate their physical origin, we have carried out a theoretical multi-approach analysis of two biologically relevant model systems, water-indole and water-uracil complexes, which we compared with the structurally similar chloride-tetracyanobenzene (TCB) complex previously shown to contain a strong charge-transfer (CT) binding component. We demonstrate that the CT component in lp-π interactions between water and indole/uracil is significantly smaller than that stabilizing the Cl(-)-TCB reference system. The strong lp(Cl(-))-π(TCB) orbital interaction is characterized by a small energy gap and an efficient lp-π* overlap. In contrast, in lp-π interactions between water and indole or uracil, the corresponding energy gap is larger and the overlap less efficient. As a result, water-uracil and water-indole interactions are weak forces composed by smaller contributions from all energy components: electrostatics, polarization, dispersion, and charge transfer. In addition, indole exhibits a negative electrostatic potential at its π-face, making lp-π interactions less favorable than O-Hπ hydrogen bonding. Consequently, some of the water-tryptophan contacts observed in X-ray structures of proteins and previously interpreted as lp-π interactions [Luisi, et al., Proteins, 2004, 57, 1-8], might in fact arise from O-Hπ hydrogen bonding.

  16. Akt1 protects against germ cell apoptosis in the post natal mouse testis following lactational exposure to 6-N-propylthiouracil

    EPA Science Inventory

    Lactational exposure to 6-propyl-2-thio-uracil (PTU), a neonatal goitrogen, leads to increased testis size and sperm production in rodents. Aktl, a gene involved in cell survival and proliferation is also phosphorylated by thyroxine (T4). Therefore, we examined the requirement f...

  17. In vivo reshaping the catalytic site of nucleoside 2'-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution.

    PubMed

    Kaminski, Pierre Alexandre; Dacher, Priscilla; Dugué, Laurence; Pochet, Sylvie

    2008-07-18

    Nucleoside 2'-deoxyribosyltransferases catalyze the transfer of 2-deoxyribose between bases and have been widely used as biocatalysts to synthesize a variety of nucleoside analogs. The genes encoding nucleoside 2'-deoxyribosyltransferase (ndt) from Lactobacillus leichmannii and Lactobacillus fermentum underwent random mutagenesis to select variants specialized for the synthesis of 2',3'-dideoxynucleosides. An Escherichia coli strain, auxotrophic for uracil and unable to use 2',3'-dideoxyuridine, cytosine, and 2',3'-dideoxycytidine as a source of uracil was constructed. Randomly mutated lactobacilli ndt libraries from two species, L. leichmannii and L. fermentum, were screened for the production of uracil with 2',3'-dideoxyuridine as a source of uracil. Several mutants suitable for the synthesis of 2',3'-dideoxynucleosides were isolated. The nucleotide sequence of the corresponding genes revealed a single mutation (G --> A transition) leading to the substitution of a small aliphatic amino acid by a nucleophilic one, A15T (L. fermentum) or G9S (L. leichmannii), respectively. We concluded that the "adaptation" of the nucleoside 2'-deoxyribosyltransferase activity to 2,3-dideoxyribosyl transfer requires an additional hydroxyl group on a key amino acid side chain of the protein to overcome the absence of such a group in the corresponding substrate. The evolved proteins also display significantly improved nucleoside 2',3'-didehydro-2',3'-dideoxyribosyltransferase activity.

  18. Formation of Nucleobases from the UV Irradiation of Pyrimidine in Astrophysical Ice Analogs

    NASA Technical Reports Server (NTRS)

    Sandford, Scott A.; Nuevo, Michel; Materese, Christopher K.

    2014-01-01

    Nucleobases are the informational subunits of DNA and RNA. They consist of Nheterocycles that belong to either the pyrimidine-base group (uracil, cytosine, and thymine) or the purinebase group (adenine and guanine). Several nucleobases, mostly purine bases, have been detected in meteorites [1-3], with isotopic signatures consistent with an extraterrestrial origin [4]. Uracil is the only pyrimidine-base compound formally reported in meteorites [2], though the presence of cytosine cannot be ruled out [5,6]. However, the actual process by which the uracil was made and the reasons for the non-detection of thymine in meteorites have yet to be fully explained. Although no N-heterocycles have ever been observed in the ISM [7,8], the positions of the 6.2-µm interstellar emission features suggest a population of such molecules is likely to be present [9]. In this work we study the formation of pyrimidine-based molecules, including the three nucleobases uracil, cytosine, and thymine from the ultraviolet (UV) irradiation of pyrimidine in ices consisting of several combinations of H(sub2)O, NH(sub3), CH(sub3)OH, and CH(sub4) at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium, in the protosolar nebula, and on icy bodies of the Solar System.

  19. 40 CFR Appendix I to Part 192 - Listed Constituents

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...-Naphthalendisulfonic acid, 3,3′- -4,4′-diyl)bis(azo)]bis(5-amino-4-hydroxy-, tetrasodium salt) Uracil mustard (2,4-(1H... vanadate (Vanadic acid, ammonium salt) Aniline (Benzenamine) Antimony and compounds, N.O.S. 1 1 The... listed by name in this appendix. Aramite (Sulfurous acid, 2-chloroethyl 2- -1-methylethyl ester)...

  20. 40 CFR Appendix I to Part 192 - Listed Constituents

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-Naphthalendisulfonic acid, 3,3′- -4,4′-diyl)bis(azo)]bis(5-amino-4-hydroxy-, tetrasodium salt) Uracil mustard (2,4-(1H... vanadate (Vanadic acid, ammonium salt) Aniline (Benzenamine) Antimony and compounds, N.O.S. 1 1 The... listed by name in this appendix. Aramite (Sulfurous acid, 2-chloroethyl 2- -1-methylethyl ester)...

  1. 40 CFR Appendix I to Part 192 - Listed Constituents

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...-Naphthalendisulfonic acid, 3,3′- -4,4′-diyl)bis(azo)]bis(5-amino-4-hydroxy-, tetrasodium salt) Uracil mustard (2,4-(1H... vanadate (Vanadic acid, ammonium salt) Aniline (Benzenamine) Antimony and compounds, N.O.S. 1 1 The... listed by name in this appendix. Aramite (Sulfurous acid, 2-chloroethyl 2- -1-methylethyl ester)...

  2. UPP1 — EDRN Public Portal

    Cancer.gov

    UPP1, or uridine phosphorylase 1, is a pyrimidine nucleoside phosphorylase. UPP1, in the presence of orthophosphate, catalyzes the reversible phosphorylytic cleavage of uridine and deoxyuridine to uracil and ribose- or deoxyribose-1-phosphate. Pyrimidine nucleoside phosphorylases can add ribose or deoxyribose to pyrimidine bases to form nucleosides that can be incorporated into RNA or DNA.

  3. Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

    PubMed Central

    Arbeithuber, Barbara; Makova, Kateryna D.; Tiemann-Boege, Irene

    2016-01-01

    The need in cancer research or evolutionary biology to detect rare mutations or variants present at very low frequencies (<10−5) poses an increasing demand on lowering the detection limits of available methods. Here we demonstrated that amplifiable DNA lesions introduce important error sources in ultrasensitive technologies such as single molecule PCR (smPCR) applications (e.g. droplet-digital PCR), or next-generation sequencing (NGS) based methods. Using templates with known amplifiable lesions (8-oxoguanine, deaminated 5-methylcytosine, uracil, and DNA heteroduplexes), we assessed with smPCR and duplex sequencing that templates with these lesions were amplified very efficiently by proofreading polymerases (except uracil), leading to G->T, and to a lesser extent, to unreported G->C substitutions at 8-oxoguanine lesions, and C->T transitions in amplified uracil containing templates. Long heat incubations common in many DNA extraction protocols significantly increased the number of G->T substitutions. Moreover, in ∼50-80% smPCR reactions we observed the random amplification preference of only one of both DNA strands explaining the known ‘PCR jackpot effect’, with the result that a lesion became indistinguishable from a true mutation or variant. Finally, we showed that artifactual mutations derived from uracil and 8-oxoguanine could be significantly reduced by DNA repair enzymes. PMID:27477585

  4. High-resolution photoelectron spectra of the pyrimidine-type nucleobases

    NASA Astrophysics Data System (ADS)

    Fulfer, K. D.; Hardy, D.; Aguilar, A. A.; Poliakoff, E. D.

    2015-06-01

    High-resolution photoelectron spectra of the gas phase pyrimidine-type nucleobases, thymine, uracil, and cytosine, were collected using synchrotron radiation over the photon energy range 17 ≤ hν ≤ 150 eV. These data provide the highest resolution photoelectron spectra of thymine, uracil, and cytosine published to date. By comparing integrated regions of the energy dependent photoelectron spectra of thymine, the ionization potentials of the first four ionic states of thymine were estimated to be 8.8, 9.8, 10.3, and 10.8 eV. The thymine data also show evidence for low energy shape resonances in three of the outermost valence electronic states. Comparing the uracil spectrum with the thymine spectrum, the four outermost valence electronic states of uracil likely begin at binding energies 9.3, 9.9, 10.5, and 11.0 eV. High-resolution spectra indicate only one tautomeric form of cytosine contributes significantly to the spectrum with the four outermost valence electronic states beginning at binding energies 8.9, 9.9, 10.4, and 10.85 eV.

  5. Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications.

    PubMed

    Arbeithuber, Barbara; Makova, Kateryna D; Tiemann-Boege, Irene

    2016-12-01

    The need in cancer research or evolutionary biology to detect rare mutations or variants present at very low frequencies (<10(-5)) poses an increasing demand on lowering the detection limits of available methods. Here we demonstrated that amplifiable DNA lesions introduce important error sources in ultrasensitive technologies such as single molecule PCR (smPCR) applications (e.g. droplet-digital PCR), or next-generation sequencing (NGS) based methods. Using templates with known amplifiable lesions (8-oxoguanine, deaminated 5-methylcytosine, uracil, and DNA heteroduplexes), we assessed with smPCR and duplex sequencing that templates with these lesions were amplified very efficiently by proofreading polymerases (except uracil), leading to G->T, and to a lesser extent, to unreported G->C substitutions at 8-oxoguanine lesions, and C->T transitions in amplified uracil containing templates. Long heat incubations common in many DNA extraction protocols significantly increased the number of G->T substitutions. Moreover, in ∼50-80% smPCR reactions we observed the random amplification preference of only one of both DNA strands explaining the known 'PCR jackpot effect', with the result that a lesion became indistinguishable from a true mutation or variant. Finally, we showed that artifactual mutations derived from uracil and 8-oxoguanine could be significantly reduced by DNA repair enzymes.

  6. A history of the DNA repair and mutagenesis field: The discovery of base excision repair.

    PubMed

    Friedberg, Errol C

    2016-01-01

    This article reviews the early history of the discovery of an DNA repair pathway designated as base excision repair (BER), since in contrast to the enzyme-catalyzed removal of damaged bases from DNA as nucleotides [called nucleotide excision repair (NER)], BER involves the removal of damaged or inappropriate bases, such as the presence of uracil instead of thymine, from DNA as free bases.

  7. High-resolution photoelectron spectra of the pyrimidine-type nucleobases.

    PubMed

    Fulfer, K D; Hardy, D; Aguilar, A A; Poliakoff, E D

    2015-06-14

    High-resolution photoelectron spectra of the gas phase pyrimidine-type nucleobases, thymine, uracil, and cytosine, were collected using synchrotron radiation over the photon energy range 17 ≤ hν ≤ 150 eV. These data provide the highest resolution photoelectron spectra of thymine, uracil, and cytosine published to date. By comparing integrated regions of the energy dependent photoelectron spectra of thymine, the ionization potentials of the first four ionic states of thymine were estimated to be 8.8, 9.8, 10.3, and 10.8 eV. The thymine data also show evidence for low energy shape resonances in three of the outermost valence electronic states. Comparing the uracil spectrum with the thymine spectrum, the four outermost valence electronic states of uracil likely begin at binding energies 9.3, 9.9, 10.5, and 11.0 eV. High-resolution spectra indicate only one tautomeric form of cytosine contributes significantly to the spectrum with the four outermost valence electronic states beginning at binding energies 8.9, 9.9, 10.4, and 10.85 eV.

  8. DNA duplex stability of the thio-iso-guanine•methyl-iso-Cytosine base pair.

    PubMed

    Lee, Dongkye; Switzer, Christopher

    2015-01-01

    We report the synthesis, incorporation into oligonucleotides, and base-pairing properties of the 2-thio-variant of iso-guanine. Iso-guanine is the purine component of a nonstandard base pair with 5-methyl-iso-cytosine. The 2-thio-iso-guanine • 5-methyl-iso-cytosine base pair is found to have similar stability to an adenine • thymine pair.

  9. Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene.

    PubMed

    Kuilenburg, André B P van; Meijer, Judith; Tanck, Michael W T; Dobritzsch, Doreen; Zoetekouw, Lida; Dekkers, Lois-Lee; Roelofsen, Jeroen; Meinsma, Rutger; Wymenga, Machteld; Kulik, Wim; Büchel, Barbara; Hennekam, Raoul C M; Largiadèr, Carlo R

    2016-04-01

    Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation.

  10. From the Primitive Atmosphere to the Prebiotic Soup to the Pre-RNA World

    NASA Technical Reports Server (NTRS)

    Miller, Stanley L.

    1996-01-01

    Organic compounds would have been produced in an earth's atmosphere that was reducing. The soup would contain amino and hydroxy acids, together with smaller amounts of purines and pyrimidines. The presence' of sugars is less likely, although they can be produced by the formose reaction from formaldehyde. However, the prebiotic synthesis of RNA has not been demonstrated. One problem is that ribose is not produced selectively over other pentoses and hexoses, except under special conditions. The second problem is that ribose is unstable, with a half-life at pH7 and 100 C of 73 minutes (44 years at 0 C). Other sugars are similarly unstable. Another problem is that there is no efficient prebiotic synthesis of polyphosphates, nor the glycosidic bond of nucleosides. This suggests that there may have been an informational macromolecule that preceded RNA. The RNA world refers to the time when RNA carried both the genetic information and the catalytic activity, and was subsequently converted to the DNA/protein world when protein synthesis began. Preceeding the RNA world was the Pre-RNA world, where a backbone different from ribose phosphate was used, and the bases may have been different from adenine, uracil, guanine and cytosine. We have shown recently that cytosine and uracil can be synthesized efficiently under prebiotic conditions using a dried lagoon model instead of the usual dilute ocean hypothesis. In addition, we have shown that uracil adds formaldehyde efficiently to give 5- hydroxymethyl uracil, which in turn adds various nucleophiles to give uracil analogs of most of the amino acids that occur in proteins. For example, the ammonia, guanidine and imidazole adducts from the analogs of lysine, arginine and histidine. This suggests that the catalytic potential of RNA may have been much more extensive than previously assumed. The major problem is finding out what was the precursor to the ribose phosphate backbone. This will be the key to developing prebiotic self

  11. Characterization of the Adsorption of Nucleic Acid Bases onto Ferrihydrite via Fourier Transform Infrared and Surface-Enhanced Raman Spectroscopy and X-ray Diffractometry.

    PubMed

    Canhisares-Filho, José E; Carneiro, Cristine E A; de Santana, Henrique; Urbano, Alexandre; da Costa, Antonio C S; Zaia, Cássia T B V; Zaia, Dimas A M

    2015-09-01

    Minerals could have played an important role in concentration, protection, and polymerization of biomolecules. Although iron is the fourth most abundant element in Earth's crust, there are few works in the literature that describe the use of iron oxide-hydroxide in prebiotic chemistry experiments. In the present work, the interaction of adenine, thymine, and uracil with ferrihydrite was studied under conditions that resemble those of prebiotic Earth. At acidic pH, anions in artificial seawater decreased the pH at the point of zero charge (pHpzc) of ferrihydrite; and at basic pH, cations increased the pHpzc. The adsorption of nucleic acid bases onto ferrihydrite followed the order adenine > uracil > thymine. Adenine adsorption peaked at neutral pH; however, for thymine and uracil, adsorption increased with increasing pH. Electrostatic interactions did not appear to play an important role on the adsorption of nucleic acid bases onto ferrihydrite. Adenine adsorption onto ferrihydrite was higher in distilled water compared to artificial seawater. After ferrihydrite was mixed with artificial seawaters or nucleic acid bases, X-ray diffractograms and Fourier transform infrared spectra did not show any change. Surface-enhanced Raman spectroscopy showed that the interaction of adenine with ferrihydrite was not pH-dependent. In contrast, the interactions of thymine and uracil with ferrihydrite were pH-dependent such that, at basic pH, thymine and uracil lay flat on the surface of ferrihydrite, and at acidic pH, thymine and uracil were perpendicular to the surface. Ferrihydrite adsorbed much more adenine than thymine; thus adenine would have been better protected against degradation by hydrolysis or UV radiation on prebiotic Earth.

  12. A method to assess genomic DNA methylation using high-performance liquid chromatography/electrospray ionization mass spectrometry.

    PubMed

    Friso, Simonetta; Choi, Sang-Woon; Dolnikowski, Gregory G; Selhub, Jacob

    2002-09-01

    Eukaryotic DNA is methylated at some cytosine residues, and this epigenetic feature performs critical functions. We developed a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The DNA was enzymatically hydrolyzed by sequential digestion with three enzymes. DNA hydrolyzates were subsequently separated by reversed-phase high-performance liquid chromatography in isocratic mode. The four major DNA bases and 5-methyl-2'-deoxycytidine were resolved and eluted in 13 min. Identification of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine was obtained by combined diode array UV spectra analysis and mass spectra of chromatographic peaks. The isotopomers [15N3]-2'-deoxycytidine and (methyl-d3,ring-6-d1)-5-methyl-2'-deoxycytidine were used as internal standards. Ions of m/z 126 and 130 were used to detect 5-methyl-2'-deoxycytidine and its isotopomer, and ions of m/z 112 and 115 were used to detect 2'-deoxycytidine and its stable isotopomer, respectively. The DNA methylation status was calculated on the basis of the amount of 5-methyl-2'-deoxycytidine per microgram of DNA with percent relative standard deviations (%RSD) for a method precision of 7.1 (within-day) and 5.7 (day-to-day). This method also allows the measurement of 5-methyl-2'-deoxycytidine expressed as a percentage of total deoxycytidine residues in genomic DNA with %RSD for method precision of 1.9 (within-day) and 1.7 (day-to-day). This LC/MS method for quantitative determination of genomic DNA methylation status is rapid, sensitive, selective, and precise.

  13. Is 5-methyltetrahydrofolate an alternative to folic acid for the prevention of neural tube defects?

    PubMed

    Obeid, Rima; Holzgreve, Wolfgang; Pietrzik, Klaus

    2013-09-01

    Women have higher requirements for folate during pregnancy. An optimal folate status must be achieved before conception and in the first trimester when the neural tube closes. Low maternal folate status is causally related to neural tube defects (NTDs). Many NTDs can be prevented by increasing maternal folate intake in the preconceptional period. Dietary folate is protective, but recommending increasing folate intake is ineffective on a population level particularly during periods of high demands. This is because the recommendations are often not followed or because the bioavailability of food folate is variable. Supplemental folate [folic acid (FA) or 5-methyltetrahydrofolate (5-methylTHF)] can effectively increase folate concentrations to the level that is considered to be protective. FA is a synthetic compound that has no biological functions unless it is reduced to dihydrofolate and tetrahydrofolate. Unmetabolized FA appears in the circulation at doses of >200 μg. Individuals show wide variations in their ability to reduce FA. Carriers of certain polymorphisms in genes related to folate metabolism or absorption can better benefit from 5-methylTHF instead of FA. 5-MethylTHF [also known as (6S)-5-methylTHF] is the predominant natural form that is readily available for transport and metabolism. In contrast to FA, 5-methylTHF has no tolerable upper intake level and does not mask vitamin B12 deficiency. Supplementation of the natural form, 5-methylTHF, is a better alternative to supplementation of FA, especially in countries not applying a fortification program. Supplemental 5-methylTHF can effectively improve folate biomarkers in young women in early pregnancy in order to prevent NTDs.

  14. Interactions of model biomolecules. Benchmark CC calculations within MOLCAS

    SciTech Connect

    Urban, Miroslav; Pitoňák, Michal; Neogrády, Pavel; Dedíková, Pavlína; Hobza, Pavel

    2015-01-22

    We present results using the OVOS approach (Optimized Virtual Orbitals Space) aimed at enhancing the effectiveness of the Coupled Cluster calculations. This approach allows to reduce the total computer time required for large-scale CCSD(T) calculations about ten times when the original full virtual space is reduced to about 50% of its original size without affecting the accuracy. The method is implemented in the MOLCAS computer program. When combined with the Cholesky decomposition of the two-electron integrals and suitable parallelization it allows calculations which were formerly prohibitively too demanding. We focused ourselves to accurate calculations of the hydrogen bonded and the stacking interactions of the model biomolecules. Interaction energies of the formaldehyde, formamide, benzene, and uracil dimers and the three-body contributions in the cytosine – guanine tetramer are presented. Other applications, as the electron affinity of the uracil affected by solvation are also shortly mentioned.

  15. New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

    PubMed Central

    Price, Alan R.; Cook, Sandra J.

    1972-01-01

    The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection. PMID:4623224

  16. Measurement of Postreplicative DNA Metabolism and Damage in the Rodent Brain.

    PubMed

    Patel, Jay P; Sowers, Mark L; Herring, Jason L; Theruvathu, Jacob A; Emmett, Mark R; Hawkins, Bridget E; Zhang, Kangling; DeWitt, Douglas S; Prough, Donald S; Sowers, Lawrence C

    2015-12-21

    The DNA of all organisms is metabolically active due to persistent endogenous DNA damage, repair, and enzyme-mediated base modification pathways important for epigenetic reprogramming and antibody diversity. The free bases released from DNA either spontaneously or by base excision repair pathways constitute DNA metabolites in living tissues. In this study, we have synthesized and characterized the stable-isotope standards for a series of pyrimidines derived from the normal DNA bases by oxidation and deamination. We have used these standards to measure free bases in small molecule extracts from rat brain. Free bases are observed in extracts, consistent with both endogenous DNA damage and 5-methylcytosine demethylation pathways. The most abundant free base observed is uracil, and the potential sources of uracil are discussed. The free bases measured in tissue extracts constitute the end product of DNA metabolism and could be used to reveal metabolic disturbances in human disease.

  17. Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis.

    PubMed

    Mourad, George S; Tippmann-Crosby, Julie; Hunt, Kevin A; Gicheru, Yvonne; Bade, Kaely; Mansfield, Tyler A; Schultes, Neil P

    2012-05-07

    Locus At5g03555 encodes a nucleobase cation symporter 1 (AtNCS1) in the Arabidopsis genome. Arabidopsis insertion mutants, AtNcs1-1 and AtNcs1-3, were used for in planta toxic nucleobase analog growth studies and radio-labeled nucleobase uptake assays to characterize solute transport specificities. These results correlate with similar growth and uptake studies of AtNCS1 expressed in Saccharomyces cerevisiae. Both in planta and heterologous expression studies in yeast revealed a unique solute transport profile for AtNCS1 in moving adenine, guanine and uracil. This is in stark contrast to the canonical transport profiles determined for the well-characterized S. cerevisiae NCS1 proteins FUR4 (uracil transport) or FCY2 (adenine, guanine, and cytosine transport).

  18. Crystallization and preliminary X-ray data analysis of β-alanine synthase from Drosophila melanogaster

    SciTech Connect

    Lundgren, Stina; Andersen, Birgit; Piškur, Jure; Dobritzsch, Doreen

    2007-10-01

    β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine. Crystals of the recombinant enzyme from D. melanogaster belong to space group C2. Diffraction data to 3.3 Å resolution were collected and analyzed. β-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 Å at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 Å, β = 125.8°) and contain 8–10 molecules per asymmetric unit.

  19. Vibrational frequencies and structure of 2-thiouracil by Hartree-Fock, post-Hartree-Fock and density functional methods.

    PubMed

    Palafox, M Alcolea; Rastogi, V K; Tanwar, R P; Mittal, Lalit

    2003-09-01

    Vibrational study of the biomolecule 2-thiouracil was carried out. Ab initio and density functional calculations were performed to assign the experimental spectra. A comparison with the uracil molecule was made, and specific scale factors were deduced and employed in the predicted frequencies of 2-thiouracil. Several scaling procedures were used. The geometry structure of the molecule was determined. The effect of sulfur substitution at C2 position in the uracil molecule, on the N1-H and N3-H frequencies and intensities reflects changes in proton donor abilities of these groups. Calculations with the 6-31 G** basis set with HF and DFT methods appear in general to be useful for interpretation of the general features of the IR and Raman spectra of the molecule. Using specific scale factors a very small error was obtained. The use of these specific scale factors resolve and correct some of the controversial assignments in the literature.

  20. Engineering of a functional thermostable kanamycin resistance marker for use in Moorella thermoacetica ATCC39073.

    PubMed

    Iwasaki, Yuki; Kita, Akihisa; Sakai, Shinsuke; Takaoka, Kazue; Yano, Shinichi; Tajima, Takahisa; Kato, Junichi; Nishio, Naomichi; Murakami, Katsuji; Nakashimada, Yutaka

    2013-06-01

    A transformation system for Moorella thermoacetica ATCC39073 was developed using thermostable kanamycin resistant gene (kanR) derived from the plasmid pJH1 that Streptococcus faecalis harbored. When kanR with its native promoter was introduced into uracil auxotrophic mutant of M. thermoacetica ATCC39073 together with a gene to complement the uracil auxotrophy as a selection marker, it did not give kanamycin resistance due to poor transcription level of kanR. However, the use of glyceraldehyde-3-phosphate dehydrogenase promoter cloned from M. thermoacetica ATCC39073 significantly improved transcription level of kanR and resulted in the cell growth in the presence of more than 150 μg mL(-1) kanamycin. It was also demonstrated that kanR with G3PD promoter can be used as a selection marker for transformation of wild-type strain of M. thermoacetica ATCC39073.

  1. Dihydropyrimidine Dehydrogenase Deficiency in Two Malaysian Siblings with Abnormal MRI Findings

    PubMed Central

    Chen, Bee Chin; Mohd Rawi, Rowani; Meinsma, Rutger; Meijer, Judith; Hennekam, Raoul C.M.; van Kuilenburg, André B.P.

    2014-01-01

    Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine metabolism. Deficiency of this enzyme leads to an accumulation of thymine and uracil and a deficiency of metabolites distal to the catabolic enzyme. The disorder presents with a wide clinical spectrum, ranging from asymptomatic to severe neurological manifestations, including intellectual disability, seizures, microcephaly, autistic behavior, and eye abnormalities. Here, we report on an 11-year-old Malaysian girl and her 6-year-old brother with DPD deficiency who presented with intellectual disability, microcephaly, and hypotonia. Brain MRI scans showed generalized cerebral and cerebellar atrophy and callosal body dysgenesis in the boy. Urine analysis showed strongly elevated levels of uracil in the girl and boy (571 and 578 mmol/mol creatinine, respectively) and thymine (425 and 427 mmol/mol creatinine, respectively). Sequence analysis of the DPYD gene showed that both siblings were homozygous for the mutation c.1651G>A (pAla551Thr). PMID:25565930

  2. The nucleoside uridine isolated in the gas phase.

    PubMed

    Peña, Isabel; Cabezas, Carlos; Alonso, José L

    2015-03-02

    Herein we present the first experimental observation of the isolated nucleoside uridine, placed in the gas phase by laser ablation and characterized by Fourier transform (FT) microwave techniques. Free from the bulk effects of their native environments, anti/C2'-endo-g+ conformation has been revealed as the most stable form of uridine. Intramolecular hydrogen bonds involving uracil and ribose moieties have been found to play an important role in the stabilization of the nucleoside.

  3. PCR (Polymerase Chain Reaction) Testing for Leishmaniasis

    DTIC Science & Technology

    1993-10-20

    reactions. Prior to temperature cycling, this moderately heat stable enzyme selectively excises uracil residues which have been incorporated into DNA... temperature . Despite these precautions, some non-specific annealing of PCR primers does occur, even with single copy gene detection, and more so with...Mallinckrodt) in slightly hypotonic saline and allowed to remain at ambient temperature for 5 minutes, during which time RBC’s are lysed. Centrifugation

  4. The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

    SciTech Connect

    Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.; Parker, William B.; Piskur, Jure; Serra, I.; Terreni, M.; Ealick, Steven E.

    2011-09-20

    Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

  5. Past and present achievements, and future direction of the Gastrointestinal Oncology Study Group (GIOSG), a Division of Japan Clinical Oncology Group (JCOG).

    PubMed

    Boku, Narikazu

    2011-12-01

    Initially, Gastrointestinal Study Group in Japan Clinical Oncology Group (GIOSG/JCOG) focused on gastric cancer. In 1980s, fluoropyrimidine, cisplatin and mitomycin C were key drugs. A randomized Phase II trial (JCOG8501) comparing futrafur plus mitomycin C and uracil plus futrafur and mitomycin C showed a higher response rate of uracil plus futrafur and mitomycin C than futrafur plus mitomycin C. From the results of two Phase II trials of etoposide, adriamycin and cisplatin, and cisplatin plus 5-fluorouracil, uracil plus futrafur and mitomycin C and cisplatin plus 5-fluorouracil were adopted for the test arms of the Phase III trial (JCOG9205) comparing with continuous infusion of 5-fluorouracil as a control arm. Neither cisplatin plus 5-fluorouracil nor uracil plus futrafur and mitomycin C showed a survival benefit over continuous infusion of 5-fluorouracil. In late 1990s, new agents, irinotecan and S-1, were developed for gastric cancer in Japan. GIOSG conducted a Phase III trial (JCOG9912) investigating superiority of irinotecan plus cisplatin and non-inferiority of monotherapy with S-1 compared with continuous infusion of 5-fluorouracil, and S-1 succeeded in showing non-inferiority. Then, SPIRITS trial showed a survival benefit of S-1 plus cisplatin over S-1, resulting in the establishment of a standard care for advanced gastric cancer in Japan. GIOSG have merged with Gastric Cancer Study Group as the Stomach Cancer Study Group (SCSG) from 2011. Recent progress in the development of new drugs has been remarkable. From the point of the roles shared with many other study groups for clinical trials, including registration trials of new drugs conducted by pharmaceutical companies, SCSG should recognize its role and conduct clinical trials with high quality for establishing new standard treatment.

  6. The structure definition of complementary pairs Ade-Ura in different phase states using IR spectra

    NASA Astrophysics Data System (ADS)

    Ten, G. N.; Glukhova, O. E.; Semagina, A. M.; Slepchenkov, M. M.; Baranov, V. I.

    2015-03-01

    The parameters of hydrogen bridges and oscillation spectra of complementary pairs of adenine-uracil formed by Watson- Crick and Hugstin and two reverse to them structures are calculated. Performed analysis shows that due to the characteristic oscillations of the IR spectra in the area of 1600-1800 and 2900-3500 cm-1 it is possible to identify uniquely each of the four pairs in the gas phase and aqueous solution.

  7. Pyrene chromophores for the photoreversal of psoralen interstrand crosslinks.

    PubMed

    Stadler, Jens M; Stafforst, Thorsten

    2014-07-28

    Applying psoralen interstrand crosslinks for the photoactivation of nucleic acids is a new concept. To find chromophores that can efficiently stimulate crosslink repair we screened several pyrenes and appended them to peptide nucleic acids for their site-selective addressing. Even though pyrenes conjugated to uracil revealed desirable spectroscopic properties they were not effective in crosslink reversal. In contrast, bare pyrenes are well suitable for crosslink repair with 350 nm light showing an uncaging efficiency similar to classical photocaging groups.

  8. Identification and Functional Characterization of the First Nucleobase Transporter in Mammals

    PubMed Central

    Yamamoto, Syunsuke; Inoue, Katsuhisa; Murata, Tomoaki; Kamigaso, Syunsuke; Yasujima, Tomoya; Maeda, Jun-ya; Yoshida, Yukihiro; Ohta, Kin-ya; Yuasa, Hiroaki

    2010-01-01

    Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 μm. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use. PMID:20042597

  9. Metabolism of Pyrimidines and Pyrimidine Nucleosides by Salmonella typhimurium

    PubMed Central

    Beck, Christoph F.; Ingraham, John L.; Neuhard, Jan; Thomassen, Elisabeth

    1972-01-01

    The pathways by which uracil, cytosine, uridine, cytidine, deoxyuridine, and deoxycytidine are metabolized by Salmonella typhimurium are established. The various 5-fluoropyrimidine analogues are shown to exert their toxic effects only after having been converted to the nucleotide level, and these conversions are shown to be catalyzed by the same enzymes which similarly convert the natural substrates. Methods for isolating mutant strains blocked in various steps of metabolism of pyrimidine bases and nucleosides are described. PMID:4259664

  10. Low energy positron interactions with uracil—Total scattering, positronium formation, and differential elastic scattering cross sections

    SciTech Connect

    Anderson, E. K.; Boadle, R. A.; Machacek, J. R.; Makochekanwa, C.; Sullivan, J. P.; Chiari, L.; Buckman, S. J.; Brunger, M. J.; Garcia, G.; Blanco, F.; Ingolfsson, O.

    2014-07-21

    Measurements of the grand total and total positronium formation cross sections for positron scattering from uracil have been performed for energies between 1 and 180 eV, using a trap-based beam apparatus. Angular, quasi-elastic differential cross section measurements at 1, 3, 5, 10, and 20 eV are also presented and discussed. These measurements are compared to existing experimental results and theoretical calculations, including our own calculations using a variant of the independent atom approach.

  11. Photosynthesis and photo-stability of nucleic acids in prebiotic extraterrestrial environments.

    PubMed

    Sandford, Scott A; Bera, Partha P; Lee, Timothy J; Materese, Christopher K; Nuevo, Michel

    2015-01-01

    Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including molecules important for biology such as amino acids, quinones, and amphiphiles. When pyrimidine is introduced into these ices, the products of irradiation include the nucleobases uracil, cytosine, and thymine, the informational sub-units of DNA and RNA, as well as some of their isomers. The formation of these compounds, which has been studied both experimentally and theoretically, requires a succession of additions of OH, NH₂, and CH₃groups to pyrimidine. Results show that H₂O ice plays key roles in the formation of the nucleobases, as an oxidant, as a matrix in which reactions can take place, and as a catalyst that assists proton abstraction from intermediate compounds. As H₂O is also the most abundant icy component in most cold astrophysical environments, it probably plays the same roles in space in the formation of biologically relevant compounds. Results also show that although the formation of uracil and cytosine from pyrimidine in ices is fairly straightforward, the formation of thymine is not. This is mostly due to the fact that methylation is a limiting step for its formation, particularly in H₂O-rich ices, where methylation must compete with oxidation. The relative inefficiency of the abiotic formation of thymine to that of uracil and cytosine, together with the fact that thymine has not been detected in meteorites, are not inconsistent with the RNA world hypothesis. Indeed, a lack of abiotically produced thymine delivered to the early Earth may have forced the choice for an RNA world, in which only uracil and cytosine are needed, but not thymine.

  12. 40 CFR Appendix Viii to Part 261 - Hazardous Constituents

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...-Naphthalenedisulfonic acid, 3,3′- -4,4′-diyl)bis(azo)]-bis[5-amino-4-hydroxy-, tetrasodium salt. 72-57-1 U236 Uracil... Ethanimidothioic acid, 2- (dimethylamino) -N-hydroxy-2-oxo-, methyl ester 30558-43-1 U394 Acetonitrile Same 75-05-8... 504-24-5 P008 Amitrole 1H-1,2,4-Triazol-3-amine 61-82-5 U011 Ammonium vanadate Vanadic acid,...

  13. 40 CFR Appendix Viii to Part 261 - Hazardous Constituents

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...-Naphthalenedisulfonic acid, 3,3′- -4,4′-diyl)bis(azo)]-bis[5-amino-4-hydroxy-, tetrasodium salt. 72-57-1 U236 Uracil... Ethanimidothioic acid, 2- (dimethylamino) -N-hydroxy-2-oxo-, methyl ester 30558-43-1 U394 Acetonitrile Same 75-05-8... 504-24-5 P008 Amitrole 1H-1,2,4-Triazol-3-amine 61-82-5 U011 Ammonium vanadate Vanadic acid,...

  14. 2-(2,4-Dioxy-1,2,3,4-Tetrahydropyrimidin-1-yl)-N-(4-Phenoxyphenyl)-Acetamides as a Novel Class of Cytomegalovirus Replication Inhibitors

    PubMed Central

    Babkov, D. A.; Paramonova, M. P.; Ozerov, A. A.; Khandazhinskaya, A. L.; Snoeck, R.; Andrei, G.; Novikov, M. S.

    2015-01-01

    A series of novel uracil derivatives, bearing N-(4-phenoxyphenyl)acetamide moiety at N3 of a pyrimidine ring, has been synthesized. Their antiviral activity has been evaluated. It has been found that the novel compounds possess high inhibitory activity against replication of human cytomegalovirus (AD-169 and Davis strains) in HEL cell cultures. In addition, some of the derivatives proved to be inhibitory against varicella zoster virus. PMID:26798502

  15. Rational Inhibitors of DNA Base Excision Repair (BER) Enzymes: New Tools for Elucidating the Role of the BER in Cancer Chemotherapy

    DTIC Science & Technology

    2005-05-01

    right of the bars. (B) Incremental change in binding free energy as UDG (Figure 2) (5). The 2’ fluorinated deoxyuridine each hydrogen bond is removed from...1996) Naphthalene, phenanthrene, and pyrene as DNA h187q Escherichia coli uracil DNA glycosylase: Electrophilic base analogues: Synthesis, structure...solution, Biochemistry 32. Drohat, A. C., Jagadeesh, J., Ferguson, E., and Stivers, J. T. (1999) 26, 5646-5650. Role of electrophilic and general base

  16. The Nucleoside Uridine Isolated in the Gas Phase**

    PubMed Central

    Peña, Isabel; Cabezas, Carlos; Alonso, José L.

    2016-01-01

    Herein we present the first experimental observation of the isolated nucleoside uridine, placed in the gas phase by laser ablation and characterized by Fourier transform microwave techniques. Free from the bulk effects of their native environments, anti/C2’-endo-g+ conformation has been revealed as the most stable form of uridine. Intramolecular hydrogen bonds involving uracil and ribose moieties have been found to play an important role in the stabilization of the nucleoside. PMID:25683559

  17. The function of oxalic acid in the human metabolism.

    PubMed

    Robertson, Daniel Stewart

    2011-09-01

    Biochemical reactions in cells which involve oxalic acid are described. It is shown that this compound is required for the formation of uracil and orotic acid. The former is a component of RNA which is common to all cells in the human metabolism. On the basis of the biochemical reactions described a possible treatment to relieve the effects of calcium oxalate renal calculi whose origin is related to the metabolic concentration of oxalic acid is proposed.

  18. Caenorhabditis elegans APN-1 plays a vital role in maintaining genome stability.

    PubMed

    Zakaria, Chadi; Kassahun, Henok; Yang, Xiaoming; Labbé, Jean-Claude; Nilsen, Hilde; Ramotar, Dindial

    2010-02-04

    We previously showed that Caenorhabditis elegans APN-1, the only metazoan apurinic/apyrimidinc (AP) endonuclease belonging to the endonuclease IV family, can functionally rescue the DNA repair defects of Saccharomyces cerevisiae mutants completely lacking AP endonuclease/3'-diesterase activities. While this complementation study provided the first evidence that APN-1 possesses the ability to act on DNA lesions that are processed by AP endonucleases/3'-diesterase activities, no former studies were conducted to examine its biological importance in vivo. Herein, we show that C. elegans knockdown for apn-1 by RNAi displayed phenotypes that are directly linked with a defect in maintaining the integrity of the genome. apn-1(RNAi) animals exhibited a 5-fold increase in the frequency of mutations at a gfp-lacZ reporter and showed sensitivities to DNA damaging agents such as methyl methane sulfonate and hydrogen peroxide that produce AP site lesions and strand breaks with blocked 3'-ends. The apn-1(RNAi) worms also displayed a delay in the division of the P1 blastomere, a defect that is consistent with the accumulation of unrepaired lesions. Longevity was only compromised, if the apn-1(RNAi) animals were challenged with the DNA damaging agents. We showed that apn-1(RNAi) knockdown suppressed formation of apoptotic corpses in the germline caused by an overburden of AP sites generated from uracil DNA glycosylase mediated removal of misincorporated uracil. Finally, we showed that depletion of APN-1 by RNAi partially rescued the lethality resulting from uracil misincorporation, suggesting that APN-1 is an important AP endonuclease for repair of misincorporated uracil.

  19. Phenotypic Changes, Signaling Pathway, and Functional Correlates of GPR17-expressing Neural Precursor Cells during Oligodendrocyte Differentiation*

    PubMed Central

    Fumagalli, Marta; Daniele, Simona; Lecca, Davide; Lee, Philip R.; Parravicini, Chiara; Fields, R. Douglas; Rosa, Patrizia; Antonucci, Flavia; Verderio, Claudia; Trincavelli, M. Letizia; Bramanti, Placido; Martini, Claudia; Abbracchio, Maria P.

    2011-01-01

    The developing and mature central nervous system contains neural precursor cells expressing the proteoglycan NG2. Some of these cells continuously differentiate to myelin-forming oligodendrocytes; knowledge of the destiny of NG2+ precursors would benefit from the characterization of new key functional players. In this respect, the G protein-coupled membrane receptor GPR17 has recently emerged as a new timer of oligodendrogliogenesis. Here, we used purified oligodendrocyte precursor cells (OPCs) to fully define the immunophenotype of the GPR17-expressing cells during OPC differentiation, unveil its native signaling pathway, and assess the functional consequences of GPR17 activation by its putative endogenous ligands, uracil nucleotides and cysteinyl leukotrienes (cysLTs). GPR17 presence was restricted to very early differentiation stages and completely segregated from that of mature myelin. Specifically, GPR17 decorated two subsets of slowly proliferating NG2+ OPCs: (i) morphologically immature cells expressing other early proteins like Olig2 and PDGF receptor-α, and (ii) ramified preoligodendrocytes already expressing more mature factors, like O4 and O1. Thus, GPR17 is a new marker of these transition stages. In OPCs, GPR17 activation by either uracil nucleotides or cysLTs resulted in potent inhibition of intracellular cAMP formation. This effect was counteracted by GPR17 antagonists and receptor silencing with siRNAs. Finally, uracil nucleotides promoted and GPR17 inhibition, by either antagonists or siRNAs, impaired the normal program of OPC differentiation. These data have implications for the in vivo behavior of NG2+ OPCs and point to uracil nucleotides and cysLTs as main extrinsic local regulators of these cells under physiological conditions and during myelin repair. PMID:21209081

  20. a Nucleoside Under Observation in the Gas Phase: a Rotational Study of Uridine

    NASA Astrophysics Data System (ADS)

    Peña, Isabel; Alonso, José L.

    2014-06-01

    The nucleoside of uridine has been placed in the gas phase by laser ablation and the most stable C2{'}-anti conformation characterized by broadband chirped pulse (CP-FTMW) and narrowband molecular beam Fourier transform microwave (LA-MB-FTMW) spectroscopies. The quadrupole hyperfine structure, originated by two 14N nuclei, has been completely resolved. Intramolecular hydrogen bonds involving uracil and ribose moieties have been found to play an important role in the stabilization of the nucleoside.

  1. Heterocyclic Salt Synthesis and Rational Properties Tailoring (PREPRINT)

    DTIC Science & Technology

    2009-06-23

    10). Altering the hydrogen atom at the 5 - ring position on uracil (12) to a fluorine atom, affords 5 - fluorouracil (13). In terms of performance...11. SPONSOR/MONITOR’S 5 Pollux Drive NUMBER(S) Edwards AFB CA 93524-70448 AFRL-RZ-ED-JA-2009-265 12. DISTRIBUTION / AVAILABILITY...bridged heterocyclium di-cation salts, demonstrates how rational structure design, and its effect on resultant predictable properties modification

  2. Loading and pre-loading processes generate a distinct siRNA population in Tetrahymena

    SciTech Connect

    Mochizuki, Kazufumi Kurth, Henriette M.

    2013-07-05

    Highlights: •The Tetrahymena Argonaute protein Twi1p binds to ∼28–30-nt siRNAs called scnRNAs. •The size of scnRNAs is determined during a pre-loading process. •The 5′ uracil bias of scnRNAs is attributed to pre-loading and loading processes. •The thermodynamic asymmetry of scnRNA duplex doesnot affect the guide strand decision. •scnRNAs may be produced non-sequentially from dsRNA substrates by Dicer. -- Abstract: The various properties of small RNAs, such as length, terminal nucleotide, thermodynamic asymmetry and duplex mismatches, can impact their sorting into different Argonaute proteins in diverse eukaryotes. The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5′ end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5′ uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3′ terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo.

  3. A source for microhydrated biomolecules

    SciTech Connect

    Förstel, M.; Hergenhahn, U.; Neustetter, M.; Denifl, S.; Lelievre, F.

    2015-07-15

    We describe the construction of an apparatus for the production of a molecular jet of microhydrated biomolecules. Our design uses a water reservoir producing water vapour, which then passes through a separate reservoir containing a vapour of a sublimated biomolecule. The mixture coexpands into a molecular beam apparatus through a conical nozzle. Mass spectra showing water-adenin and water-uracil complexes are shown as typical examples. Suitable expansion conditions are reached without the use of an inert carrier gas.

  4. High resolution mapping of modified DNA nucleobases using excision repair enzymes

    PubMed Central

    Bryan, D. Suzi; Ransom, Monica; Adane, Biniam; York, Kerri

    2014-01-01

    The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. We describe methods for mapping positions and local content of modified DNA nucleobases in genomic DNA. We combined in vitro nucleobase excision with massively parallel DNA sequencing (Excision-seq) to determine the locations of modified nucleobases in genomic DNA. We applied the Excision-seq method to map uracil in E. coli and budding yeast and discovered significant variation in uracil content, wherein uracil is excluded from the earliest and latest replicating regions of the genome, possibly driven by changes in nucleotide pool composition. We also used Excision-seq to identify sites of pyrimidine dimer formation induced by UV light exposure, where the method could distinguish between sites of cyclobutane and 6-4 photoproduct formation. These UV mapping data enabled analysis of local sequence bias around pyrimidine dimers and suggested a preference for an adenosine downstream from 6-4 photoproducts. The Excision-seq method is broadly applicable for high precision, genome-wide mapping of modified nucleobases with cognate repair enzymes. PMID:25015380

  5. Identification and antifungal activity of novel organic compounds found in cuticular and internal lipids of medically important flies.

    PubMed

    Gołębiowski, Marek; Cerkowniak, Magdalena; Urbanek, Aleksandra; Dawgul, Małgorzata; Kamysz, Wojciech; Boguś, Mieczysława I; Stepnowski, Piotr

    2015-01-01

    Novel organic compounds found in the cuticular and internal lipids of medically important flies were identified. Uracil, 9-tricosene, 1-oleoyl glycerol, dimethyl suberate and butyl stearate were tested for their potential antifungal activity. Minimal inhibitory concentrations of the compounds against reference strains of fungi were determined. Uracil and dimethyl suberate slightly inhibited the growth of entomopathogenic fungi. The cuticular and internal lipids of Calliphora vicina, Calliphora vomitoria, Sarcophaga carnaria and Musca domestica were studied by gas chromatography (GC) combined with mass spectrometry (GC/MS). A comparison of the lipid extracts between the preimaginal and mature stages showed adults flies contained a higher total content of the identified components. Furthermore, their amounts distinctly predominated in the internal lipids of all the species. The amount of 9-tricosene was the highest in adults of C. vicina, while the larvae and pupae had a definitively lower amount of this compound. Uracil was found to be the most abundant component in extracts obtained from C. vomitoria especially in the internal lipids of adults. 1-oleoyl glycerol was detected in all of the examined species of flies. It was most abundant in the internal extracts isolated from the larvae of C. vicina and the pupae of C. vomitoria. Suberic acid dimethyl ester was found in the larval and pupal internal lipids of C. vicina and S. carnaria in low amounts. Butyl stearate was identified only in the internal lipids of the larvae and adults of houseflies.

  6. The transport of uric acid across mouse small intestine in vitro.

    PubMed Central

    Bronk, J R; Shaw, M I

    1986-01-01

    The in vitro recirculation technique was used to study the uptake and transport of uric acid by the jejunum of mouse small intestine. Three components of the serosal secretions appeared to be endogenously derived nucleic acid derivatives; two of these were identified as uric acid and uracil. There was no detectable metabolism of uric acid by the intestine. Uric acid transported from the lumen appeared in the serosal fluid at a concentration higher than that in the lumen. The final serosal/luminal concentration ratio of about 1.18 for exogenous uric acid was found to be constant over the concentration range studied (0.01-0.1 mM). The presence of exogenous uric acid in the lumen did not affect the production of endogenous uric acid by the intestine and its release into the serosal secretions. Mucosal concentration of exogenous uric acid was below, but the total mucosal concentration (exogenous+endogenous) was above, that in the lumen. There was no evidence for the secretion of endogenous uric acid into the lumen. Oxypurinol significantly decreased the rate of serosal appearance of exogenous uric acid. Allopurinol did not affect the transport of exogenous uric acid from the lumen and there was negligible metabolism of allopurinol to oxypurinol by the tissue. Uracil did not affect the transport of exogenous uric acid from the lumen, or the serosal appearance of endogenous uric acid. Likewise uracil transport was unaffected by luminal uric acid. PMID:3795104

  7. Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy

    PubMed Central

    Pukáncsik, Mária; Orbán, Ágnes; Nagy, Kinga; Matsuo, Koichi; Gekko, Kunihiko; Maurin, Damien; Hart, Darren; Kézsmárki, István; Vertessy, Beata G.

    2016-01-01

    A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive α-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of α-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies. PMID:27273007

  8. Designing DNA interstrand lock for locus-specific methylation detection in a nanopore

    PubMed Central

    Kang, Insoon; Wang, Yong; Reagan, Corbin; Fu, Yumei; Wang, Michael X.; Gu, Li-Qun

    2013-01-01

    DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg2+) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5′-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer. PMID:24135881

  9. Response of biological uv dosimeters to the simulated extraterrestrial uv radiation

    NASA Astrophysics Data System (ADS)

    Bérces, A.; Rontó, G.; Kerékgyártó, T.; Kovács, G.; Lammer, H.

    In the Laboratory polycrystalline uracil thin layer and bacteriophage T7 detectors have been developed for UV dosimetry on the EarthSs surface. Exponential response of the uracil polycrystal has been detected both by absorption spectroscopy and measurements of the refractive index under the influence of terrestrial solar radiation or using UV-C sources. In UV biological dosimetry the UV dose scale is additive starting at a value of zero according to the definition of CIE (Technical Report TC-6-18). The biological dose can be defined by a measured end-effect. In our dosimeters (phage T7 and uracil dosimeter) exposed to natural (terrestrial) UV radiation the proportion of pyrimidin photoproducts among the total photoproducts is smaller than 0.1 and the linear correlation between the biological and physical dose is higher than 0.9. According to the experimental data this linear relationship is often not valid. We observed that UV radiation did not only induce dimerisation but shorter wavelengths caused monomerisation of pyrimidin dimers. Performing the irradiation in oxygen free environment and using a Deuterium lamp as UV source, we could increase monomerisation against dimerisation thus the DNA-based dosimetrySs additivity rule is not fulfilled in these conditions. In this study we will demonstrate those non-linear experiments which constitute the basis of our biological experiments on the International Space Station.

  10. The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

    PubMed

    Garavaglia, Marco; Rossi, Elio; Landini, Paolo

    2012-01-01

    Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

  11. Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD).

    PubMed

    Zhang, Xin; Zhao, Yuan; Yan, Honggao; Cao, Zexing; Mo, Yirong

    2016-05-15

    Extensive combined quantum mechanical (B3LYP/6-31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton-transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2-pyrimidinone. The rate-determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc.

  12. Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection

    SciTech Connect

    Ramsey, R.S.; Ho, C. )

    1989-11-01

    Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.

  13. Ultraviolet Irradiation of Pyrimidine in Interstellar Ice Analogs: Formation and Photo-Stability of Nucleobases

    NASA Technical Reports Server (NTRS)

    Nuevo, Michel; Milam, Stefanie N.; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

    2010-01-01

    Astrochemistry laboratory experiments recently showed that molecules of prebiotic interest can potentially form in space, as supported by the detection of amino acids in organic residues formed by the UV photolysis of ices simulating interstellar and cometary environments (H2O, CO, CO2, CH3OH, NH3, etc.). Although the presence of amino acids in the interstellar medium (ISM) is still under debate, experiments and the detection of amino acids in meteorites both support a scenario in which prebiotic molecules could be of extraterrestrial origin, before they are delivered to planets by comets, asteroids, and interplanetary dust particles. Nucleobases, the informational subunits of DNA and RNA, have also been detected in meteorites, although they have not yet been observed in the ISM. Thus, these molecules constitute another family of prebiotic compounds that can possibly form via abiotical processes in astrophysical environments. Nucleobases are nitrogen-bearing cyclic aromatic species with various functional groups attached, which are divided into two classes: pyrimidines (uracil, cytosine, and thymine) and purines (adenine and guanine). In this work, we study how UV irradiation affects pyrimidine mixed in interstellar ice analogs (H2O, NH3, CH3OH). In particular, we show that the UV irradiation of H2O:pyrimidine mixtures leads to the production of oxidized compounds including uracil, and show that both uracil and cytosine are formed upon irradiation of H2O:NH3:pyrimidine mixtures. We also study the photostability of pyrimidine and its photoproducts formed during these experiments.

  14. The PUR Experiment on the EXPOSE-R facility: biological dosimetry of solar extraterrestrial UV radiation

    NASA Astrophysics Data System (ADS)

    Bérces, A.; Egyeki, M.; Fekete, A.; Horneck, G.; Kovács, G.; Panitz, C.

    2015-01-01

    The aim of our experiment Phage and Uracil Response was to extend the use of bacteriophage T7 and uracil biological dosimeters for measuring the biologically effective ultraviolet (UV) dose in the harsh extraterrestrial radiation conditions. The biological detectors were exposed in vacuum-tightly cases in the European Space Agency (ESA) astrobiological exposure facility attached to the external platform of Zvezda (EXPOSE-R). EXPOSE-R took off to the International Space Station (ISS) in November 2008 and was installed on the External platform of the Russian module Zvezda of the ISS in March 2009. Our goal was to determine the dose-effect relation for the formation of photoproducts (i.e. damage to phage DNA and uracil, respectively). The extraterrestrial solar UV radiation ranges over the whole spectrum from vacuum-UV (λ<200 nm) to UVA (315 nm<λ<400 nm), which causes photolesions (photoproducts) in the nucleic acids/their components either by photoionization or excitation. However, these wavelengths cause not only photolesions but in a wavelength-dependent efficiency the reversion of some photolesions, too. Our biological detectors measured in situ conditions the resultant of both reactions induced by the extraterrestrial UV radiation. From this aspect the role of the photoreversion in the extension of the biological UV dosimetry are discussed.

  15. First-principles study of interaction of serine with nucleobases of DNA and RNA.

    PubMed

    Abbas, Haider

    2017-03-01

    The nature of interaction between serine-a vital molecule for cancer cell proliferation and nucleic acid bases-adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) is investigated within the framework of Møller-Plesset perturbation theory (MP2) and density functional theory (DFT). To quantify the interaction strength between serine and nucleobases, the corresponding binding energies were computed, showing energetic ordering such that G > C > T > A > U. This shows that the interaction energy of serine with guanine is the highest, while with uracil it is the lowest. The amount of charge transferred is the lowest in case of the serine-guanine complex and highest in case of the serine-uracil complex. The results show the serine-guanine complex to be more stable and to have a salt bridge structure involving the -COOH group. Theoretical analysis based on MP2 and DFT shows that the interaction between the serine and nucleobases is mainly determined by hydrogen bonding.

  16. Designing DNA interstrand lock for locus-specific methylation detection in a nanopore

    NASA Astrophysics Data System (ADS)

    Kang, Insoon; Wang, Yong; Reagan, Corbin; Fu, Yumei; Wang, Michael X.; Gu, Li-Qun

    2013-10-01

    DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg2+) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5'-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer.

  17. Effects of hypoxanthine substitution in peptide nucleic acids targeting KRAS2 oncogenic mRNA molecules: theory and experiment.

    PubMed

    Sanders, Jeffrey M; Wampole, Matthew E; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D; Thakur, Mathew L; Wickstrom, Eric

    2013-10-03

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multimutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick base pairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA:PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA:PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition.

  18. The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease

    PubMed Central

    Georg, Jens; Schomacher, Lars; Chong, James P. J.; Majerník, Alan I.; Raabe, Monika; Urlaub, Henning; Müller, Sabine; Ciirdaeva, Elena; Kramer, Wilfried; Fritz, Hans-Joachim

    2006-01-01

    The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity. PMID:17012282

  19. Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1.

    PubMed

    Córdoba-Cañero, Dolores; Roldán-Arjona, Teresa; Ariza, Rafael R

    2011-11-01

    Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.

  20. Rev1 is essential in generating G to C transversions downstream of the Ung2 pathway but not the Msh2+Ung2 hybrid pathway.

    PubMed

    Krijger, Peter Hugo Lodewijk; Tsaalbi-Shtylik, Anastasia; Wit, Niek; van den Berk, Paul Cornelius Maria; de Wind, Niels; Jacobs, Heinz

    2013-10-01

    Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes are initiated by the enzymatic deamination of cytosine (C) to uracil (U). Uracil-DNA-glycosylase (Ung2) converts uracils into apyrimidinic (AP) sites, which is essential for the generation of transversions (TVs) at G/C basepairs during SHM and for efficient DNA break formation during CSR. Besides Ung2, the mismatch repair protein Msh2 and the translesion synthesis (TLS) DNA polymerase (Pol) Rev1 are implicated in SHM and CSR. To further unravel the role of Rev1, we studied WT, Rev1-deficient, Msh2-deficient, and Rev1, Msh2 double-deficient B cells. Loss of Rev1 only slightly reduced CSR. During SHM G/C to C/G TVs are generated in both Ung2- and Ung+Msh2-dependent fashions. We found that Rev1 is essential for the Msh2-independent generation of these TVs downstream of Ung2-induced AP sites. In the Ung+Msh2 hybrid pathway, Rev1 is not essential and can be substituted by an alternative TLS Pol, especially when Rev1 is lacking.

  1. Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog

    NASA Technical Reports Server (NTRS)

    Yamauchi, Keiko; Hales, Nathan W.; Robinson, Sandra M.; Niehoff, Michael L.; Ramesh, Vani; Pellis, Neal R.; Kulkarni, Anil D.

    2002-01-01

    Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions.

  2. In Situ Biodosimetric Experiment for Space Applications

    NASA Astrophysics Data System (ADS)

    Goldschmidt, Gergely; Kovaliczky, Éva; Szabó, József; Rontó, Györgyi; Bérces, Attila

    2012-06-01

    This paper presents the principles and application of DNA based biological UV dosimeters, as developed by Research Group for Biophysics (RGB). These dosimeters are used for assessing the biological hazard of living systems on the Earth's surface and in different waters (rivers, lakes, seas, etc.). The UV dosimetry system has also been used in the space. In dosimeters a bacterial virus, bacteriophage T7 and polycrystalline uracil thin layers have been used as biological detectors. On the Earth's surface the UV radiation induces dimer formation in phage T7 and in the uracil detector, which was evaluated by loss of viability of the phage particles and by the decrease of the characteristic optical density (OD) of uracil thin layers. Recently the development of human space activities has also increased the need to measure the biological effect of extraterrestrial solar radiation, too. The evaluation of the space samples occurred on ground, thus only the starting and the final state were taken into account. A new improved, automated method is presented below which makes data collection more efficient and also makes the dynamics of the process observable.

  3. Mechanism of mutation by thymine starvation in Escherichia coli: clues from mutagenic specificity.

    PubMed Central

    Kunz, B A; Glickman, B W

    1985-01-01

    To probe the mechanisms of mutagenesis induced by thymine starvation, we examined the mutational specificity of this treatment in strains of Escherichia coli that are wild type (Ung+) or deficient in uracil-DNA-glycosylase (Ung-). An analysis of Ung+ his-4 (ochre) revertants revealed that the majority of induced DNA base substitution events were A:T----G:C transitions. However, characterization of lacI nonsense mutations induced by thymine starvation demonstrated that G:C----A:T transitions and all four possible transversions also occurred. In addition, thymineless episodes led to reversion of the trpE9777 frameshift allele. Although the defect in uracil-DNA-glycosylase did not appear to affect the frequency of total mutations induced in lacI by thymine deprivation, the frequency of nonsense mutations was reduced by 30%, and the spectrum of nonsense mutations was altered. Furthermore, the reversion of trpE9777 was decreased by 90% in the Ung- strain. These findings demonstrate that in E. coli, thymine starvation can induce frameshift mutations and all types of base substitutions. The analysis of mutational specificity indicates that more than a single mechanism is involved in the induction of mutation by thymine depletion. We suggest that deoxyribonucleoside triphosphate pool imbalances, the removal of uracil incorporated into DNA during thymine starvation, and the induction of recA-dependent DNA repair functions all may play a role in thymineless mutagenesis. PMID:3888966

  4. UPRT, a suicide-gene therapy candidate in higher eukaryotes, is required for Drosophila larval growth and normal adult lifespan

    PubMed Central

    Ghosh, Arpan C.; Shimell, MaryJane; Leof, Emma R.; Haley, Macy J.; O’Connor, Michael B.

    2015-01-01

    Uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage pathway enzyme that catalyzes the conversion of uracil to uridine monophosphate (UMP). The enzyme is highly conserved from prokaryotes to humans and yet phylogenetic evidence suggests that UPRT homologues from higher-eukaryotes, including Drosophila, are incapable of binding uracil. Purified human UPRT also do not show any enzymatic activity in vitro, making microbial UPRT an attractive candidate for anti-microbial drug development, suicide-gene therapy, and cell-specific mRNA labeling techniques. Nevertheless, the enzymatic site of UPRT remains conserved across the animal kingdom indicating an in vivo role for the enzyme. We find that the Drosophila UPRT homologue, krishah (kri), codes for an enzyme that is required for larval growth, pre-pupal/pupal viability and long-term adult lifespan. Our findings suggest that UPRT from all higher eukaryotes is likely enzymatically active in vivo and challenges the previous notion that the enzyme is non-essential in higher eukaryotes and cautions against targeting the enzyme for therapeutic purposes. Our findings also suggest that expression of the endogenous UPRT gene will likely cause background incorporation when using microbial UPRT as a cell-specific mRNA labeling reagent in higher eukaryotes. PMID:26271729

  5. Grepafloxacin, a dimethyl derivative of ciprofloxacin, acts preferentially through gyrase in Streptococcus pneumoniae: role of the C-5 group in target specificity.

    PubMed

    Morris, Julia E; Pan, Xiao-Su; Fisher, L Mark

    2002-02-01

    Grepafloxacin, a 5-methyl-7-piperazinyl-3"-methyl analogue of ciprofloxacin, was used to obtain stepwise-selected mutants of Streptococcus pneumoniae 7785. Analysis of the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes in these mutants revealed that gyrA mutations preceded those in parC. Given that ciprofloxacin (5-H,7-piperazinyl) and AM-1121 (5-H,7-piperazinyl-3"-methyl) both act through topoisomerase IV, we conclude that the 5-methyl group of grepafloxacin favors gyrase in S. pneumoniae.

  6. A representative of p,p,p-trihalogenylides: synthesis and structure.

    PubMed

    Marchenko, Anatoliy P; Koidan, Georgyi N; Kostyuk, Aleksandr N; Tolmachev, Andrei A; Kapustin, Evgenij G; Pinchuk, Aleksandr M

    2006-10-27

    The representative of P,P,P-trichloroylides-5-methyl-2-phenyl-4-(trichlorophosphoranylidene)-2,4-dihydro-3H-pyrazol-3-one-was synthesized. Its constitution was confirmed by 1H, 13C, and 31P NMR spectroscopy and by X-ray analysis. Some chemical properties were studied and compared with ones of P,P,P-trimethylylide-5-methyl-2-phenyl-4-(trimethylphosphoranylidene)-2,4-dihydro-3H-pyrazol-3-one. DFT calculations of the model molecules were carried out.

  7. Localized Disruption of Narp in Medial Prefrontal Cortex Blocks Reinforcer Devaluation Performance

    ERIC Educational Resources Information Center

    Johnson, Alexander W.; Han, Sungho; Blouin, Ashley M.; Saini, Jasjit; Worley, Paul F.; During, Matthew J.; Holland, Peter C.; Baraban, Jay M.; Reti, Irving M.

    2010-01-01

    Neuronal activity regulated pentraxin (Narp) is a secreted protein that regulates [alpha]-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPAR) aggregation and synaptogenesis. Mapping of Narp-positive neurons in brain has revealed it is prominently expressed in several limbic system projection pathways. Consistent with this…

  8. Isolation and characterization of secondary metabolites from Asphodelus microcarpus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioassay guided fractionation of the ethanolic extract of Asphodelus microcarpus Salzm.et Vivi (Asphodelaceae) resulted in the isolation of two hitherto unknown compounds , methy-1, 4, 5-trihydroxy-7-methyl-9,10-dioxo-9,10dihydroanthracene-2-carboxylate (1) and (1R) 3, 10-dimethoxy-5-methyl-1H-1,4-e...

  9. Retrieval Is Not Necessary to Trigger Reconsolidation of Object Recognition Memory in the Perirhinal Cortex

    ERIC Educational Resources Information Center

    Santoyo-Zedillo, Marianela; Rodriguez-Ortiz, Carlos J.; Chavez-Marchetta, Gianfranco; Bermudez-Rattoni, Federico; Balderas, Israela

    2014-01-01

    Memory retrieval has been considered as requisite to initiate memory reconsolidation; however, some studies indicate that blocking retrieval does not prevent memory from undergoing reconsolidation. Since N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors in the perirhinal cortex have…

  10. Synthesis and photochemistry of pH-sensitive GFP chromophore analogues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nobel GFP chromophore analogues containing 2-thienyl-, 5-methyl-2-furyl-, 2-pyrryl, and 6-methyl-2-pyridyl-groups were synthesized, and their fluorescence spectra were recorded across pH range of 1 to 7. The GFP chromophores prevent photoisomerizaiton in acidic media and increase their fluorescent a...

  11. Coantagonism of Glutamate Receptors and Nicotinic Acetylcholinergic Receptors Disrupts Fear Conditioning and Latent Inhibition of Fear Conditioning

    ERIC Educational Resources Information Center

    Gould, Thomas J.; Lewis, Michael C.

    2005-01-01

    The present study investigated the hypothesis that both nicotinic acetylcholinergic receptors (nAChRs) and glutamate receptors ([alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) and N-methyl-D-aspartate glutamate receptors (NMDARs)) are involved in fear conditioning, and may modulate similar processes. The effects of the…

  12. TRANSFORMATION AND TRANSPORT OF SEMI-VOLATILE ORGANIC COMPOUNDS FROM SOIL: MEASURING DICARBOXIMIDES IN A CHAMBER

    EPA Science Inventory

    A laboratory chamber was used to determine transport of a suspected anti-androgenic dicarboximide fungicide, vinclozolin (3,5-dichlorophenyl)-5-methyl-5-vinyl-oxzoli-dine-2,4-dione) and three degradation products from a North Carolina Piedmont aquic hapludult soil following a s...

  13. TRANSFORMATION AND TRANSPORT OF VINCLOZOLIN FROM SOIL TO AIR

    EPA Science Inventory

    A laboratory chamber was designed and used to determine the headspace flux of the fungicide vinclozolin (3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-oxzoli-dine-2.4-dione) and its three degradation products from chamber surfaces, 20-30 mesh Ottawa sand, and sterilized and nonsteril...

  14. Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

  15. Synthesis and photophysical studies of oxazole rings containing compounds as electron accepting units.

    PubMed

    Zhang, Caihong; Li, Lifeng; Wu, Hongjuan; Liu, Zhixun; Li, Junfen; Zhang, Guomei; Wen, Guangming; Shuang, Shaomin; Dong, Chuan; Choi, Martin M F

    2013-02-01

    A series of oxazole derivatives, 5-methyl-2-(p-methylphenyl)-4-acetyl oxazole (MMPAO), 5-methyl-2-(p-methoxyphenyl)-4-acetyl oxazole (MOPAO), 5-methyl-2-(p-N,N'-dimethylamino-phenyl)-4-acetyl oxazole (MDMAPAO) and 5-methyl-2-(p-N,N'-diphenylaminophenyl)-4-acetyl oxazole (MDPAPAO) have been synthesized and studied to compare their photophysical properties. The UV-visible absorption spectra of MDMAPAO and MDPAPAO are bathochromatically shifted as compared to that of MMPAO and MOPAO. The fluorescence emission of MDPAPAO is very sensitive to the polarity of solvents. The magnitude of change in the dipole moment was calculated using the Lippert-Mataga equation. MDPAPAO shows the highest change in the dipole moment (Δu=13.3D) than that of the other three oxazole derivatives. The spectral properties including fluorescence quantum yield and lifetime were determined in solvents with different polarities. MDPAPAO displays the highest fluorescence quantum yield and lifetime, following a bi-exponential fluorescent decay fashion. Our result demonstrates that the excited state of MDPAPAO possesses the property of intramolecular charge transfer.

  16. The Nuclear Transcription Factor RAR Associates with Neuronal RNA Granules and Suppresses Translation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    All-trans-retinoic acid stimulates dendritic growth in hippocampal neurons within minutes by activating mitogen-activated protein kinase and mTOR and increasing dendritic translation of calcium calmodulin-dependent protein kinase II alpha and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionat...

  17. An Easy Student Synthesis of a Substituted 1,3-Dioxane by Use of an Ion-Exchange Resin as Catalyst: Clean Illustration of the Prins Reaction.

    ERIC Educational Resources Information Center

    Delmas, Michael; And Others

    1982-01-01

    Background information and experimental procedures are provided for a Prins reaction (condensation of an aldehyde with an alkene). The preparation of 4-(4-hydroxy, 3-methoxy-phenyl) 5-methyl, 1,3-dioxane realized from isoeugenol (natural plant product, commercially available) can be completed in a three-hour laboratory period. (Author/JN)

  18. Probing DNA by 2-OG-dependent dioxygenase

    PubMed Central

    Tsai, Chi-Lin; Tainer, John A.

    2014-01-01

    TET-mediated 5-methyl cytosine (5mC) oxidation acts in epigenetic regulation, stem cell development, and cancer. Hu et al. now determine the crystal structure of the TET2 catalytic domain bound to DNA, shedding light on 5mC-DNA substrate recognition and the catalytic mechanism of 5mC oxidation. PMID:24360270

  19. Synthesis and photophysical studies of oxazole rings containing compounds as electron accepting units

    NASA Astrophysics Data System (ADS)

    Zhang, Caihong; Li, Lifeng; Wu, Hongjuan; Liu, Zhixun; Li, Junfen; Zhang, Guomei; Wen, Guangming; Shuang, Shaomin; Dong, Chuan; Choi, Martin M. F.

    2013-02-01

    A series of oxazole derivatives, 5-methyl-2-(p-methylphenyl)-4-acetyl oxazole (MMPAO), 5-methyl-2-(p-methoxyphenyl)-4-acetyl oxazole (MOPAO), 5-methyl-2-(p-N,N'-dimethylamino-phenyl)-4-acetyl oxazole (MDMAPAO) and 5-methyl-2-(p-N,N'-diphenylaminophenyl)-4-acetyl oxazole (MDPAPAO) have been synthesized and studied to compare their photophysical properties. The UV-visible absorption spectra of MDMAPAO and MDPAPAO are bathochromatically shifted as compared to that of MMPAO and MOPAO. The fluorescence emission of MDPAPAO is very sensitive to the polarity of solvents. The magnitude of change in the dipole moment was calculated using the Lippert-Mataga equation. MDPAPAO shows the highest change in the dipole moment (Δu = 13.3D) than that of the other three oxazole derivatives. The spectral properties including fluorescence quantum yield and lifetime were determined in solvents with different polarities. MDPAPAO displays the highest fluorescence quantum yield and lifetime, following a bi-exponential fluorescent decay fashion. Our result demonstrates that the excited state of MDPAPAO possesses the property of intramolecular charge transfer.

  20. Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine forming Met, which is then used for the syn...

  1. 76 FR 17374 - Receipt of Several Pesticide Petitions Filed for Residues of Pesticide Chemicals in or on Various...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-29

    ...]-5-methyl-3-pyridinecarboxylic acid, in or on soybean at 0.5 parts per million (ppm). The proposed... (CL 189,215) in soybean seed and processed fractions is a liquid chromatography-tandem mass..., except soybean, subgroup 6C; soybean, forage; soybean, hay; and soybean, seed to support the use...

  2. [The molecular structure of iso-aloesin isolated from the leaves of Aloe vera L. var. chinensis (Haw.) Berge].

    PubMed

    Yuan, A X

    1993-10-01

    A new constituent iso-aloesin was isolated from the leaves of Aloe vera var. chinensis found in the Province of Guangxi. The molecular formula of iso-aloesin is C19H22O9, which is 2-acetonyl-6-C-beta-D-glucopyranosyl-7-hydroxy-5-methyl-chromone.

  3. Reactivation-Dependent Amnesia for Appetitive Memories Is Determined by the Contingency of Stimulus Presentation

    ERIC Educational Resources Information Center

    Lee, Jonathan L. C.; Everitt, Barry J.

    2008-01-01

    Previously acquired aversive and appetitive memories are not stable and permanent. The reactivation of such memories by re-exposure to training stimuli renders them vulnerable to disruption by amnestic agents such as the noncompetitive N-methyl-"D"-aspartate receptor antagonist (+)-5-methyl-10,11-dihydro-"SH"-dibenzo{a,d}cyclohepten-5,10imine…

  4. 76 FR 47579 - Notice of Receipt of Requests To Voluntarily Cancel Certain Pesticide Registrations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-05

    ... Glyphosate-isopropylammonium. Concentrate. 087650-00001 Fipronil Technical Fipronil CA920028 Devrinol 50-DF... Bromide 99.5%....... Methyl bromide. ID070004 MBC Concentrate Soil Methyl bromide. Fumigant. Table 3...., except for export consistent with FIFRA section 17 or for proper disposal. Persons other than...

  5. The loss of 5-methyltetrahydrofolate in human serum under suboptimal preanalytical conditions can only partially be recovered by an oxidation product1,2,3

    PubMed Central

    Fazili, Zia; Sternberg, Maya R.; Paladugula, Neelima; Whitehead, Ralph D.; Chen, Huiping; Pfeiffer, Christine M.

    2017-01-01

    Background Maintaining folate stability during sample handling is important, yet challenging. Objective We investigated the effects of suboptimal preanalytical conditions on serum folate stability. Design Using an HPLC-tandem MS method we measured folates [5-methyltetrahydrofolate (5-methylTHF), folic acid, and MeFox (5-methylTHF oxidation product), minor forms at or below limit of detection] in human serum exposed to suboptimal conditions. Results Whole blood samples (n = 21) stored at 32°C for ≤3 d (experiment 1 – delayed processing) showed significant decreases in serum total folate [(tFOL; sum of folate forms) 11–32%, 5.5–15.9 nmol/L] and 5-methylTHF (36–62%, 14.5–25.1 nmol/L) and a significant increase in MeFox (346–415%, 7.17–8.63 nmol/L). Serum samples (n = 21) stored at 11°C for 7–14 d (experiment 2 – delayed freezing) also showed significant decreases in tFOL (4.6–10.4%, 2.3–5.1 nmol/L) and 5-methylTHF (8.4–29%, 3.4–11.6 nmol/L) and significant increases in MeFox (88–320%, 1.82–6.62 nmol/L). The molar loss in 5-methylTHF exceeded the gain in MeFox in these 2 experiments. When we exposed 3 serum pools (tFOL 16.7–58.3 nmol/L) for 24 h to 37°C elevated temperature (experiment 3), the significant decrease in 5-methylTHF (33% on average) was compensated by an equimolar gain in MeFox. Repeated freeze/thawing (≤3 cycles) of serum [closed (experiment 4) and open (experiment 5) vials] showed generally stable folates with small (<1 nmol/L) changes. Long-term (≤12 mo) exposure of 3 serum pools (tFOL 17.5–63.7 nmol/L) to suboptimal (−20°C) freezing temperature (experiment 6) showed significant decreases in tFOL (5% on average) already after 3 mo. The molar loss in 5-methylTHF exceeded the gain in MeFox. Folic acid generally showed good stability. Conclusions To avoid folate losses, unprocessed whole blood should be protected from elevated temperature and serum should not be refrigerated for >2 d or long-term stored at

  6. Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells: tandem mutations and enhancement of G-->T transversions.

    PubMed

    Kalam, M Abul; Basu, Ashis K

    2005-08-01

    Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T > C > A > G, the same at the GUC site was T > A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% G-->T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated approximately 60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo)-->T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with deltadeltaG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than

  7. Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

    PubMed Central

    Swinnen, Steve; Goovaerts, Annelies; Schaerlaekens, Kristien; Dumortier, Françoise; Verdyck, Pieter; Souvereyns, Kris; Van Zeebroeck, Griet; Foulquié-Moreno, María R.

    2015-01-01

    Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress. PMID:26116212

  8. Crystal structures of the ribonuclease MC1 from bitter gourd seeds, complexed with 2'-UMP or 3'-UMP, reveal structural basis for uridine specificity.

    PubMed

    Suzuki, A; Yao, M; Tanaka, I; Numata, T; Kikukawa, S; Yamasaki, N; Kimura, M

    2000-08-28

    Ribonuclease MC1 (RNase MC1) isolated from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and is characterized by a preferential cleavage at the 5'-side of uridine. This uridine specificity distinguishes RNase MC1 from other enzymes belonging to the RNase T2 family. The three-dimensional structures of RNase MC1, in a complex with either 2'-UMP or 3'-UMP, were determined at 1.48 and 1.77 A resolutions, respectively. The side chains of Gln9 and Asn71 interact with O4 and N3, respectively, of the uracil base by hydrogen bondings. In addition, the uracil base is sandwiched by the hydrophobic side chains of Leu73 and Phe80. Compared with these amino acid residues and corresponding residues in RNases in the RNase T2 family, Gln9 and Phe80 are highly conserved in the RNases in T2 family, while Asn71 and Leu73 in RNase MC1 are variant in sequences. It is thus likely that interactions of the side chains of Asn71 and Leu73 with the uracil base are responsible for the absolute uridine specificity of RNase MC1. Site-directed mutagenesis experiments showed that replacement of Asn by Thr decreased both the catalytic efficiency and the binding affinity by 2.3- and 7.0-fold, respectively, and substitution of Leu73 for Ala predominantly decreased the binding affinity by 14. 5-fold, compared with findings in case of wild-type RNase MC1. It is thus demonstrated that Asn71 and Leu73 play an essential role in uridine preference for RNase MC1.

  9. Pathophysiology of B-cell intrinsic immunoglobulin class switch recombination deficiencies.

    PubMed

    Durandy, Anne; Taubenheim, Nadine; Peron, Sophie; Fischer, Alain

    2007-01-01

    B-cell intrinsic immunoglobulin class switch recombination (Ig-CSR) deficiencies, previously termed hyper-IgM syndromes, are genetically determined conditions characterized by normal or elevated serum IgM levels and an absence or very low levels of IgG, IgA, and IgE. As a function of the molecular mechanism, the defective CSR is variably associated to a defect in the generation of somatic hypermutations (SHMs) in the Ig variable region. The study of Ig-CSR deficiencies contributed to a better delineation of the mechanisms underlying CSR and SHM, the major events of antigen-triggered antibody maturation. Four Ig-CSR deficiency phenotypes have been so far reported: the description of the activation-induced cytidine deaminase (AID) deficiency (Ig-CSR deficiency 1), caused by recessive mutations of AICDA gene, characterized by a defect in CSR and SHM, clearly established the role of AID in the induction of the Ig gene rearrangements underlying CSR and SHM. A CSR-specific function of AID has, however, been detected by the observation of a selective CSR defect caused by mutations affecting the C-terminus of AID. Ig-CSR deficiency 2 is the consequence of uracil-N-glycosylase (UNG) deficiency. Because UNG, a molecule of the base excision repair machinery, removes uracils from DNA and AID deaminates cytosines into uracils, that observation indicates that the AID-UNG pathway directly targets DNA of switch regions from the Ig heavy-chain locus to induce the CSR process. Ig-CSR deficiencies 3 and 4 are characterized by a selective CSR defect resulting from blocks at distinct steps of CSR. A further understanding of the CSR machinery is expected from their molecular definition.

  10. Environmental and Nutritional Factors That Affect Growth and Metabolism of the Pneumococcal Serotype 2 Strain D39 and Its Nonencapsulated Derivative Strain R6

    PubMed Central

    Carvalho, Sandra M.; Kuipers, Oscar P.; Neves, Ana Rute

    2013-01-01

    Links between carbohydrate metabolism and virulence in Streptococcus pneumoniae have been recurrently established. To investigate these links further we developed a chemically defined medium (CDM) and standardized growth conditions that allowed for high growth yields of the related pneumococcal strains D39 and R6. The utilization of the defined medium enabled the evaluation of different environmental and nutritional factors on growth and fermentation patterns under controlled conditions of pH, temperature and gas atmosphere. The same growth conditions impacted differently on the nonencapsulated R6, and its encapsulated progenitor D39. A semi-aerobic atmosphere and a raised concentration of uracil, a fundamental component of the D39 capsule, improved considerably D39 growth rate and biomass. In contrast, in strain R6, the growth rate was enhanced by strictly anaerobic conditions and uracil had no effect on biomass. In the presence of oxygen, the difference in the growth rates was mainly attributed to a lower activity of pyruvate oxidase in strain D39. Our data indicate an intricate connection between capsule production in strain D39 and uracil availability. In this study, we have also successfully applied the in vivo NMR technique to study sugar metabolism in S. pneumoniae R6. Glucose consumption, end-products formation and evolution of intracellular metabolite pools were monitored online by 13C-NMR. Additionally, the pools of NTP and inorganic phosphate were followed by 31P-NMR after a pulse of glucose. These results represent the first metabolic profiling data obtained non-invasively for S. pneumoniae, and pave the way to a better understanding of regulation of central metabolism. PMID:23505518

  11. Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation.

    PubMed

    Stavnezer, Janet; Linehan, Erin K; Thompson, Mikayla R; Habboub, Ghaith; Ucher, Anna J; Kadungure, Tatenda; Tsuchimoto, Daisuke; Nakabeppu, Yusaku; Schrader, Carol E

    2014-06-24

    Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase β. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.

  12. Environmental and nutritional factors that affect growth and metabolism of the pneumococcal serotype 2 strain D39 and its nonencapsulated derivative strain R6.

    PubMed

    Carvalho, Sandra M; Kuipers, Oscar P; Neves, Ana Rute

    2013-01-01

    Links between carbohydrate metabolism and virulence in Streptococcus pneumoniae have been recurrently established. To investigate these links further we developed a chemically defined medium (CDM) and standardized growth conditions that allowed for high growth yields of the related pneumococcal strains D39 and R6. The utilization of the defined medium enabled the evaluation of different environmental and nutritional factors on growth and fermentation patterns under controlled conditions of pH, temperature and gas atmosphere. The same growth conditions impacted differently on the nonencapsulated R6, and its encapsulated progenitor D39. A semi-aerobic atmosphere and a raised concentration of uracil, a fundamental component of the D39 capsule, improved considerably D39 growth rate and biomass. In contrast, in strain R6, the growth rate was enhanced by strictly anaerobic conditions and uracil had no effect on biomass. In the presence of oxygen, the difference in the growth rates was mainly attributed to a lower activity of pyruvate oxidase in strain D39. Our data indicate an intricate connection between capsule production in strain D39 and uracil availability. In this study, we have also successfully applied the in vivo NMR technique to study sugar metabolism in S. pneumoniae R6. Glucose consumption, end-products formation and evolution of intracellular metabolite pools were monitored online by (13)C-NMR. Additionally, the pools of NTP and inorganic phosphate were followed by (31)P-NMR after a pulse of glucose. These results represent the first metabolic profiling data obtained non-invasively for S. pneumoniae, and pave the way to a better understanding of regulation of central metabolism.

  13. Photochemistry of Pyrimidine in Astrophysical Ices: Formation of Nucleobases and Other Prebiotic Species

    NASA Technical Reports Server (NTRS)

    Nuevo, Michel; Sandford, Scott A.; Materese, Christopher K.; Milam, Stefanie N.

    2012-01-01

    Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA. They are divided into two molecular groups: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites, and their extraterrestrial origin confirmed by isotopic measurements. Although no N-heterocycles have ever been observed in the ISM, the positions of the 6.2- m interstellar emission features suggest a population of such molecules is likely to be present. However, laboratory experiments have shown that the ultraviolet (UV) irradiation of pyrimidine in ices of astrophysical relevance such as H2O, NH3, CH3OH, CH4, CO, or combinations of these at low temperature (less than or equal to 20 K) leads to the formation of several pyrimidine derivatives including the nucleobases uracil and cytosine, as well as precursors such as 4(3H)-pyrimidone and 4-aminopyrimidine. Quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways.10 In those residues, other species of prebiotic interest such as urea as well as the amino acids glycine and alanine could also be identified. However, only very small amounts of pyrimidine derivatives containing CH3 groups could be detected, suggesting that the addition of methyl groups to pyrimidine is not an efficient process. For this reason, the nucleobase thymine was not observed in any of the samples. In this work, we study the formation of nucleobases and other photo-products of prebiotic interest from the UV irradiation of pyrimidine in ices containing H2O, NH3, CH3OH, and CO, mixed in astrophysical proportions.

  14. Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: implications for the nature of the first genetic material

    NASA Technical Reports Server (NTRS)

    Stribling, R.; Miller, S. L.

    1991-01-01

    Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system. Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5'-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template. Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3 x 10(-6) M), as well as the difficulty of prebiotic activation of phosphates.

  15. Effects of seven chemicals on DNA damage in the rat urinary bladder: a comet assay study.

    PubMed

    Wada, Kunio; Yoshida, Toshinori; Takahashi, Naofumi; Matsumoto, Kyomu

    2014-07-15

    The in vivo comet assay has been used for the evaluation of DNA damage and repair in various tissues of rodents. However, it can give false-positive results due to non-specific DNA damage associated with cell death. In this study, we examined whether the in vivo comet assay can distinguish between genotoxic and non-genotoxic DNA damage in urinary bladder cells, by using the following seven chemicals related to urinary bladder carcinogenesis in rodents: N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), glycidol, 2,2-bis(bromomethyl)-1,3-propanediol (BMP), 2-nitroanisole (2-NA), benzyl isothiocyanate (BITC), uracil, and melamine. BBN, glycidol, BMP, and 2-NA are known to be Ames test-positive and they are expected to produce DNA damage in the absence of cytotoxicity. BITC, uracil, and melamine are Ames test-negative with metabolic activation but have the potential to induce non-specific DNA damage due to cytotoxicity. The test chemicals were administered orally to male Sprague-Dawley rats (five per group) for each of two consecutive days. Urinary bladders were sampled 3h after the second administration and urothelial cells were analyzed by the comet assay and subjected to histopathological examination to evaluate cytotoxicity. In the urinary bladders of rats treated with BBN, glycidol, and BMP, DNA damage was detected. In contrast, 2-NA induced neither DNA damage nor cytotoxicity. The non-genotoxic chemicals (BITC, uracil, and melamine) did not induce DNA damage in the urinary bladders under conditions where some histopathological changes were observed. The results indicate that the comet assay could distinguish between genotoxic and non-genotoxic chemicals and that no false-positive responses were obtained.

  16. Effect of adenosine on the supramolecular architecture and activity of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Singh, Udai P.; Kashyap, Sujata; Singh, Hari Ji; Mishra, Bhupesh Kumar; Roy, Partha; Chakraborty, Ajanta

    2012-04-01

    The reactions of adenosine (Ad) with 5-halouracils (5XU where X = F for 1, Cl for 2, Br for 3 and I for 4) resulted in the formation of co-crystals 1-4 in monoclinic with P21 space group. Despite of great variation in the halo substituent at the 5th position of the uracil, each structure contains the same number and same type of non-covalent interactions i.e., primary N-H⋯N, N-H⋯O, O-H⋯N, O-H⋯O hydrogen bonds and secondary C-H⋯O and X⋯O interactions within these motifs as well as with neighboring molecules. As compared to Ad the size of cavity increases in co-crystal 1 to accommodate the 5FU as a guest. With the variation of halogen from fluoro to iodo on the uracil, the orientation of the molecules remains the same with a slight difference in the dihedral angle in all the co-crystals 1-4. This study demonstrates that hydrogen-bonded interactions between adenosine and halouracils provide a supramolecular assembly to these co-crystals. Computational studies illustrate that the size of the halo substituents on uracil has no effect on the hydrogen bond interaction energy. It further reveals that the orientation of molecules remain same in both solid phase as well as in the gaseous phase. The antitumor and DNA cleavage activity studies show that the antitumor activity of 5-fluorouracil against MCF-7 breast cancer decreases in the presence of adenosine.

  17. Exploring the Fate of Nitrogen Heterocycles in Complex Prebiotic Mixtures

    NASA Technical Reports Server (NTRS)

    Smith, Karen E.; Callahan, Michael P.; Cleaves, Henderson J.; Dworkin, Jason P.; House, Christopher H.

    2011-01-01

    A long standing question in the field of prebiotic chemistry is the origin of the genetic macromolecules DNA and RNA. DNA and RNA have very complex structures with repeating subunits of nucleotides, which are composed of nucleobases (nitrogen heterocycles) connected to sugar-phosphate. Due to the instability of some nucleobases (e.g. cytosine), difficulty of synthesis and instability of D-ribose, and the likely scarcity of polyphosphates necessary for the modern nucleotides, alternative nucleotides have been proposed for constructing the first genetic material. Thus, we have begun to investigate the chemistry of nitrogen heterocycles in plausible, complex prebiotic mixtures in an effort to identify robust reactions and potential alternative nucleotides. We have taken a complex prebiotic mixture produced by a spark discharge acting on a gas mixture of N2, CO2, CH4, and H2, and reacted it with four nitrogen heterocycles: uracil, 5-hydroxymethyluracil, guanine, and isoxanthopterin (2-amino-4,7-dihydroxypteridine). The products of the reaction between the spark mixture and each nitrogen heterocycle were characterized by liquid chromatography coupled to UV spectroscopy and Orbitrap mass spectrometry. We found that the reaction between the spark mixtUl'e and isoxanthopterin formed one major product, which was a cyanide adduct. 5-hydroxymethyluracil also reacted with the spark mixture to form a cyanide adduct, uracil-5-acetonitrile, which has been synthesized previously by reacting HCN with S-hydroxymethyluracil. Unlike isoxanthopterin, the chromatogram of the 5-hydroxymethyluracil reaction was much more complex with multiple products including spark-modified dimers. Additionally, we observed that HMU readily self-polymerizes in solution to a variety of oligomers consistent with those suggested by Cleaves. Guanine and uracil, the biological nucleobases, did not react with the spark mixture, even at high temperature (100 C). This suggests that there are alternative

  18. Complete recovery from acute encephalopathy of late-onset ornithine transcarbamylase deficiency in a 3-year-old boy.

    PubMed

    Mak, C M; Siu, T-S; Lam, C-W; Chan, G C-F; Poon, G W-K; Wong, K-Y; Low, L C-K; Tang, N L; Li, S K; Lau, K-Y; Kwong, N-S; Tam, S

    2007-11-01

    Ornithine transcarbamylase deficiency is the commonest urea cycle disorder which is transmitted in X-linked inheritance. It is mainly characterized in males by acute encephalopathy and hyperammonaemia with fatal outcomes in both classical neonatal and late-onset types. We report a 3-year-old healthy Hong Kong Chinese boy who presented with acute encephalopathy and coma after three days of gastroenteritis. He had no focal neurological deficit and brain CT imaging was normal. His plasma ammonia (54 micromol/L) and glutamine (747 micromol/L) concentrations were normal. The only biochemical abnormalities detected were marked orotic aciduria (700 micromol/mmol creatinine) and elevated urinary uracil. He regained consciousness spontaneously after three days under intensive care with parenteral fluid therapy. He recovered completely without any neurological deficits. Five months after discharge, urinary uracil concentration remained elevated despite normalized orotic acid concentration. Finally, ornithine transcarbamylase deficiency was diagnosed by DNA analysis. A missense mutation of arginine-to-glutamine substitution on amino acid 277 (p.R277Q) was revealed to be a late-onset mutant. Our case strengthens the argument that in any child with coma or acute encephalopathy of undetermined cause, genetic analysis of the OTC gene and the measurement of urinary uracil concentration remain the most reliable indicators of late-onset OTCD during acute and even quiescent phases. Existing neonatal screening programmes for inheritable metabolic disorders fail to detect late-onset variants. Therefore, a high clinical suspicion is a key to correct and timely diagnosis, especially in those patients with atypical presentations.

  19. Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress.

    PubMed

    Swinnen, Steve; Goovaerts, Annelies; Schaerlaekens, Kristien; Dumortier, Françoise; Verdyck, Pieter; Souvereyns, Kris; Van Zeebroeck, Griet; Foulquié-Moreno, María R; Thevelein, Johan M

    2015-09-01

    Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.

  20. Studies in pharmacokinetics and tolerance of substances temporarily retained in the liver by microsphere embolization.

    PubMed Central

    Lindell, B; Aronsen, K F; Nosslin, B; Rothman, U

    1978-01-01

    Earlier investigation has shown that a temporary arrest of arterial blood flow can be achieved by embolization with degradable microspheres. This study was made to investigate the change in pharmacokinetics, and drug tolerance which takes place when a substance is retained in the liver by a microsphere embolization. 14C-labelled inulin and 5-fluorouracil were studied. The administration of these substances with microspheres led to a delay in their systemic distribution. Furthermore there was an increased tolerance to 5-fluoro-uracil, probably due to a prolonged first pass effect when the substance was temporarily retained in the liver by a microsphere embolization. PMID:619805