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Sample records for 5-methyl uracil

  1. [Uracil-DNA glycosylases].

    PubMed

    Pytel, Dariusz; Słupianek, Artur; Ksiazek, Dominika; Skórski, Tomasz; Błasiak, Janusz

    2008-01-01

    Uracil is one of four nitrogen bases, most frequently found in normal RNA. Uracyl can be found also in DNA as a result of enzymatic or non-enzymatic deamination of cytosine as well as misincorporation of dUMP instead of dTMP during DNA replication. Uracil from DNA can be removed by DNA repair enzymes with apirymidine site as an intermediate. However, if uracil is not removed from DNA a pair C:G in parental DNA can be changed into a T:A pair in the daughter DNA molecule. Therefore, uracil in DNA may lead to a mutation. Uracil in DNA, similarly to thymine, forms energetically most favorable hydrogen bonds with adenine, therefore uracil does not change the coding properties of DNA. Uracil in DNA is recognized by uracil DNA glycosylase (UDGs), which initiates DNA base excision repair, leading to removing of uracil from DNA and replacing it by thymine or cytosine, when arose as a result of cytosine deamination. Eukaryotes have at least four nuclear UDGs: UNG2, SMUG1, TDG i MBD4, while UNG1 operates in the mitochondrium. UNG2 is involved in DNA repair associated with DNA replication and interacts with PCNA and RPA proteins. Uracil can also be an intermediate product in the process of antigen-dependent antibody diversification in B lymphocytes. Enzymatic deamination of viral DNA by host cells can be a defense mechanism against viral infection, including HIV-1. UNG2, MBD4 and TDG glycosylases may cooperate with mismatch repair proteins and TDG can be involved in nucleotide excision repair system.

  2. 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.

  3. 2-Amino-5-methyl-pyridinium nicotinate.

    PubMed

    Hemamalini, Madhukar; Fun, Hoong-Kun

    2010-02-20

    In the title compound, C(6)H(9)N(2) (+)·C(6)H(4)NO(2) (-), the 2-amino-5-methyl-pyridinium cation is essentially planar, with a maximum deviation of 0.023 (2) Å. In the crystal, the cations and anions are linked via strong N-H⋯O hydrogen bonds, forming a two dimensional network parallel to (100). In addition, π⋯π inter-actions involving the pyridinium and pyridine rings, with centroid-centroid distances of 3.6383 (8) Å, are observed.

  4. A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

    PubMed Central

    Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh

    2015-01-01

    Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. PMID:26304551

  5. Quantification of 5-methyl-2'-deoxycytidine in the DNA.

    PubMed

    Giel-Pietraszuk, Małgorzata; Insińska-Rak, Małgorzata; Golczak, Anna; Sikorski, Marek; Barciszewska, Mirosława; Barciszewski, Jan

    2015-01-01

    Methylation at position 5 of cytosine (Cyt) at the CpG sequences leading to formation of 5-methyl-cytosine (m(5)Cyt) is an important element of epigenetic regulation of gene expression. Modification of the normal methylation pattern, unique to each organism, leads to the development of pathological processes and diseases, including cancer. Therefore, quantification of the DNA methylation and analysis of changes in the methylation pattern is very important from a practical point of view and can be used for diagnostic purposes, as well as monitoring of the treatment progress. In this paper we present a new method for quantification of 5-methyl-2'deoxycytidine (m(5)C) in the DNA. The technique is based on conversion of m(5)C into fluorescent 3,N(4)-etheno-5-methyl-2'deoxycytidine (εm(5)C) and its identification by reversed-phase high-performance liquid chromatography (RP-HPLC). The assay was used to evaluate m(5)C concentration in DNA of calf thymus and peripheral blood of cows bred under different conditions. This approach can be applied for measuring of 5-methylcytosine in cellular DNA from different cells and tissues. PMID:26098716

  6. Structures and fragmentation of [Cu(uracil-H)(uracil)]+ in the gas phase.

    PubMed

    Ali, Osama Y; Fridgen, Travis D

    2012-02-01

    Complexes of copper (II) ions and uracil were studied using tandem mass spectrometry (Fourier transform ion cyclotron resonance, FTICR, mass spectrometry) including extensive isotopic labeling as well as theoretical calculations. Positive ion electrospray mass spectra of aqueous solutions of CuCl(2) and uracil show that the [Cu(Ura-H)(Ura)](+) ion is the most abundant ion even at low concentrations of uracil. Sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments show that the lowest energy decomposition pathway for [Cu(Ura-H)(Ura)](+) , surprisingly, is not the loss of uracil, but the loss of HNCO followed by HCN as the most abundant secondary fragmentation product. MS(n) studies identified primary, secondary and tertiary fragmentation products. Extensive isotopic labeling studies, as well as computational studies allowed for a detailed fragmentation scheme for the [Cu(Ura-H)(Ura)](+) ion, beginning with the lowest energy structure. PMID:22183913

  7. 2-Amino-5-methyl­pyridinium nicotinate

    PubMed Central

    Hemamalini, Madhukar; Fun, Hoong-Kun

    2010-01-01

    In the title compound, C6H9N2 +·C6H4NO2 −, the 2-amino-5-methyl­pyridinium cation is essentially planar, with a maximum deviation of 0.023 (2) Å. In the crystal, the cations and anions are linked via strong N—H⋯O hydrogen bonds, forming a two dimensional network parallel to (100). In addition, π⋯π inter­actions involving the pyridinium and pyridine rings, with centroid–centroid distances of 3.6383 (8) Å, are observed. PMID:21580410

  8. Uracil within DNA: an actor of antiviral immunity

    PubMed Central

    Sire, Joséphine; Quérat, Gilles; Esnault, Cécile; Priet, Stéphane

    2008-01-01

    Uracil is a natural base of RNA but may appear in DNA through two different pathways including cytosine deamination or misincorporation of deoxyuridine 5'-triphosphate nucleotide (dUTP) during DNA replication and constitutes one of the most frequent DNA lesions. In cellular organisms, such lesions are faithfully cleared out through several universal DNA repair mechanisms, thus preventing genome injury. However, several recent studies have brought some pieces of evidence that introduction of uracil bases in viral genomic DNA intermediates during genome replication might be a way of innate immune defence against some viruses. As part of countermeasures, numerous viruses have developed powerful strategies to prevent emergence of uracilated viral genomes and/or to eliminate uracils already incorporated into DNA. This review will present the current knowledge about the cellular and viral countermeasures against uracils in DNA and the implications of these uracils as weapons against viruses. PMID:18533995

  9. 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.

  10. 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.

  11. Real time monitoring uracil excision using uracil-containing molecular beacons.

    PubMed

    Li, Chen; Long, Ying; Liu, Bin; Xiang, Dan; Zhu, Haizhen

    2014-03-28

    As a highly conserved damage repair protein, UDG excises uracil bases through its glycosylase activity. We report here an alternative fluorescence method for UDG assay with high accuracy and sensitivity by applying uracil-modified molecular beacons as substrates. The detection limit of UDG is 0.005 U mL(-1). The KM and kcat are 0.89±0.1 μM and 210±10 min(-1), respectively. The method is applied to screening inhibitors and the results indicate that both of the 5-FU and cisplatin can inhibit UDG activity with the IC50 values of 6.1±0.52 mM and 3.2±0.24 mM, respectively. Furthermore, the combination of uracil-modified molecular beacons and nuclease inhibitor makes the new method possible to specifically detect UDG activity in cell-free extracts and serum. Taken together, the simple, rapid and sensitive method has potential relevance for a variety of applications, such as molecular diagnosis and screening of UDG inhibitors.

  12. [Express evaluation of antioxidant activity of uracil derivatives].

    PubMed

    Gimadieva, A R; Khazimullina, Yu Z; Belaya, E A; Zimin, Yu S; Abdrakhmanov, I B; Mustafin, A G

    2015-01-01

    Using photometric methods the antioxidant activity of 19 uracil derivatives has been analyzed. The test using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals can be applied for the rapid assessment of antioxidant activity of uracils. Among uracil derivatives studied the compounds possesing a proton-donor group in C-5 position--free or alkylated amino group, as well as hydroxyl group were the most active: 5-aminouracil (IC50 3 mg/ml), 5-amino-6-methyluracil (IC50 of 5 mg/ml), 5-hydroxy-6-methyluracil (IC50 of 15 mg/ml), 5-hydroxy-1,3,6-trimethyluracil (IC50 of 15 mg/ml), 5-ethylamino-6-methyluracil (IC50 of 20 mg/ml), 5-methylamino-6-methyluracil (IC50 of 20 mg/ml), 5-allylaminouracil (IC50 of 20 mg/ml), 5-amino-1,3,6- trimethyluracil (IC50 of 25 mg/ml). These uracil derivatives were more active than the reference compounds ionol (IC50 of 30 mg/ml) and a-naphthylamine (IC50 of 45 mg/ml), but less active than ascorbic acid (IC50 0.8 mg/ml). There was a correlation between the results of DPPH test (IC50) and coupling constants of uracil derivatives with peroxide radicals of 1,4-dioxane (fk7). Uracil with proton-donor group at C-5 also showed high ferrum-reducing activity as determined by FRAP.

  13. 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.

  14. 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

  15. DFT studies of CNT-functionalized uracil-acetate hybrids

    NASA Astrophysics Data System (ADS)

    Mirzaei, Mahmoud; Gulseren, Oguz

    2015-09-01

    Calculations based on density functional theory (DFT) have been performed to investigate the stabilities and properties of hybrid structures consisting of a molecular carbon nanotube (CNT) and uracil acetate (UA) counterparts. The investigated models have been relaxed to minimum energy structures and then various physical properties and nuclear magnetic resonance (NMR) properties have been evaluated. The results indicated the effects of functionalized CNT on the properties of hybrids through comparing the results of hybrids and individual structures. The oxygen atoms of uracil counterparts have been seen as the detection points of properties for the CNT-UA hybrids.

  16. 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 ...

  17. Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

    PubMed

    Villela, Anne Drumond; Ducati, Rodrigo Gay; Rosado, Leonardo Astolfi; Bloch, Carlos Junior; Prates, Maura Vianna; Gonçalves, Danieli Cristina; Ramos, Carlos Henrique Inacio; Basso, Luiz Augusto; Santos, Diogenes Santiago

    2013-01-01

    Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.

  18. Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

    PubMed Central

    Villela, Anne Drumond; Ducati, Rodrigo Gay; Rosado, Leonardo Astolfi; Bloch, Carlos Junior; Prates, Maura Vianna; Gonçalves, Danieli Cristina; Ramos, Carlos Henrique Inacio; Basso, Luiz Augusto; Santos, Diogenes Santiago

    2013-01-01

    Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis. PMID:23424660

  19. 5'-Methyl-cytosine in the macronuclear DNA of Blepharisma japonicum.

    PubMed

    Salvini, M; Durante, M; Citti, L; Nobili, R

    1984-12-15

    Brief report on the presence of 5'-methyl-cytosine as a minor base (0.56%) in the macronuclear DNA of the ciliate protozoan Blepharisma japonicum. The evidence comes from electrophoresis of macronuclear DNA digested by appropriate restriction endonucleases and high-performance liquid chromatography.

  20. 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.

  1. 2-Amino-5-methyl­pyridinium picolinate 0.63-hydrate

    PubMed Central

    Hemamalini, Madhukar; Fun, Hoong-Kun

    2010-01-01

    The asymmetric unit of the title compound, C6H9N2 +·C6H4NO2 −·0.63H2O, contains two crystallographically independent 2-amino-5-methyl­pyridinium cations, a pair of picolinate anions and two water mol­ecules, one with an occupancy of 0.25. Both the 2-amino-5-methyl­pyridine mol­ecules are protonated at the pyridine N atoms. In the crystal structure, the cations, anions and water mol­ecules are linked via N—H⋯O, N—H⋯N and O—H⋯O hydrogen bonds, as well as by C—H⋯O contacts, forming a chain along the b axis. In addition, weak π–π inter­actions are observed between pyridinium rings, with centroid–centroid distances of 3.5306 (13) Å. PMID:21579497

  2. Primary fragmentation pathways of gas phase [M(uracil-H)(uracil)]+ complexes (M=Zn, Cu, Ni, Co, Fe, Mn, Cd, Pd , Mg, Ca, Sr, Ba, and Pb): loss of uracil versus HNCO.

    PubMed

    Ali, Osama Y; Randell, Nicholas M; Fridgen, Travis D

    2012-04-23

    Complexes formed between metal dications, the conjugate base of uracil, and uracil are investigated by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Positive-ion electrospray spectra show that [M(Ura-H)(Ura)](+) (M=Zn, Cu, Ni, Co, Fe, Mn, Cd, Pd, Mg, Ca, Sr, Ba, or Pb) is the most abundant ion even at low concentrations of uracil. SORI-CID experiments show that the main primary decomposition pathway for all [M(Ura-H)(Ura)](+) , except where M=Ca, Sr, Ba, or Pb, is the loss of HNCO. Under the same SORI-CID conditions, when M is Ca, Sr, Ba, or Pb, [M(Ura-H)(Ura)](+) are shown to lose a molecule of uracil. Similar results were observed under infrared multiple-photon dissociation excitation conditions, except that [Ca(Ura-H)(Ura)](+) was found to lose HNCO as the primary fragmentation product. The binding energies between neutral uracil and [M(Ura-H)](+) (M=Zn, Cu, Ni, Fe, Cd, Pd ,Mg, Ca, Sr Ba, or Pb) are calculated by means of electronic-structure calculations. The differences in the uracil binding energies between complexes which lose uracil and those which lose HNCO are consistent with the experimentally observed differences in fragmentation pathways. A size dependence in the binding energies suggests that the interaction between uracil and [M(Ura-H)](+) is ion-dipole complexation and the experimental evidence presented supports this. PMID:22447672

  3. Enzymatic Excision of Uracil Residues in Nucleosomes Depends on Local DNA Structure and Dynamics†

    PubMed Central

    Ye, Yu; Stahley, Mary R.; Xu, Jianqing; Friedman, Joshua I.; Sun, Yan; McKnight, Jeffrey N.; Gray, Jeffrey J.; Bowman, Gregory D.; Stivers, James T.

    2012-01-01

    The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from X. laevis histones and the high-affinity “Widom 601” positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple turnover conditions was generally decreased as compared to free 601 DNA, mostly due to anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations, or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multi-phasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism where some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP. PMID:22784353

  4. Regulation of Tryptophan Biosynthesis in Saccharomyces cerevisiae: Mode of Action of 5-Methyl-Tryptophan and 5-Methyl-Tryptophan-Sensitive Mutants

    PubMed Central

    Schürch, A.; Miozzari, J.; Hütter, R.

    1974-01-01

    In a wild-type strain of Saccharomyces cerevisiae the tryptophan analogue dl-5-methyl-tryptophan (5MT) causes only a slight reduction of the growth rate. Uptake experiments indicate that the limited inhibition is partly due to low levels of 5MT inside the cell. On the other hand, this low concentration of 5MT leads to an increase in the activity of the tryptophan-biosynthetic enzymes. Evidence is presented that suggests that 5MT acts primarily through feedback inhibition of anthranilate synthase, the first enzyme of the pathway. A number of 5MT-sensitive mutants have been isolated, characterized, and assigned to one of the following three classes: class I, strains with altered activity and/or feedback sensitivity of anthranilate synthase; class II, strains with elevated uptake of 5MT; class III, mutants with altered regulation of the tryptophan-biosynthetic enzymes, which do not exhibit increases in activity in the presence of 5MT. This failure to exhibit increased enzyme activities in mutants of class III can also be observed after tryptophan starvation. Two mutants of class III show high sensitivity towards 3-amino-1,2,4-triazole. They can not exhibit derepression of some histidine- and arginine-biosynthetic enzymes under conditions that lead to an increase in these same enzymes in the wild-type strain. PMID:4360539

  5. 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.

  6. Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats.

    PubMed

    Pogribny, I P; Muskhelishvili, L; Miller, B J; James, S J

    1997-11-01

    Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair

  7. Intermolecular associations of some biologically interesting pyrimidines in aqueous solution: caffeine-uracil

    NASA Astrophysics Data System (ADS)

    Iza, N.; Gil, M.; Montero, J. L.; Morcillo, J.

    1986-03-01

    Ultraviolet absorption spectra of aqueous solutions of caffeine-uracil mixt ures at C T=0.004 M have been obtained. An apparent hyperchromic effect has been observed for both bases at two bands. The hyperchromic effect attributed solely to caffeine has been evaluated at 290 nm, where uracil absorption is minimum. From this value, uracil contribution has been determinated. It results about 10% relative hyperchromism for caffeine and about -7, 5% relative hypochromism for uracil. The data obtained indicate the presence of complexes having caffeine-uracil ratio of 1:1 and higher complexes. The association constant K (CAF-U) is found to be 76± 12 M -1 from an equation where K (CAF-CAF) = 102± 6 M -1 and K (U-U) = =68±5 M -1, have been enclosed.

  8. Urea-acetylene dicarboxylic acid reaction: A likely pathway for prebiotic uracil formation

    NASA Astrophysics Data System (ADS)

    Subbaraman, A. S.; Kazi, Z. A.; Choughuley, A. S. U.; Chadha, M. S.

    1980-12-01

    A number of routes have been suggested for the prebiotic synthesis of uracil involving the reaction of urea with malic acid, propiolic acid, cyanoacetylene and others. Cyanoacetylene has been detected in the interstellar medium as well as simulated prebiotic experiments. It is therefore plausible that dicyanoacetylene and its hydrolytic product acetylene dicarboxylic acid, (ADCA) may have played a role in chemical evolution. This aspect has been examined in the present work for the synthesis of uracil from ADCA and urea reaction. It was found that when ADCA reacted with urea, uracil was formed only in the presence of phosphoric acid and phosphates. Ammonium phosphates gave higher yields of uracil than other phosphates. In the absence of phosphoric acid or phosphates no uracil formation took place. This type of synthesis could have taken place in prebiotic oceans which contained ammonium phosphates and other salts.

  9. 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

    2014-01-01

    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.

  10. 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.

  11. 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. PMID:26220618

  12. [Theoretical study of hydrophobicity and hydrophilicity of uracil and its dimers].

    PubMed

    Ten, G N; Kadrov, D M; Baranov, V I

    2014-01-01

    The influence of hydrophilic and hydrophobic properties of the uracil elementary nucleic acids bases on its solubility and structure in aqueous solution was studied. Complexes of uracil with water molecules (from 1 to 14) were then calculated. The geometrical parameters of the hydrogen bridge of uracil and the changes in the frequency of valence vibrations of the bonds participating directly in hydrogen bond formation were calculated. It is shown that for the hydrogen bonds O(w)...HN(1) and O(w)...HN3 the hydrogen atom can tear, it may lead to tautomeric transformation of uracil. The results obtained having calculated the structure of uracil dimers, formed with the hydrogen bonds, in an isolated state and water solution, energy, dipole moments and the hydrogen bridge parameters made it possible to explain low solubility of uracil in water at room temperature. It is shown that water molecules with increase in their number are located mainly at one side of the plane of a pyrimidine uracil ring, that leads to the formation of stacking. Of two possible variants of stacking formation, the most profitable grouping is when a dipole moment of the formed dimer is equal to zero (anti-parallel stacking). PMID:25707232

  13. 2-Amino-5-methyl­pyridinium 4-carb­oxy­butano­ate

    PubMed Central

    Hemamalini, Madhukar; Fun, Hoong-Kun

    2010-01-01

    In the title salt, C6H9N2 +·C5H7O4 −, the 2-amino-5-methyl­pyridinium cation is essentially planar, with a maximum deviation of 0.008 (1) Å. In the crystal, the protonated N atom and the 2-amino group 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 4-carb­oxy­butano­ate anions are linked via O—H⋯O hydrogen bonds. The crystal structure is further stabilized by weak C—H⋯O inter­actions. PMID:21588042

  14. Influence of local duplex stability and N6-methyladenine on uracil recognition by mismatch-specific uracil-DNA glycosylase (Mug).

    PubMed

    Valinluck, Victoria; Liu, Pingfang; Burdzy, Artur; Ryu, Junichi; Sowers, Lawrence C

    2002-12-01

    To maintain genomic integrity, DNA repair enzymes continually remove damaged bases and lesions resulting from endogenous and exogenous processes. These repair enzymes must distinguish damaged bases from normal bases to prevent the inadvertent removal of normal bases, which would promote genomic instability. The mechanisms by which this high level of specificity is accomplished are as yet unresolved. One member of the uracil-DNA glycosylase family of repair enzymes, Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug), is reported to distinguish U:G mispairs from U:A base pairs based upon specific contacts with the mispaired guanine after flipping the target uracil out of the duplex. However, recent studies suggest other mechanisms for base selection, including local duplex stability. In this study, we used the modified base N6-methyladenine to probe the effect of local helix perturbation on Mug recognition of uracil. N6-Methyladenine is found in E. coli as part of both the mismatch repair and restriction-modification systems. In its cis isomer, N6-methyladenine destabilizes hydrogen bonding by interfering with pseudo-Watson-Crick base pairing. It is observed that the selection of uracil by Mug is sequence dependent and that uracil residues in sequences of reduced thermostability are preferentially removed. The replacement of adenine by N6-methyladenine increases the frequency of removal of the uracil residue paired opposite the modified adenine. These results are in accord with suggestions that local helix stability is an important determinant of base recognition by some DNA repair enzymes and provide a potential strategy for identifying the sequence location of modified bases in DNA. PMID:12482242

  15. Lewis Acid Triggered Regioselective Magnesiation and Zincation of Uracils, Uridines, and Cytidines.

    PubMed

    Klier, Lydia; Aranzamendi, Eider; Ziegler, Dorothée; Nickel, Johannes; Karaghiosoff, Konstantin; Carell, Thomas; Knochel, Paul

    2016-03-01

    The Lewis acid MgCl2 allows control of the metalation regioselectivity of uracils and uridines. In the absence of the Lewis acid, metalation of uracil and uridine derivatives with TMPMgCl·LiCl occurs at the position C(5). In the presence of MgCl2, zincation using TMP2Zn·2LiCl·2MgCl2 occurs at the position C(6). This metalation method provides easy access to functionalized uracils and uridines. Using TMP2Zn·2LiCl·2MgCl2 also allows to functionalize cytidine derivatives at the position C(6).

  16. Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase

    PubMed Central

    Chen, Cheng-Yao; Mosbaugh, Dale W.; Bennett, Samuel E.

    2011-01-01

    To investigate the role of Arginine 276 in the conserved leucine-loop of human uracil-DNA glycosylase (UNG), the effects of six R276 amino acid substitutions (C, E, H, L, W, and Y) on nucleotide flipping and enzyme conformational change were determined using transient and steady state, fluorescence-based, kinetic analysis. Relative to UNG, the mutant proteins exhibited a 2.6- to 7.7-fold reduction in affinity for a doubled-stranded oligonucleotide containing a pseudouracil residue opposite 2-aminopurine, as judged by steady-state DNA binding-base flipping assays. An anisotropy binding assay was utilized to determine the Kd of UNG and the R276 mutants for carboxyfluorescein-labeled uracil-containing single- and double-stranded oligonucleotides; the binding affinities varied 11-fold for single-stranded uracil-DNA, and 43-fold for double-stranded uracil-DNA. Productive uracil-DNA binding was monitored by rapid quenching of UNG intrinsic protein fluorescence. Relative to UNG, the rate of intrinsic fluorescence quenching of five mutant proteins for binding double-stranded uracil-DNA was reduced approximately 50%; the R276E mutant exhibited 1% of the rate of fluorescence quenching of UNG. When reacted with single-stranded uracil-DNA, the rate of UNG fluorescence quenching increased. Moreover, the rate of fluorescence quenching for all the mutant proteins, except R276E, was slightly faster than UNG. The kcat of the R276 mutants was comparable to UNG on single-stranded DNA and differentially affected by NaCl; however, kcat on double-stranded DNA substrate was reduced 4–12-fold and decreased sharply at NaCl concentrations as low as 20 mM. Taken together, these results indicate that the effects of mutations at Arg276 were largely limited to enzyme interactions with double-stranded uracil-containing DNA, and suggested that mutations at Arg276 effectively transformed UNG into a single-stranded DNA-specific uracil-DNA glycosylase. PMID:15970468

  17. 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

    2011-06-01

    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.

  18. The pyrimidine biosynthesis operon of the thermophile Bacillus caldolyticus includes genes for uracil phosphoribosyltransferase and uracil permease.

    PubMed Central

    Ghim, S Y; Neuhard, J

    1994-01-01

    A 3-kb DNA segment of the Bacillus caldolyticus genome including the 5' end end of the pyr cluster has been cloned and sequenced. The sequence revealed the presence of two open reading frames, pyrR and pyrP, located immediately upstream of the previously sequenced pyrB gene encoding the pyrimidine biosynthesis enzyme aspartate transcarbamoylase. The pyrR and pyrP genes encoded polypeptides with calculated molecular masses of 19.9 and 45.2 kDa, respectively. Expression of these ORFs was confirmed by analysis of plasmid-encoded polypeptides in minicells. Sequence alignment and complementation analyses identified the pyrR gene product as a uracil phosphoribosyltransferase and the pyrP gene product as a membrane-bound uracil permease. By using promoter expression vectors, a 650-bp EcoRI-HincII fragment, including the 5' end of pyrR and its upstream region, was found to contain the pyr operon promoter. The transcriptional start point was located by primer extension at a position 153 bp upstream of the pyrR translation initiation codon, 7 bp 3' of a sequence resembling a sigma A-dependent Bacillus subtilis promoter. This established the following organization of the ten cistrons within the pyr operon: promoter-pyrR-pyrP-pyrB-pyrC-pyrAa-pyrA b-orf2-pyrD-pyrF-pyrE. The nucleotide sequences of the region upstream of pyrR and of the pyrR-pyrP and pyrP-pyrB intercistronic regions indicated that the transcript may form two mutually exclusive secondary structures within each of these regions. One of these structures resembled a rho-independent transcriptional terminator. The possible implication of these structures for pyrimidine regulation of the operon is discussed. Images PMID:8206848

  19. 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.

  20. 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. PMID:25638501

  1. Hydrogen abstraction from deoxyribose by a neighboring 3'-uracil peroxyl radical.

    PubMed

    Schyman, Patric; Eriksson, Leif A; Laaksonen, Aatto

    2009-05-01

    Theoretical examination of the reactivity of the uracil-5-peroxyl radical when abstracting a hydrogen atom from a neighboring 5'-deoxyribose in 5'-ApU-5-peroxyl-3' has been performed using density functional theory with the MPWB1K functional. Halogenated uracils are often used as radiosensitizers in DNA since the reactive uracil-5-yl radical is formed upon radiation and is known to create strand break and alkali-labile sites. Under aerobic conditions, such as in the cell, it has been proposed that the uracil-5-peroxyl radical is formed and would be the damaging agent. Our results show low reactivity for the uracil-5-peroxyl radical, determined by calculating the activation and reaction energies for the plausible hydrogen abstraction sites C1', C2', and C3' of the neighboring 5'-deoxyribose. These findings support the hypothesis that hydrogen abstraction primarily occurs by the uracil-5-yl radical, also under aerobic conditions, prior to formation of the peroxyl radical. PMID:19402732

  2. Hydrogen abstraction from deoxyribose by a neighboring 3'-uracil peroxyl radical.

    PubMed

    Schyman, Patric; Eriksson, Leif A; Laaksonen, Aatto

    2009-05-01

    Theoretical examination of the reactivity of the uracil-5-peroxyl radical when abstracting a hydrogen atom from a neighboring 5'-deoxyribose in 5'-ApU-5-peroxyl-3' has been performed using density functional theory with the MPWB1K functional. Halogenated uracils are often used as radiosensitizers in DNA since the reactive uracil-5-yl radical is formed upon radiation and is known to create strand break and alkali-labile sites. Under aerobic conditions, such as in the cell, it has been proposed that the uracil-5-peroxyl radical is formed and would be the damaging agent. Our results show low reactivity for the uracil-5-peroxyl radical, determined by calculating the activation and reaction energies for the plausible hydrogen abstraction sites C1', C2', and C3' of the neighboring 5'-deoxyribose. These findings support the hypothesis that hydrogen abstraction primarily occurs by the uracil-5-yl radical, also under aerobic conditions, prior to formation of the peroxyl radical.

  3. 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

  4. 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

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

    PubMed

    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. PMID:27644592

  6. Antioxidant and Antimicrobial Activity of 5-methyl-2-(5-methyl-1,3-diphenyl-1H-pyrazole-4-carbonyl)-2,4-dihydro-pyrazol-3-one

    PubMed Central

    Umesha, K. B.; Rai, K. M. L.; Harish Nayaka, M. A.

    2009-01-01

    Cycloaddition of nitrile imines 4 generated in situ by the catalytic dehydrogenation of diphenyl hydrazones 3 using Chloramine-T (CAT) as oxidant in glacial acetic acid with enolic form of ethyl acetoacetate 5 afforded Ethyl 3-aryl-5-methyl-1-phenyl-1H-pyrazol-4-carboxylate 6 in 80% yield. The said pyrazoles 6 refluxed with 80% hydrazine hydrate using absolute alcohol as solvent for about 2–3 hours to produce the respective 5-methyl-1,3-diphenyl-1H-pyrazole-4-carboxylic acid hydrazide 7. The alcoholic solution of pyrazole acid hydrazides on heating with ethyl acetoacetate 5 to give the 5-methyl-2-(5-methyl-1,3-diphenyl-1H-pyrazole-4-carbonyl)-2,4-dihydro-pyrazol-3-one 8. The synthesized compounds were found to exhibit good antimicrobial and antioxidant activity as evaluated by 1,1-diphenyl-2-picryl Hydrazyl (DPPH) radical scavenging, reducing power and DNA protection assays. PMID:23675159

  7. Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo

    PubMed Central

    Bande, Omprakash; Abu El Asrar, Rania; Braddick, Darren; Dumbre, Shrinivas; Pezo, Valérie; Schepers, Guy; Pinheiro, Vitor B; Lescrinier, Eveline; Holliger, Philipp; Marlière, Philippe; Herdewijn, Piet

    2015-01-01

    The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoCMe) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)–DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoCMe bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoCMe misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoCMe/isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality. PMID:25684598

  8. Molecular geometry of 5-methyl-2-furaldehyde from gas electron diffraction

    NASA Astrophysics Data System (ADS)

    Shishkov, Igor F.; Vilkov, Lev V.; Hargittai, István

    1995-06-01

    An electron diffraction study of 5-methyl-2-furaldehyde (MFA) has been carried out to obtain information on its conformational properties and molecular geometry. Two conformers, with syn and anti orientations of the CO and CC bonds, were found to coexist with the composition 74(11) and 26%, respectively, at 333 K. The differences between the parameters in the syn and anti forms were assumed from ab initio calculations (MP2/6-31G∗) in the analysis. The following bond lengths ( rg, Å) and bond angles (deg) were obtained for the syn form with estimated total errors parenthesized in units of the last digit of the parameter: CC(mean) = 1.366(14), CO = 1.223(4), OCC(Me) = 117.1(13), CO(mean) = 2.378(12), CH(Me) = 1.116(15), CCO = 110.5(5), CC(Me) = 1.507(14), COC = 105.7(11), CCO = 121.5(14), CC(ald) = 1.454(12), OCC(ald) = 115.8(18). The conformationals properties of MFA are similar to those of 2-furaldehyde while for acrolein the anti form was found to be the most stable.

  9. 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.

  10. Biosynthesis of 3-hydroxy-5-methyl-o-methyltyrosine in the saframycin/ safracin biosynthetic pathway.

    PubMed

    Fu, Cheng-Yu; Tang, Man-Cheng; Peng, Chao; Li, Lei; He, Yan-Ling; Liu, Wen; Tang, Gong-Li

    2009-05-01

    The biosynthesis study of antibiotics saframycin (SFM) in Streptomyces lavendulae and safracin (SAC) in Pseudomonas fluorescens demonstrated that 3-hydroxy-5-methyl-Omethyltyrosine (3h5mOmTyr), a nonproteinogenic amino acid, is the precursor of the tetrahydroisoquinoline molecular core. In the biosynthetic gene cluster of SAC/SFM, sacD/ sfmD encodes a protein with high homology to each other but no sequence similarity to other known enzymes; sacF/ sfmM2 and sacG/sfmM3 encode methyltransferases for Cmethylation and O-methylation; and sacE/sfmF encodes a small protein with significant sequence similarity to the MbtH-like proteins, which are frequently found in the biosynthetic pathways of nonribosomal peptide antibiotics and siderophores. To address their function, the biosynthetic cassette of 3h5mOmTyr was heterologously expressed in S. coelicolor and P. putida, and an in-frame deletion and complementation in trans were carried out. The results revealed that (i) SfmD catalyzes the hydroxylation of aromatic rings;(ii) sacD/sacF/sacG in the SAC gene cluster and sfmD/sfmM2/sfmM3 in the SFM cluster are sufficient for the biosynthesis of 3h5mOmTyr; and (iii) the mbtH-like gene is not required for the biosynthesis of the 3h5mOmTyr precursor. PMID:19494690

  11. 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.

  12. 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.

  13. 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.

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

    DOE PAGESBeta

    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 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

  15. Chemical transformations drive complex self-assembly of uracil on close-packed coinage metal surfaces.

    PubMed

    Papageorgiou, Anthoula C; Fischer, Sybille; Reichert, Joachim; Diller, Katharina; Blobner, Florian; Klappenberger, Florian; Allegretti, Francesco; Seitsonen, Ari P; Barth, Johannes V

    2012-03-27

    We address the interplay of adsorption, chemical nature, and self-assembly of uracil on the Ag(111) and Cu(111) surfaces as a function of molecular coverage (0.3 to 1 monolayer) and temperature. We find that both metal surfaces act as templates and the Cu(111) surface acts additionally as a catalyst for the resulting self-assembled structures. With a combination of STM, synchrotron XPS, and NEXAFS studies, we unravel a distinct polymorphism on Cu(111), in stark contrast to what is observed for the case of uracil on the more inert Ag(111) surface. On Ag(111) uracil adsorbs flat and intact and forms close-packed two-dimensional islands. The self-assembly is driven by stable hydrogen-bonded dimers with poor two-dimensional order. On Cu(111) complex structures are observed exhibiting, in addition, a strong annealing temperature dependence. We determine the corresponding structural transformations to be driven by gradual deprotonation of the uracil molecules. Our XPS study reveals unambiguously the tautomeric signature of uracil in the contact layer and on Cu(111) the molecule's deprotonation sites. The metal-mediated deprotonation of uracil and the subsequent electron localization in the molecule determine important biological reactions. Our data show a dependence between molecular coverage and molecule-metal interaction on Cu(111), as the molecules tilt at higher coverages in order to accommodate a higher packing density. After deprotonation of both uracil N atoms, we observe an adsorption geometry that can be understood as coordinative anchoring with a significant charge redistribution in the molecule. DFT calculations are employed to analyze the surface bonding and accurately describe the pertaining electronic structure.

  16. Analysis of nuclear uracil-DNA glycosylase (nUDG) turnover during the cell cycle.

    PubMed

    Fischer, Jennifer A; Caradonna, Salvatore

    2011-01-01

    Uracil-DNA glycosylases (UDG/UNG) are enzymes that remove uracil from DNA and initiate base-excision repair. These enzymes play a key role in maintaining genomic integrity by reducing the mutagenic events caused by G:C to A:T transition mutations. The recent finding that a family of RNA editing enzymes (AID/APOBECs) can deaminate cytosine in DNA has raised the interest in these base-excision repair enzymes. The methodology presented here focuses on determining the regulation of the nuclear isoform of uracil-DNA glycosylase (nUDG), a 36,000 Da protein. In synchronized HeLa cells, nUDG protein levels decrease to barely detectable levels during the S phase of the cell cycle. Immunoblot analysis of immunoprecipitated or affinity-isolated nUDG reveals ubiquitin-conjugated nUDG when proteolysis is inhibited by agents that block proteasomal-dependent protein degradation. PMID:21755446

  17. Dissociative Electron Attachment Dynamics in the Nucleobase Uracil and Related Molecules

    NASA Astrophysics Data System (ADS)

    Slaughter, Daniel; Kuriyama, Yosuke; Kawarai, Yu; Azuma, Yoshiro; Winstead, Carl; McKoy, Vincent; Weber, Thorsten; Belkacem, Ali

    2015-05-01

    We report the dynamics of dissociative electron attachment (DEA) in the biologically-relevant molecule uracil and the diazines pyrazine and pyrimidine. Our DEA reaction microscope consists of a 3D momentum-imaging spectrometer, a pulsed low-energy electron gun and an effusive gas target. The experimental approach allows the measurement of kinetic energy and angular distributions of ionic fragments produced by DEA, in some cases elucidating the total kinetic energy release following two-body breakup. By comparison of calculations of the electron attachment probability in the molecular frame with measured ion angular distributions, we determine that one of the uracil anion resonances could be a Feshbach resonance and we compare the dynamics of the dissociation of the uracil anion with the diazines. Work supported by Chemical Sciences, Geosciences and Biosciences division of BES/DOE

  18. 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...

  19. A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

    PubMed Central

    Stuart, D T; Upton, C; Higman, M A; Niles, E G; McFadden, G

    1993-01-01

    Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability. Images PMID:8474156

  20. Synthesis and crystal structure of new heterocyles derived from saccharin and uracil carrying 1,2,4-oxadiazolylmethyl group.

    PubMed

    Dürüst, Yaşar; Özer, Besra; Kariuki, Benson M; Cariuki, Benson M

    2015-05-01

    Saccharin, uracil, and 1,2,4-oxadiazole heterocyles are important in terms of exhibiting various biological acitivities. In this work, four series of 1,2,4-oxadiazolylmethyl-substituted saccharin, and uracil derivatives are synthesized and their structures are identified by means of spectral/physical characteristics. The first series are oxadiazolylmethyl-substituted saccharins. The second one is oxadiazole-substituted uracils which are obtained as a separable mixture of both mono- and bis-substituted end products. Third series is obtained from 5-amino uracil and chloromethyl oxadiazoles. The fourth group is oxadiazolyl methyl-substituted imino uracils. The structures of some title compounds are also confirmed by X-ray diffraction data.

  1. Highly charged ion impact on uracil: Cross sections measurements and scaling

    NASA Astrophysics Data System (ADS)

    Agnihotri, A. N.; Kasthurirangan, S.; Champion, C.; Rivarola, R. D.; Tribedi, L. C.

    2014-04-01

    Absolute total ionization cross sections (TCS) of uracil in collisions with highly charge C, O and F ions are measured. The scaling properties of cross sections are obtained as a function of projectile charge state and energy. The measurements are compared with the CDW-EIS, CB1 and CTMC calculations. The absolute double differential cross sections (DDCS) of secondary electron emission from uracil in collisions with bare MeV energy C and O ions are also measured. Large enhancement in forward emission is observed.

  2. Computational rationale for the selective inhibition of the herpes simplex virus type 1 uracil-DNA glycosylase enzyme.

    PubMed

    Hendricks, Umraan; Crous, Werner; Naidoo, Kevin J

    2014-12-22

    The herpes simplex virus uracil-DNA glycosylase (hsvUNG) enzyme is responsible for the reactivation of the virus from latency and efficient viral replication in nerve tissue. The lack of uracil-DNA glycosylase enzyme in human neurons and the continuous deamination of cytosine create an environment where the presence of viral uracil-DNA glycosylase is a necessity for the proliferation of the virus. A series of 6-(4-alkylanilino)-uracil inhibitors has been developed that selectively and strongly binds to the hsvUNG enzyme while weakly binding to human uracil-DNA glycosylase (hUNG). Here, by using a combination of sequence and structural comparisons between the two enzymes along with free energy of binding computations and principal component analysis of the ligands, we investigate and rationalize the inhibitory effect of the 6-(4-alkylanilino)-uracil series as a function of alkyl chain length on the hsvUNG. The results of these computations corroborate the experimental finding that the inhibitor with an octyl aliphatic chain selectively binds hsvUNG best. More importantly we find that 6-(4-octylanilino)-uracil's selective inhibition of hsvUNG over hUNG is due to the combination of the solution preconfigured bent conformation of that specific chain length and the position of HIS92 (absent in hUNG) just outside hsvUNG's hydrophobic gorge lying adjacent to its uracil binding pocket. The similarities between the uracil binding pockets in hsvUNG and hUNG obfuscate an understanding of the preferential inhibition of the virus enzyme. However, the differences in the enzymes' shallow hydrophobic grooves adjacent to the binding pockets, such as the gorge we identify here, rationalizes 6-(4-alkylanilino)-uracil with an octyl chain length as an excellent pharmacophore template for hsvUNG inhibitor design.

  3. 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...

  4. Characterizing the dark state in thymine and uracil by double resonant spectroscopy and quantum computation.

    PubMed

    Ligare, M; Siouri, F; Bludsky, O; Nachtigallová, D; de Vries, M S

    2015-10-01

    We report on gas phase double resonant spectroscopy of both the ground state and the dark excited state in isolated uracil and thymine. We also report lifetimes of the dark state for different excitation wavelengths. In combination with ab initio calculations the results suggest that the dark state is of triplet ((3)ππ*) character. PMID:26325364

  5. 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.

  6. 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.

  7. HPCE quantification of 5-methyl-2'-deoxycytidine in genomic DNA: methodological optimization for chestnut and other woody species.

    PubMed

    Hasbún, Rodrigo; Valledor, Luís; Rodríguez, José L; Santamaria, Estrella; Ríos, Darcy; Sanchez, Manuel; Cañal, María J; Rodríguez, Roberto

    2008-01-01

    Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2'-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2'-deoxycytidine. PMID:18538578

  8. Expanding Targets of DNAzyme-based Sensors through Deactivation and Activation of DNAzymes by Single Uracil Removal: Sensitive Fluorescent Assay of Uracil-DNA Glycosylase

    PubMed Central

    Xiang, Yu

    2012-01-01

    Although deoxyribozymes (DNAzymes) have been widely used as biosensors for the detection of their cofactors and the targets of related aptazymes, it is desirable to expand their range of analytes to take advantage of the DNAzyme-based signal amplification for more sensitive detections. In this study, the activity of uracil-DNA glycosylase (UNG) was successfully detected and quantified by deoxyuridine-modified DNAzymes that underwent UNG-dependent deactivation or activation. In one design, the indispensable thymidine T2.1 in the 8–17 DNAzyme was replaced with a deoxyuridine, resulting in minimal change of the DNAzyme’s activity. Since UNG is capable of removing uracils from single- or double-stranded DNAs, the modified DNAzyme was deactivated when the uracil at the indispensable thymidine site was eliminated by UNG. In another design, introducing a deoxyuridine to the 3′ position of the deoxycytidine C13 in the catalytic core of the same DNAzyme caused significant decrease of the activity. However, the removal of the interfering deoxyuridine by UNG activated the DNAzyme. By monitoring the activity change of the DNAzymes through the fluorescence enhancement from the DNAzyme-catalyzed cleavage of DNA substrates labeled by a fluorophore and quencher pair, the UNG activity was measured based on UNG-dependent deactivation and activation of the DNAzymes. The method was found to be able to detect UNG activity as low as 0.0034 U/mL. Such a method can be applied to the detection of other nucleotide-modifying enzymes and expand the analyte range of DNAzyme-based biosensors. PMID:23072386

  9. Pd(OAc)2-catalyzed dehydrogenative C–H activation: An expedient synthesis of uracil-annulated β-carbolinones

    PubMed Central

    Mondal, Biplab; Hazra, Somjit; Panda, Tarun K

    2015-01-01

    Summary An intramolecular dehydrogenative C–H activation enabled an efficient synthesis of an uracil-annulated β-carbolinone ring system. The reaction is simple, efficient and high yielding (85–92%). PMID:26425190

  10. Synthesis and antiprotozoal activity of mono- and bis-uracil isatin conjugates against the human pathogen Trichomonas vaginalis.

    PubMed

    Kumar, Kewal; Liu, Nicole; Yang, Donald; Na, Daniel; Thompson, John; Wrischnik, Lisa A; Land, Kirkwood M; Kumar, Vipan

    2015-08-15

    A library of mono- and bis-uracil isatin conjugates were synthesized and subjected for the assessment of their in vitro activity against the protozoal pathogen Trichomonas vaginalis. The structure activity studies (SAR) revealed that the bis-uracil-isatin based conjugates were more effective than their corresponding mono conjugates in inhibiting the growth of T. vaginalis at approximately 10 μM with no visual effect on mammalian cells at the same concentration.

  11. 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.

  12. 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.

  13. Structural and vibrational studies on 1-(5-methyl-[1,3,4] thiadiazol-2-yl)-pyrolidin-2-ol

    NASA Astrophysics Data System (ADS)

    Ramesh Babu, N.; Saleem, H.; Subashchandrabose, S.; Padusha, M. Syed Ali; Bharanidharan, S.

    2016-01-01

    FT-Raman and FT-IR spectra were recorded for1-(5-methyl-[1,3,4]thiadiazol-2-yl)-pyrolidin-2-ol (MTPN) sample in solid state. The equilibrium geometries, harmonic vibrational frequencies, IR and the Raman scattering intensities were computed using DFT/6-311++G (d,p) level. Results obtained at this level of theory were used for a detailed interpretation of the IR and Raman spectra, based on the TED of the normal modes. Molecular parameters such as bond lengths, bond angles and dihedral angles were calculated. The intra-molecular charge transfer was calculated by means of NBO. Hyperconjugative interaction energy was more during the π-π∗ transition. Energy gap of the molecule has been found using HOMO and LUMO calculation, hence the less band gap, which seems to be more stable.

  14. Bis(2-amino-5-methyl-1,3,4-thia­diazole-κN 3)dichloridocobalt(II)

    PubMed Central

    Song, Ye; Ji, Yu-Fei; Kang, Min-Yan; Liu, Zhi-Liang

    2012-01-01

    In the monomeric title complex, [CoCl2(C3H5N3S)2], the CoII atom is tetra­coordinated by two chloride anions and two N atoms from two monodentate 2-amino-5-methyl-1,3,4-thia­diazole ligands, giving a slightly distorted tetra­hedral stereochemistry [bond angle range about Co = 105.16 (12)–112.50 (10)°]. In the complex, the dihedral angle between the 1,3,4-thia­diazole planes in the two ligands is 72.8 (1)°. There are two intra­molecular N—H⋯Cl inter­actions in the complex unit, while in the crystal, inter­molecular N—H⋯N and N—H⋯Cl hydrogen bonds link these units into a two-dimensional layered structure parallel to (011). PMID:22719326

  15. Synthesis, potential anticonvulsant and antidepressant effects of 2-(5-methyl-2,3-dioxoindolin-1-yl)acetamide derivatives

    PubMed Central

    Zhen, Xinghua; Peng, Zhou; Zhao, Shuilian; Han, Yan; Jin, Qinghao; Guan, Liping

    2015-01-01

    A new series of 2-(5-methyl-2,3-dioxoindolin-1-yl)acetamide derivatives were synthesized and evaluated for their anticonvulsive activity in a pentylenetetrazole (PTZ)-evoked convulsion model and antidepressant activity in the forced swimming test (FST) model. Eleven synthesized compounds were found to be protective against PTZ-induced seizure and showed the anticonvulsant activity. In addition, four of the synthesized compounds (4l, 4m, 4p and 4q) showed potent antidepressant-like activity. Among these compounds, compound 4l was found to have the most potent antidepressant-like activity, and significantly reduced the duration of immobility time at 100 mg/kg dose level when compared to the vehicle control, which is similar to the reference drug fluoxetine. PMID:26579465

  16. Even-odd alternation in mass spectrum of thymine and uracil clusters: Evidence of intracluster photodimerization

    PubMed Central

    Kim, Nam Joon; Kang, Hyuk; Jeong, Gawoon; Kim, Yung Sam; Lee, Kang Taek; Kim, Seong Keun

    2001-01-01

    Multiphoton ionization of thymine and uracil clusters generated by a supersonic molecular beam gave rise to a remarkable alternation of mass spectral intensities between even- and odd-numbered clusters. Such alternation was observed in clusters of up to 30 molecules. Excitation to the two lowest electronically excited states seemed to be a strong prerequisite. In view of the well known photodimerization reaction of thymine and uracil in the bulk phase, it is proposed that such alternation in the mass spectral intensity resulted from formation of photodimer units within the cluster on intense UV irradiation. Several analogues of thymine with no known propensity for photodimerization in the bulk phase did not exhibit any sign of such alternation in the cluster mass spectrum. The intrinsic UV window for photodimerization, and hence photoinduced mammalian mutagenesis, was estimated to be approximately 210–280 nm, significantly narrower than the previously reported bulk values of 150–300 nm. PMID:11296267

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

    PubMed

    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(')-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. PMID:24832276

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

    PubMed

    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(')-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.

  19. The study of interaction between PFOA/PFOS and uracil by topology quality and spectroscopic analysis

    NASA Astrophysics Data System (ADS)

    Xu, Hui-Ying; Zhu, Jian-Qing; Wang, Wei; Xu, Xiao-Lu; Lu, Yin

    2014-02-01

    It has been established that perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) can be considered as emerging persistent organic pollutants. In recent years, there was increasing distribution of PFOA/PFOS in environmental systems, and accumulation and toxic effects of PFOA/PFOS in human body. In this paper, quantum chemistry methods were employed to study the interaction between perfluorinated organic pollutants and base (uracil). The results showed that there were four stable binding modes between the two perfluorinated compounds with uracil, especially the second mode which caused the most detrimental physiological functional response. NBO analysis showed that reactive hydrogen in the two perfluorinated compounds had the greatest effect on the hydrogen bond. The nature of the hydrogen bond formed between the two perfluorinated compounds and base was investigated using the AIM theory. The changes of spectroscopic properties in complexes were analyzed by IR and NMR spectra.

  20. Synthesis of 1-[3-deoxy-β-D-psicofuranosyl]uracil and related compounds

    PubMed Central

    Holý, A.

    1974-01-01

    D-Fructose affords on treatment with cyanamide 2-amino-β-D-fructofuro[2′,3′:3,4]-oxazoline which is not isolated but transformed directly by the reaction with ethyl propiolate into O2,3-anhydro-2-[β-D-fructofuranosyl]uracil. This compound is benzoylated to the 1′,4′,6′-tri-O-benzoyl derivative by the action of benzoyl cyanide and triethylamine. On treatment with hydrogen chloride/dimethylformamide, the latter intermediate is converted to the 1-[1,4,6-tri-O-benzoyl-3-chloro-3-deoxy-β-D-psicofuranosyl]uracil 1′,4′,5′-tribenzoate from which the title nucleoside derivative is obtained by methanolysis. PMID:4370426

  1. 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.

  2. 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. PMID:19004346

  3. Solid State 2H NMR Analysis of Furanose Ring Dynamics in DNA Containing Uracil

    PubMed Central

    Kinde-Carson, Monica N.; Ferguson, Crystal; Oyler, Nathan A.; Harbison, Gerard S.; Meints, Gary A.

    2010-01-01

    DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. Single-base lesions, such as uracil, in DNA can be cytotoxic or mutagenic and are recognized by a DNA glycosylase during the process of base excision repair. Increased dynamic properties in lesion-containing DNAs have been suggested to assist recognition and specificity. Deuterium solid-state nuclear magnetic resonance (SSNMR) has been used to directly observe local dynamics of the furanose ring within a uracil: adenine (U:A) base pair and compared to a normal thymine:adenine (T:A) base pair. Quadrupole echo lineshapes, ⟨T1Z⟩, and ⟨T2e⟩ relaxation data were collected, and computer modeling was performed. The results indicate that the relaxation times are identical within the experimental error, the solid lineshapes are essentially indistinguishable above the noise level, and our lineshapes are best fit with a model that does not have significant local motions. Therefore, U:A base pair furanose rings appear to have essentially identical dynamic properties as a normal T:A base pair, and the local dynamics of the furanose ring are unlikely to be the sole arbiter for uracil recognition and specificity in U:A base pairs. PMID:20151717

  4. Revisiting the Reactivity of Uracil During Collision Induced Dissociation: Tautomerism and Charge-Directed Processes

    NASA Astrophysics Data System (ADS)

    Beach, Daniel G.; Gabryelski, Wojciech

    2012-05-01

    In our recent work towards the nontarget identification of products of nucleic acid (NA) damage in urine, we have found previous work describing the dissociation of NA bases not adequate to fully explain their observed reactivity. Here we revisit the gas-phase chemistry of protonated uracil (U) during collision induced dissociation (CID) using two modern tandem mass spectrometry techniques; quadrupole ion trap (QIT) and quadrupole time of flight (Q-TOF). We present detailed mechanistic proposals that account for all observed products of our experiments and from previous isotope labeling data, and that are supported by previous ion spectroscopy results and theoretical work. The diverse product-ions of U cannot be explained adequately by only considering the lowest energy form of protonated U as a precursor. The tautomers adopted by U during collisional excitation make it possible to relate the complex reactivity observed to reasonable mechanistic proposals and feasible product-ion structures for this small highly conjugated heterocycle. These reactions proceed from four different stable tautomers, which are excited to a specific activated precursor from which dissociation can occur via a charge-directed process through a favorable transition state to give a stabilized product. Understanding the chemistry of uracil at this level will facilitate the identification of new modified uracil derivatives in biological samples based solely on their reactivity during CID. Our integrated approach to describing ion dissociation is widely applicable to other NA bases and similar classes of biomolecules.

  5. 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.

  6. Folate Deficiency Induces Neurodegeneration and Brain Dysfunction in Mice Lacking Uracil DNA Glycosylase

    PubMed Central

    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

    2008-01-01

    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. PMID:18614692

  7. The stability of tryptophan, 5-methyl-tryptophan and α-methyl-tryptophan during NaOH hydrolysis of selected foods.

    PubMed

    Rutherfurd, Shane M; Richardson, Russell K; Moughan, Paul J

    2015-12-01

    This study evaluated the use of 5-methyl-tryptophan, α-methyl-tryptophan or synthetic tryptophan to correct for the losses of protein-bound tryptophan in foods during NaOH hydrolysis. Synthetic tryptophan and each protein source was incubated in 4.5M NaOH containing 5-methyl-tryptophan and α-methyl-tryptophan in nitrogen gas-sparged Teflon vials for 0-144 h at 110 °C. The hydrolysis and loss rates of protein-bound tryptophan, 5-methyl-tryptophan, α-methyl-tryptophan and synthetic tryptophan were predicted using least-squares nonlinear regression. Using 5-methyl-tryptophan or synthetic tryptophan to correct for hydrolytic losses of tryptophan overestimated the tryptophan content by 8.2-19% and -0.3-8.8% respectively, while correction using α-methyl-tryptophan underestimated tryptophan by between 0.2% and 8.1% across the protein sources. Correction using α-methyl-tryptophan or synthetic tryptophan was more accurate than using 5-methyl-tryptophan, but when highly accurate tryptophan composition data are required, least-squares nonlinear regression is the best approach as it removes the need for a hydrolysis correction factor.

  8. 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

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

    PubMed

    Young-Gonzales, Amanda R; Richert, Ranko

    2016-08-21

    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. PMID:27544115

  10. 2-Amino-5-methyl­pyridinium 1H-pyrazole-3,5-dicarboxyl­ate trihydrate

    PubMed Central

    Shahani, Tara; Fun, Hoong-Kun; Hemamalini, Madhukar

    2010-01-01

    In the title compound, 2C6H9N2 +·C5H2N2O4 2−·3H2O, the 1H-pyrazole-3,5-dicarboxyl­ate anion is close to planar [maximum deviation = 0.208 (1) Å]. The two distinct 2-amino-5-methyl­pyridinium cations are also almost planar, with maximum deviations of 0.018 (2) and 0.014 (2) Å. In the crystal, pairs of inter­molecular N—H⋯O and O—H⋯O hydrogen bonds connect neighbouring mol­ecules into dimers, generating R 2 2(8) and R 2 4(8) ring motifs, respectively. Further inter­molecular N—H⋯O, O—H⋯O and C—H⋯O hydrogen bonds link the mol­ecules into a three-dimensional network. PMID:21589058

  11. 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 SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices § 73.3122 4-...

  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, 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 SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices § 73.3122 4-...

  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, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 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 SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Medical Devices § 73.3122 4-...

  14. Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae

    SciTech Connect

    Chen, Jau-Der; Lacks, S.A. )

    1991-01-01

    Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A {center dot}T{r arrow}G {center dot} C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G {center dot} C{r arrow}A {center dot} T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G {center dot} C{r arrow}A {center dot} T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A {center dot} T{r arrow}G {center dot} C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This hot spot was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.

  15. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.

  16. Biological evaluation of some uracil derivatives as potent glutathione reductase inhibitors

    NASA Astrophysics Data System (ADS)

    Güney, Murat; Ekinci, Deniz; Ćavdar, Huseyin; Şentürk, Murat; Zilbeyaz, Kani

    2016-04-01

    Discovery of glutathione reductase (GR) inhibitors has become very popular recently due to antimalarial and anticancer activities. In this study, GR inhibitory capacities of some uracil derivatives (UDCs) (1-4) were reported. Some commercially available molecules (5-6) were also tested for comparison reasons. The novel UDCs were obtained in high yields using simple chemical procedures and exhibited much potent inhibitory activities against GR at low nanomolar concentrations with IC50 values ranging from 2.68 to 166.6 nM as compared with well-known agents.

  17. Action of uracil analogs on human immunodeficiency virus type 1 and its reverse transcriptase.

    PubMed Central

    Piras, G; Dutschman, G E; Im, G J; Pan, B C; Chu, S H; Cheng, Y C

    1995-01-01

    Three structural analogs of 5-ethyl-1-benzyloxymethyl-6-(phenylthio)uracil (E-BPU) inhibited human immunodeficiency virus type 1 (HIV-1) replication without cytotoxicity in vitro and were more potent than azidothymidine and were as potent as E-BPU. The target of these compounds is HIV-1 reverse transcriptase. Reverse transcriptases resistant to nevirapine (tyrosine at position 181 to cysteine) and TIBO R82150 (leucine at position 100 to isoleucine) are cross resistant to E-BPU analogs. Nevirapine- or TIBO R82150-resistant HIV-1 were cross resistant to E-BPU analogs but were inhibited at concentrations 11- to 135-fold lower than the cytotoxic doses. PMID:7537030

  18. Vibrational studies, barrier height and thermodynamic functions for biomolecules: 5-Trifluoromethyl uracil

    NASA Astrophysics Data System (ADS)

    Shanker, R.; Yadav, R. A.; Singh, I. S.

    1994-07-01

    Laser Raman (50-4000 cm -1) and IR (200-4000 cm -1) spectra of 5-trifluoromethyl uracil have been recorded and analysed. It has been possible to assign all the 39 (26a'+13a″) normal modes of vibration. Consistent assignments have been made for the internal modes of the CF 3 group, especially for the antisymmetric CF 3 stretching and bending modes. Using thus assigned vibrational frequencies and assumed structural parameters, thermodynamic functions, in the temperature range 100-1000 K, have been computed and the barrier to the internal rotation for the CF 3 top has been determined.

  19. Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.

    PubMed

    Pedersen, Hege Lynum; Johnson, Kenneth A; McVey, Colin E; Leiros, Ingar; Moe, Elin

    2015-10-01

    Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues

  20. Antimalarial and antimicrobial activities of 8-Aminoquinoline-Uracils metal complexes

    PubMed Central

    Phopin, Kamonrat; Sinthupoom, Nujarin; Treeratanapiboon, Lertyot; Kunwittaya, Sarun; Prachayasittikul, Supaluk; Ruchirawat, Somsak; Prachayasittikul, Virapong

    2016-01-01

    8-Aminoquinoline (8AQ) derivatives have been reported to have antimalarial, anticancer, and antioxidant activities. This study investigated the potency of 8AQ-5-substituted (iodo and nitro) uracils metal (Mn, Cu, Ni) complexes (1-6) as antimalarial and antimicrobial agents. Interestingly, all of these metal complexes (1-6) showed fair antimalarial activities. Moreover, Cu complexes 2 (8AQ-Cu-5Iu) and 5 (8AQ-Cu-5Nu) exerted antimicrobial activities against Gram-negative bacteria including P. shigelloides and S. dysenteriae. The results reveal application of 8AQ and its metal complexes as potential compounds to be further developed as novel antimalarial and antibacterial agents. PMID:27103894

  1. Cellular Plasticity Induced by Anti–α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid (AMPA) Receptor Encephalitis Antibodies

    PubMed Central

    Peng, Xiaoyu; Hughes, Ethan G; Moscato, Emilia H; Parsons, Thomas D; Dalmau, Josep; Balice-Gordon, Rita J

    2015-01-01

    Objective Autoimmune-mediated anti–α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis is a severe but treatment-responsive disorder with prominent short-term memory loss and seizures. The mechanisms by which patient antibodies affect synapses and neurons leading to symptoms are poorly understood. Methods The effects of patient antibodies on cultures of live rat hippocampal neurons were determined with immunostaining, Western blot, and electrophysiological analyses. Results We show that patient antibodies cause a selective decrease in the total surface amount and synaptic localization of GluA1- and GluA2-containing AMPARs, regardless of receptor subunit binding specificity, through increased internalization and degradation of surface AMPAR clusters. In contrast, patient antibodies do not alter the density of excitatory synapses, N-methyl-D-aspartate receptor (NMDAR) clusters, or cell viability. Commercially available AMPAR antibodies directed against extracellular epitopes do not result in a loss of surface and synaptic receptor clusters, suggesting specific effects of patient antibodies. Whole-cell patch clamp recordings of spontaneous miniature postsynaptic currents show that patient antibodies decrease AMPAR-mediated currents, but not NMDAR-mediated currents. Interestingly, several functional properties of neurons are also altered: inhibitory synaptic currents and vesicular γ-aminobutyric acid transporter (vGAT) staining intensity decrease, whereas the intrinsic excitability of neurons and short-interval firing increase. Interpretation These results establish that antibodies from patients with anti-AMPAR encephalitis selectively eliminate surface and synaptic AMPARs, resulting in a homeostatic decrease in inhibitory synaptic transmission and increased intrinsic excitability, which may contribute to the memory deficits and epilepsy that are prominent in patients with this disorder. PMID:25369168

  2. 5-Methyl Salicylic Acid-Induced Thermo Responsive Reversible Transition in Surface Active Ionic Liquid Assemblies: A Spectroscopic Approach.

    PubMed

    Roy, Arpita; Dutta, Rupam; Banerjee, Pavel; Kundu, Sangita; Sarkar, Nilmoni

    2016-07-19

    This article describes the formation of stable unilamellar vesicles involving surface active ionic liquid (SAIL), 1-hexadecyl-3-methylimidazolium chloride (C16mimCl), and 5-methyl salicylic acid (5mS). Turbidity, dynamic light scattering (DLS), transmission electron microscopy (TEM), and viscosity measurements suggest that C16mimCl containing micellar aggregates are transformed to elongated micelle and finally into vesicular aggregates with the addition of 5mS. Besides, we have also investigated the photophysical aspects of a hydrophobic (coumarin 153, C153) and a hydrophilic molecule (rhodamine 6G (R6G) perchlorate) during 5mS-induced micelle to vesicle transition. The rotational motion of C153 becomes slower, whereas faster motion is observed for R6G during micelle to vesicle transition. Moreover, the fluorescence correlation spectroscopy (FCS) measurements suggest that the translational diffusion of hydrophobic probe becomes slower in C16mimCl-5mS aggregates in comparison to C16mimCl micelle. However, a reverse trend in translational diffusion motion of hydrophilic molecule has been observed in C16mimCl-5mS aggregates. Moreover, we have also found that the C16mimCl-5mS containing vesicles are transformed into micelles upon enhanced temperature, and it is further confirmed by turbidity, DLS measurements that this transition is a reversible one. Finally, temperature-induced rotational motion of C153 and R6G has been monitored in C16mimCl-5mS aggregates to get a complete scenario regarding the temperature-induced vesicle to micelle transition. PMID:27345738

  3. Photodissociation dynamics of the iodide-uracil (I-U) complex

    NASA Astrophysics Data System (ADS)

    Li, Wei-Li; Kunin, Alice; Matthews, Edward; Yoshikawa, Naruo; Dessent, Caroline E. H.; Neumark, Daniel M.

    2016-07-01

    Photofragment action spectroscopy and femtosecond time-resolved photoelectron imaging are utilized to probe the dissociation channels in iodide-uracil (I- ṡ U) binary clusters upon photoexcitation. The photofragment action spectra show strong I- and weak [U—H]- ion signal upon photoexcitation. The action spectra show two bands for I- and [U—H]- production peaking around 4.0 and 4.8 eV. Time-resolved experiments measured the rate of I- production resulting from excitation of the two bands. At 4.03 eV and 4.72 eV, the photoelectron signal from I- exhibits rise times of 86 ± 7 ps and 36 ± 3 ps, respectively. Electronic structure calculations indicate that the lower energy band, which encompasses the vertical detachment energy (4.11 eV) of I-U, corresponds to excitation of a dipole-bound state of the complex, while the higher energy band is primarily a π-π∗ excitation on the uracil moiety. Although the nature of the two excited states is very different, the long lifetimes for I- production suggest that this channel results from internal conversion to the I- ṡ U ground state followed by evaporation of I-. This hypothesis was tested by comparing the dissociation rates to Rice-Ramsperger-Kassel-Marcus calculations.

  4. Classical treatment of the electron emission from collisions of uracil molecules with fast protons

    NASA Astrophysics Data System (ADS)

    Sarkadi, L.

    2015-12-01

    The electron emission from the uracil molecule induced by fast proton impact has been investigated using the classical-trajectory Monte Carlo (CTMC) method. Applying the independent-particle model, the full three-body dynamics of the projectile, an active electron, and the molecule core is considered. The interactions with the molecule core are described by a multicenter potential built from screened atomic potentials. Double and single differential, as well as total ionization cross sections are calculated and compared with the predictions of the first Born approximation with correct boundary conditions (CB1), the continuum-distorted-wave-eikonal-initial-state (CDW-EIS) approach, as well as the combined classical-trajectory Monte Carlo-classical over-the-barrier (CTMC-COB) model. The effect of the molecular treatment of the ionization by the multicenter potential is analyzed by simplified CTMC calculations in which the ionization cross section of the uracil is determined as a linear combination of the contributions of the constituent atoms of the molecule.

  5. Investigation of aromatase inhibitory activity of metal complexes of 8-hydroxyquinoline and uracil derivatives

    PubMed Central

    Prachayasittikul, Veda; Pingaew, Ratchanok; Nantasenamat, Chanin; Prachayasittikul, Supaluk; Ruchirawat, Somsak; Prachayasittikul, Virapong

    2014-01-01

    Purpose Estrogens play important roles in the pathogenesis and progression of breast cancer as well as estrogen-related diseases. Aromatase is a key enzyme in the rate-limiting step of estrogen production, in which its inhibition is one strategy for controlling estrogen levels to improve prognosis of estrogen-related cancers and diseases. Herein, a series of metal (Mn, Cu, and Ni) complexes of 8-hydroxyquinoline (8HQ) and uracil derivatives (4–9) were investigated for their aromatase inhibitory and cytotoxic activities. Methods The aromatase inhibition assay was performed according to a Gentest™ kit using CYP19 enzyme, wherein ketoconazole and letrozole were used as reference drugs. The cytotoxicity was tested on normal embryonic lung cells (MRC-5) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results Only Cu complexes (6 and 9) exhibited aromatase inhibitory effect with IC50 0.30 and 1.7 μM, respectively. Cytotoxicity test against MRC-5 cells showed that Mn and Cu complexes (5 and 6), as well as free ligand 8HQ, exhibited activity with IC50 range 0.74–6.27 μM. Conclusion Cu complexes (6 and 9) were found to act as a novel class of aromatase inhibitor. Our findings suggest that these 8HQ–Cu–uracil complexes are promising agents that could be potentially developed as a selective anticancer agent for breast cancer and other estrogen-related diseases. PMID:25152615

  6. 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. PMID:27012237

  7. Suppressed catalytic activity of base excision repair enzymes on rotationally positioned uracil in nucleosomes.

    PubMed

    Beard, Brian C; Wilson, Samuel H; Smerdon, Michael J

    2003-06-24

    The majority of DNA in eukaryotic cells exists in the highly condensed structural hierarchy of chromatin, which presents a challenge to DNA repair enzymes in that recognition, incision, and restoration of the original sequence at most sites must take place within these structural constraints. To test base excision repair (BER) activities on chromatin substrates, an in vitro system was developed that uses human uracil DNA glycosylase (UDG), apyrimidinic/apurinic endonuclease (APE), and DNA polymerase beta (pol beta) on homogeneously damaged, rotationally positioned DNA in nucleosomes. We find that UDG and APE carry out their combined catalytic activities with reduced efficiency on nucleosome substrates ( approximately 10% of that on naked DNA). Furthermore, these enzymes distinguish between two different rotational settings of the lesion on the histone surface, showing a 2- to 3-fold difference in activity between uracil facing "toward" and "away from" the histones. However, UDG and APE will digest such substrates to completion in a concentration-dependent manner. Conversely, the synthesis activity of pol beta is inhibited completely by nucleosome substrates and is independent of enzyme concentration. These results suggest that the first two steps of BER, UDG and APE, may occur "unassisted" in chromatin, whereas downstream factors in this pathway (i.e., pol beta) may require nucleosome remodeling for efficient DNA BER in at least some regions of chromatin in eukaryotic cells.

  8. Photoinduced electron transfer in a Watson-Crick base-paired, 2-aminopurine:uracil-C60 hydrogen bonding conjugate.

    PubMed

    D'Souza, Francis; Gadde, Suresh; Islam, D-M Shafiqul; Pang, Siew-Cheng; Schumacher, Amy Lea; Zandler, Melvin E; Horie, Rumiko; Araki, Yasuyaki; Ito, Osamu

    2007-02-01

    A fluorescent reporter molecule, 2-aminopurine was self-assembled via Watson-Crick base-pairing to a uracil appended fullerene to form a donor-acceptor conjugate; efficient photoinduced charge separation was confirmed by time-resolved emission and transient absorption spectral studies.

  9. Replacement of Lys by Glu in a transmembrane segment strongly impairs the function of the uracil permease from Saccharomyces cerevisiae.

    PubMed Central

    Urban-Grimal, D; Pinson, B; Chevallier, J; Haguenauer-Tsapis, R

    1995-01-01

    The co-transport of uracil and protons through the plasma membrane of the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. The uracil permease is a multi-spanning membrane protein that follows the secretory pathway to the plasma membrane. Recent experimental data led to the proposal of a two-dimensional model of its topology. A spontaneous mutant corresponding to the substitution of Lys-272 by glutamic acid was obtained. The influence of this mutation was studied by comparing the wild-type and mutant permeases produced in a strain carrying a chromosomal deletion of the FUR4 gene. The mutant permease is correctly targeted to the plasma membrane and its stability is similar to that of the wild-type permease. The uptake parameters for the mutant permease were impaired and showed an approximately 65-fold increase of apparent K(m) and a decrease in apparent Vmax. Equilibrium binding measurements with enriched plasma membrane preparations showed an approximately 70-fold increase in apparent Kd in the mutant, whereas its Bmax. was similar to that of the wild type. Lys-272 is fully conserved in the uracil permease family and is predicted to lie in the fourth transmembrane segment of the protein. It seems to be essential for both efficient uracil binding and translocation. Images Figure 2 PMID:8948441

  10. 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.

  11. Is uracil aromatic? The enthalpies of hydrogenation in the gaseous and crystalline phases, and in aqueous solution, as tools to obtain an answer.

    PubMed

    Galvão, Tiago L P; Rocha, Inês M; da Silva, Maria D M C Ribeiro; da Silva, Manuel A V Ribeiro

    2013-07-18

    The enthalpy of hydrogenation of uracil was derived from the experimental enthalpies of formation, in the gaseous phase, of uracil and 5,6-dihydrouracil, in order to analyze its aromaticity. The enthalpy of formation of 5,6-dihydrouracil was obtained from combustion calorimetry, Knudsen effusion technique and Calvet microcalorimetry results. High-level computational methods were tested for the enthalpy of hydrogenation of uracil, but only with G3 was possible to obtain results in agreement with the experimental ones. It was found that uracil possesses 30.0% of aromatic character in the gaseous phase. Using both implicit, explicit, and hybrid solvation methods, it was possible to obtain a reference value for the enthalpy of hydrogenation of uracil in the aqueous solution and the effect of polarity and hydrogen bonds on the aromaticity of uracil was analyzed. The value of the hydrogenation enthalpy of uracil in aqueous solution was compared with the experimental value in the crystal phase, also dominated by polarity and hydrogen bonds, derived from combustion calorimetry results. The supramolecular effects on the crystal lattice were explored by the computational simulation of π-π staking dimers and hydrogen bonded dimers.

  12. Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4.

    PubMed

    Nishino, Kohei; Kushima, Misaki; Matsuo, Yuzy; Matsuo, Yasuhiro; Kawamukai, Makoto

    2015-01-01

    Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.

  13. 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.

  14. Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4

    PubMed Central

    Nishino, Kohei; Kushima, Misaki; Matsuo, Yuzy; Matsuo, Yasuhiro; Kawamukai, Makoto

    2015-01-01

    Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background. PMID:26536126

  15. Characterization of GM-CSF-inhibitory factor and Uracil DNA glycosylase encoding genes from camel pseudocowpoxvirus.

    PubMed

    Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Narnaware, S D; Mehta, S C; Singh, P K; Singh, Raghvendar; Tuteja, F C; Patil, N V

    2015-06-01

    The present study describes the PCR amplification of GM-CSF-inhibitory factor (GIF) and Uracil DNA glycosylase (UDG) encoding genes of pseudocowpoxvirus (PCPV) from the Indian Dromedaries (Camelus dromedarius) infected with contagious ecthyma using the primers based on the corresponding gene sequences of human PCPV and reindeer PCPV, respectively. The length of GIF gene of PCPV obtained from camel is 795 bp and due to the addition of one cytosine residue at position 374 and one adenine residue at position 516, the open reading frame (ORF) got altered, resulting in the production of truncated polypeptide. The ORF of UDG encoding gene of camel PCPV is 696 bp encoding a polypeptide of 26.0 kDa. Comparison of amino acid sequence homologies of GIF and UDG of camel PCPV revealed that the camel PCPV is closer to ORFV and PCPV (reference stains of both human and reindeer), respectively. PMID:25816930

  16. 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.

  17. Momentum Imaging of the Dynamics of Dissociative Electron Attachment to Uracil

    NASA Astrophysics Data System (ADS)

    Slaughter, Dan; Kawarai, Yu; Weber, Thorsten; Azuma, Yoshiro; Winstead, Carl; McKoy, Vince; Belkacem, Ali

    2013-05-01

    Observation of the dynamics of dissociative electron attachment (DEA) in biomolecules has recently become possible by momentum imaging of the fragments resulting from the dissociating transient anion resonance. A momentum spectrometer featuring a 4 π solid angle of detection is combined with a pulsed electron beam and effusive molecular beam in a crossed geometry to measure the full 3D momentum distribution of dissociating negative ions. Guided by electronic structure calculations that indicate the most likely orientation of the molecule at the time of attachment, we present key aspects of the dynamics of ring-breaking dissociation of the transient anion formed upon DEA to the nucleobase uracil. Performed under the auspices of the US DOE by LBNL under Contract DE-AC02-05CH11231.

  18. Sites of Adsorption of Adenine, Uracil, and Their Corresponding Derivatives on Sodium Montmorillonite

    NASA Astrophysics Data System (ADS)

    Perezgasga, L.; Serrato-Díaz, A.; Negrón-Mendoza, A.; Gal'N, L. De Pablo; Mosqueira, F. G.

    2005-04-01

    Clay minerals are considered important to chemical evolution processes due to their properties, ancient origin, and wide distribution. To extend the knowledge of their role in the prebiotic epoch, the adsorption sites of adenine, adenosine, AMP, ADP, ATP, Poly A, uracil, uridine, UMP, UDP, UTP and Poly U on sodium montmorillonite are investigated. X-ray diffraction, ultraviolet and infrared spectroscopy studies indicate that these molecules distribute into the interlamellar channel and the edge of the clay crystals. Monomers are adsorbed predominantly in the interlamellar channel, whereas polymers adsorb along the crystal edges. Such behavior is discussed mainly in terms of bulk pH, pKa of the adsorbate, and Van der Waals interactions.

  19. Cloning and characterization of uracil-DNA glycosylase and the biological consequences of the loss of its function in the nematode Caenorhabditis elegans.

    PubMed

    Nakamura, Nobuya; Morinaga, Hironobu; Kikuchi, Masahiro; Yonekura, Shin-Ichiro; Ishii, Naoaki; Yamamoto, Kazuo; Yonei, Shuji; Zhang, Qiu-Mei

    2008-09-01

    Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO(3)-inducing cytosine deamination than wild-type strain.

  20. Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

    PubMed Central

    Ishino, Sonoko; Connolly, Bernard A.

    2016-01-01

    In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil); Endonuclease V (EndoV, which recognises hypoxanthine); and Endonuclease Q (EndoQ), (which recognises both uracil and hypoxanthine). Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations. PMID:27721668

  1. 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.

  2. Label-free fluorescence turn-on detection of uracil DNA glycosylase activity based on G-quadruplex formation.

    PubMed

    Ma, Changbei; Wu, Kefeng; Liu, Haisheng; Xia, Kun; Wang, Kemin; Wang, Jun

    2016-11-01

    We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research. PMID:27591637

  3. Potential derived point charge model study of electrostatic interaction energies in some complexes of water with uracil, thymine, and cytosine.

    PubMed

    Ray, N K; Bolis, G; Shibata, M; Rein, R

    1984-01-01

    Potential derived (PD) point charges and segmental multipole moments are calculated for water, uracil, thymine, and cytosine using STO-3G quality wave functions. The PD point charges are used to estimate the electrostatic interaction energies for a series of complexes of water with these nucleic acid bases. It is shown here that the results obtained using simple PD charge model is very similar to those obtained from more elaborate segmental multipole moment analysis.

  4. 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.

  5. Multi-photon ionization and fragmentation of uracil: Neutral excited-state ring opening and hydration effects

    SciTech Connect

    Barc, B.; Ryszka, M.; Spurrell, J.; Dampc, M.; Limão-Vieira, P.; Parajuli, R.; Mason, N. J.; Eden, S.

    2013-12-28

    Multi-photon ionization (MPI) of the RNA base uracil has been studied in the wavelength range 220–270 nm, coinciding with excitation to the S{sub 2}(ππ*) state. A fragment ion at m/z = 84 was produced by 2-photon absorption at wavelengths ≤232 nm and assigned to C{sub 3}H{sub 4}N{sub 2}O{sup +} following CO abstraction. This ion has not been observed in alternative dissociative ionization processes (notably electron impact) and its threshold is close to recent calculations of the minimum activation energy for a ring opening conical intersection to a σ(n-π)π* closed shell state. Moreover, the predicted ring opening transition leaves a CO group at one end of the isomer, apparently vulnerable to abstraction. An MPI mass spectrum of uracil-water clusters is presented for the first time and compared with an equivalent dry measurement. Hydration enhances certain fragment ion pathways (particularly C{sub 3}H{sub 3}NO{sup +}) but represses C{sub 3}H{sub 4}N{sub 2}O{sup +} production. This indicates that hydrogen bonding to water stabilizes uracil with respect to neutral excited-state ring opening.

  6. BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability

    PubMed Central

    Slupianek, Artur; Falinski, Rafal; Znojek, Pawel; Stoklosa, Tomasz; Flis, Sylwia; Doneddu, Valentina; Pytel, Dariusz; Synowiec, Ewelina; Blasiak, Janusz; Bellacosa, Alfonso; Skorski, Tomasz

    2013-01-01

    Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of CML-CP. Unfortunately, 25% of TKI-naive patients and 50–90% of TKI-responding patients carry CML clones expressing TKI resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species (ROS), which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil-DNA glycosylase UNG2 were inhibited in BCR-ABL1 –transformed cell lines and CD34+ CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na+/K+ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI-resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML. PMID:23047475

  7. Multi-photon ionization and fragmentation of uracil: Neutral excited-state ring opening and hydration effects

    NASA Astrophysics Data System (ADS)

    Barc, B.; Ryszka, M.; Spurrell, J.; Dampc, M.; Limão-Vieira, P.; Parajuli, R.; Mason, N. J.; Eden, S.

    2013-12-01

    Multi-photon ionization (MPI) of the RNA base uracil has been studied in the wavelength range 220-270 nm, coinciding with excitation to the S2(ππ*) state. A fragment ion at m/z = 84 was produced by 2-photon absorption at wavelengths ≤232 nm and assigned to C3H4N2O+ following CO abstraction. This ion has not been observed in alternative dissociative ionization processes (notably electron impact) and its threshold is close to recent calculations of the minimum activation energy for a ring opening conical intersection to a σ(n-π)π* closed shell state. Moreover, the predicted ring opening transition leaves a CO group at one end of the isomer, apparently vulnerable to abstraction. An MPI mass spectrum of uracil-water clusters is presented for the first time and compared with an equivalent dry measurement. Hydration enhances certain fragment ion pathways (particularly C3H3NO+) but represses C3H4N2O+ production. This indicates that hydrogen bonding to water stabilizes uracil with respect to neutral excited-state ring opening.

  8. Formation of uracil from the ultraviolet photo-irradiation of pyrimidine in pure H2O ices.

    PubMed

    Nuevo, Michel; Milam, Stefanie N; Sandford, Scott A; Elsila, Jamie E; Dworkin, Jason P

    2009-09-01

    The detection of nucleobases in carbonaceous chondrites such as Murchison supports the scenario in which extraterrestrial organic molecules could have contributed to the origin of life on Earth. However, such large molecules have not been observed to date in astrophysical environments, in particular, comets and the interstellar medium (ISM). The physico-chemical conditions under which nucleobases and, more generally, N-heterocycles were formed are unknown, as are their mechanisms of formation. In this work, H2O:pyrimidine ice mixtures were irradiated with UV photons under interstellar/cometary-relevant conditions to study the formation of pyrimidine derivatives, including the nucleobase uracil. Liquid and gas chromatography analyses of the samples produced in our experiments revealed the presence of numerous photoproducts among which 4(3H)-pyrimidone and uracil could be conclusively identified. The photostability of pyrimidine against UV photons was also studied, and we showed that it would survive from the ISM to the solar nebula if formed and preserved in ice mantles on the surface of cold grains. We propose pathways for the formation of 4(3H)-pyrimidone and uracil under astrophysically relevant conditions and discuss the possibility for such molecules to survive from the ISM to their delivery to Earth and other Solar System bodies. PMID:19778279

  9. Properties and Applications of Sodium (5-methyl-2-alkyl-1,3-dioxane-5-yl)-Carboxylate Synthesized with Nanosolid Superacid.

    PubMed

    Yuan, Lin; Jia, Guo Kai; Li, Zhong Yan; Zhang, Min; Yuan, Xian You

    2016-01-01

    A series of novel sodium (5-methyl-2-alkyl-1,3-dioxane-5-yl) carboxylate surfactants were synthesized using nanosolid superacid SO₄²⁻/Fe₂O₃as a catalyst and characterized by ¹H NMR, IR and elemental analysis. The critical micelle concentration (CMC) of surfactants was determined and the results showed that the CMC values were less than 2.0 x 10⁻³ mol/L. Other relevant surface properties (Krafft point, emulsion stability, foam ability, degradability) were also evaluated. It was suggested that with respect to emulsion formation, foam stability and the range of application temperature, compared with traditional surfactants, the new surfactants could give better results and showed better properties when used as an emulsifier in emulsion polymerization. In addition, the surfactants were stable under neutral and alkaline conditions, and could form solid under acid condition. The solid will generate the original surfactants for reuse with alkali. Sodium (5-methyl-2-alkyl-1,3-dioxane-5-yl) carboxylate is likely to be a new type of 'environmentally friendly' surfactant. PMID:27398572

  10. Synthesis and anti-HIV-1 activity of a series of 1-alkoxy-5-alkyl-6-(arylthio)uracils.

    PubMed

    Kim, D K; Gam, J; Kim, Y W; Lim, J; Kim, H T; Kim, K H

    1997-07-18

    A series of 1-alkoxy-5-alkyl-6-(arylthio)uracils was synthesized and tested for their ability to inhibit HIV-1 replication. Treatment of 2-alkyl-3,3-bis(methylthio)acryloyl chlorides (5a-e) with AgOCN in benzene followed by reaction of the resulting isocyanates 6a-e with an appropriate alkoxyamine gave N-alkoxy-N'-((2-alkyl-3,3-bis(methylthio)acryloyl)ureas (10a-z) in good to excellent yields. Cyclization of 10a-z in AcOH containing a catalytic amount of p-TsOH produced 1-alkoxy-5-alkyl-6-(methylthio)uracils (11a-z). Oxidation of 11a-z with 3-chloroperoxybenzoic acid in CH2Cl2 resulted in high yields of 1-alkoxy-5-alkyl-6-(methylsulfonyl)uracils (12a-x and 12z) and 1-(benzyloxy)-6-(methylsulfinyl)thymine (12y), which were subsequently reacted with an appropriate arenethiol in ethanolic NaOH solution to afford 1-alkoxy-5-alkyl-6-(arylthio)uracils (14-49). Substitution at the 3- and 5-positions of the C-6-(phenylthio) ring by two methyl groups significantly increased its original anti-HIV-1 activity (EC50: 6-((3,5-dimethylphenyl)thio)-5-isopropyl-1-propoxyuracil (18), 0.064 microM; 6-((3,5-dimethylphenyl)thio)-1-(3-hydroxypropoxy)-5-isopropyluracil++ + (23), 0.19 microM). Among the various alkoxy substituents at the N-1, the propoxy group was the most beneficial for improving the anti-HIV-1 activity. The 1-propoxy derivative 18 proved to be the most potent inhibitor of HIV-1 replication, followed by the 1-(3-hydroxypropoxy) derivative 23. Introduction of an isopropyl group at C-5 of the uracil base also remarkably enhanced the activity. When compound 18 was incubated with a rat liver homogenate preparation, no metabolite was observed, thus confirming the metabolic stability of the N-O bond in these 1-alkoxyuracils.

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

    PubMed

    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

  12. INHIBITION OF A THYMINE-DEFICIENT MUTANT OF ESCHERICHIA COLI BY 5-SUBSTITUTED URACILS

    PubMed Central

    Shapira, Jacob; Lowden, Lois; Hale, Ralph

    1962-01-01

    Shapira, Jacob (Consolidated Veterans Administration Hospital, Little Rock, Ark.), Lois Lowden, and Ralph Hale. Inhibition of a thymine-deficient mutant of Escherichia coli by 5-substituted uracils. J. Bacteriol. 83:919–923. 1962.—Small inocula of well-washed cells of a thymine-requiring mutant of Escherichia coli were incubated in a thymine-containing glucose-salts medium with a variety of 5-substituted pyrimidines and pyrimidine ribosides. After a lag phase, the turbidity of the cultures increased appreciably which, in the case of 5-ethyluracil and 5-ethyluridine, was primarily due to an elongation of the cells. 5-Ethyluracil at low thymine concentrations increased the lag phase and decreased the rate and final amount of growth. At high thymine concentrations, it had less effect on the final turbidity of the cultures. The inhibition index for this compound was relatively constant, suggesting competitive inhibition. Several other pyrimidine analogues inhibited growth. The nucleosides of 5-bromouracil and 5-aminouracil were no more effective than the free bases. The ribosides of 5-ethyluracil and 5-butyluracil were appreciably more inhibitory than the free bases and were the most potent compounds tested. It is likely that the inhibition of growth is a reflection of the effect of these compounds on ribonucleic acid synthesis by the cells. PMID:13911280

  13. Effects of the ionization in the tautomerism of uracil: A reaction electronic flux perspective.

    PubMed

    Lamsabhi, Al Mokhtar; Gutiérrez-Oliva, Soledad; Mó, Otilia; Toro-Labbé, Alejandro; Yáñez, Manuel

    2015-10-30

    The one-step tautomerization processes of uracil and its radical cation and radical anion have been investigated in the light of the reaction force and reaction electronic flux (REF) formalisms. The relative energies of the different tautomers as well as the corresponding tautomerization barriers have been obtained through the use of the G4 high-level ab initio method and by means of B3LYP/6-311+G(3df,2p)//B3LYP/6-311+G(d, p) calculations. Systematically, the enol radical cations are more stable in relative terms than the neutral, due to the higher ionization energy of the diketo forms with respect to the enolic ones. Conversely, the enol radical anions, with the only exception of the 2-keto-N1 anion, are found to be less stable than the neutral. The effects of the ionization are also sizable on the tautomerization barriers although this effect also depends on the particular tautomerization process. The reaction force analysis shows that all reactions are mainly activated through structural rearrangements that initiate the electronic activity. This electronic activity is monitored along the reaction coordinate through the REF that obeys a delicate balance between the acid and basic character of the atoms involved in the hydrogen transfer. PMID:26285048

  14. Further evidence for involvement of a noncanonical function of uracil DNA glycosylase in class switch recombination.

    PubMed

    Begum, Nasim A; Stanlie, Andre; Doi, Tomomitsu; Sasaki, Yoko; Jin, Hai Wei; Kim, Yong Sung; Nagaoka, Hitoshi; Honjo, Tasuku

    2009-02-24

    Activation-induced cytidine deaminase (AID) introduces DNA cleavage in the Ig gene locus to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. The DNA deamination model assumes that AID deaminates cytidine (C) on DNA and generates uridine (U), resulting in DNA cleavage after removal of U by uracil DNA glycosylase (UNG). Although UNG deficiency reduces CSR efficiency to one tenth, we reported that catalytically inactive mutants of UNG were fully proficient in CSR and that several mutants at noncatalytic sites lost CSR activity, indicating that enzymatic activity of UNG is not required for CSR. In this report we show that CSR activity by many UNG mutants critically depends on its N-terminal domain, irrespective of their enzymatic activities. Dissociation of the catalytic and CSR activity was also found in another UNG family member, SMUG1, and its mutants. We also show that Ugi, a specific peptide inhibitor of UNG, inhibits CSR without reducing DNA cleavage of the S (switch) region, confirming dispensability of UNG in DNA cleavage in CSR. It is therefore likely that UNG is involved in a repair step after DNA cleavage in CSR. Furthermore, requirement of the N terminus but not enzymatic activity of UNG mutants for CSR indicates that the UNG protein structure is critical. The present findings support our earlier proposal that CSR depends on a noncanonical function of the UNG protein (e.g., as a scaffold for repair enzymes) that might be required for the recombination reaction after DNA cleavage.

  15. 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

  16. Relationship of in vivo antitumor activities of fluorinated pyrimidines to thymidylate synthase activity and intratumoral concentrations of 5-fluorouracil and uracil.

    PubMed

    Nio, Y; Shiraishi, T; Tsubono, M; Morimoto, H; Tseng, C C; Kawabata, K; Imai, S; Tobe, T

    1991-01-01

    MOPC-104E plasmacytomas were subcutaneously transplanted into BALB/c mice and after 7 days the mice were administered different fluorinated pyrimidines at 4 times the clinical doses, 5-fluorouracil (5-FU, 15 mg/kg), tegafur (FT, 100 mg/kg) or UFT (FT, 20 mg/kg + uracil, 44.8 mg/kg) daily for 7 days. Tumor growth was most effectively inhibited in the UFT group. The % inhibition of tumor growth on day 14, while not correlating with the concentration of 5-FU in the tumor, negatively correlated with the concentration of uracil, which was lowest in the UFT group. The activity of thymidylate synthase (TS) was measured using a 5-fluoro-deoxyuridine monophosphate (FdUMP) binding assay. The total and free TS activities in the tumor negatively correlated with the % inhibition of tumor growth, and were lowest in the UFT group. However, the % inhibition of TS activity in the tumor, which was about 80% in all 3 groups, did not correlate with the tumor-inhibitory effect. These results suggest that uracil in the tumor may play an important role in the metabolism of fluorinated pyrimidines, and that exogeneously administered uracil may decrease the amounts of uracil and TS in the tumor, and subsequently cause 5-FU accumulation.

  17. Altered trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) in the striatum leads to behavioral changes in emotional responses.

    PubMed

    Lee, Young; Lee, Hojin; Kim, Hyung-Wook; Yoon, Bong-June

    2015-01-01

    The striatum receives and integrates multiple inputs from diverse areas in the brain and plays a critical role in the regulation of motor activity. However, whether the striatum is involved in the alteration of behavior in the presence of emotional challenges is unknown. Here, we examined whether alterations in the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) in the dorsal striatum would affect anxiety-related behaviors. We found that the transient expression of G1CT or G2CT, AMPAR-derived peptides, in the dorsomedial striatum led to decreased mobility in high-anxiety circumstances; however, the expression of these peptides in the dorsolateral striatum did not affect anxiety-related behavior. These data suggest that excitatory connections within the dorsomedial striatum play important roles in the control of motor actions in the presence of emotional challenges.

  18. 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.

  19. 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.

  20. Synthesis and herbicidal activity evaluation of novel α-amino phosphonate derivatives containing a uracil moiety.

    PubMed

    Che, Jian-yi; Xu, Xiao-yun; Tang, Zi-long; Gu, Yu-cheng; Shi, De-qing

    2016-02-15

    A series of novel α-amino phosphonate derivatives containing a uracil moiety 3a-3l were designed and synthesized by a Lewis acid (magnesium perchlorate) catalyzed the Kabachnik-Fields reaction. The bioassays {in vitro, in vivo [Glass House 1 (GH1) and Glass House 2 (GH2)]} showed that most of compounds 3 exhibited excellent and selective herbicidal activities; for example, in GH1 test, compounds 3b, 3d, 3f, 3h and 3j showed excellent and wide spectrum herbicidal activities at the dose of 1000 g/ha, and compounds 3b and 3j exhibited 100% inhibition activities against the four plants in both post- and pre-emergence treatments. Moreover, most of compounds 3 showed higher inhibition against Amaranthus retroflexus and Digitaria sanguinalis than Glyphosate did in pre-emergence treatment. In GH2 test, the four compounds (3b, 3d, 3h and 3j) exhibited 100% inhibition against Solanum nigrum, Amaranthus retroflexus and Ipomoea hederacea in post-emergence treatment and displayed 100% inhibition against Solanum nigrum, Amaranthus retroflexus in pre-emergence treatment at the rate of 250 g/ha, and compound 3b showed the best and broad spectrum herbicidal activities against the six test plants. However, the four compounds displayed weaker herbicidal activities against Lolium perenne and Echinochloa crus-galli than the other four plants at the rate of 250 g/ha in both pre- and post-emergence treatments. So, compounds 3 can be used as a lead compound for further structure optimization for developing potential selective herbicidal agent. Their preliminary structure-activity relationships were also investigated. PMID:26786699

  1. Cyclophosphamide augments the anti-tumor efficacy of uracil and tegafur by inhibiting dihydropyrimidine dehydrogenase.

    PubMed

    Nio, Yoshinori; Iguchi, Chikage; Kodama, Hiroshi; Itakura, Masayuki; Hashimoto, Koji; Koike, Makoto; Toga, Tomoko; Maruyama, Riruke; Fukushima, Masakazu

    2007-01-01

    The present study assesses the effects of neo-adjuvant chemotherapy (NAC) with uracil and tegafur (UFT) alone vs UFT plus cyclophosphamide (CPA), on the activity of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) in breast cancer tissues. Breast cancer patients were randomly assigned to 3 groups; the control (no-treatment) group (n=13), the UFT (5-8 mg/kg/day) alone group (n=10) and the UFT plus CPA (1 mg/kg/one day interval) (UC) group (n=9), and they received NAC for 2-4 weeks. A total of 32 invasive ductal breast carcinomas were used to assay for TS and DPD activity. There were no statistically significant differences in tumor size or stage classification between the 3 groups. The DPD activity was inversely and significantly correlated with the tumor size and pT, but the TS activity was not correlated with these clinicopathological factors. The TS activity was decreased by NAC with UFT, and the addition of CPA resulted in an increased inhibition of TS activity. In contrast, DPD activity was increased by NAC with UFT administration, but its increased activity was significantly inhibited by the addition of CPA. Multiple regression analyses demonstrated that the total dose of UFT was a significant variable for inhibiting TS activity, and that CPA was a significant variable for inhibiting DPD activity. The DPD activity increased by UFT can be inhibited by CPA, and this may represent one of the possible mechanisms responsible for the anti-tumor activity of 5-FU or its derivatives as enhanced by CPA.

  2. BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability.

    PubMed

    Slupianek, A; Falinski, R; Znojek, P; Stoklosa, T; Flis, S; Doneddu, V; Pytel, D; Synowiec, E; Blasiak, J; Bellacosa, A; Skorski, T

    2013-03-01

    Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

  3. SEIRA studies of uracil adsorbed on wet-chemically prepared gold nanoparticles film on glass substrate - Effect of morphology of film

    NASA Astrophysics Data System (ADS)

    Kumar, Naveen; Thomas, S.; Tokas, R. B.; Kshirsagar, R. J.

    2014-08-01

    Surface-enhanced infrared absorption (SEIRA) studies of uracil adsorbed on wet-chemically prepared gold nanoparticles (AuNp) immobilized on silanised glass substrate were carried out using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The deposition time dependent evolution of morphological changes in AuNp films and its influence on the SEIRA spectra of uracil were investigated. The morphological changes were examined by atomic force microscopy (AFM). The spectrum of uracil adsorbed on AuNp film obtained with ½ an hour deposition time showed a clear enhancement than 2 and 4 h deposition times. The small shift seen in SEIRA spectra indicates weak interaction of the molecules with AuNp film.

  4. Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil- DNA glycosylase.

    PubMed

    Do, Hongdo; Dobrovic, Alexander

    2012-05-01

    Non-reproducible sequence artefacts are frequently detected in DNA from formalinfixed and paraffin-embedded (FFPE) tissues. However, no rational strategy has been developed for reduction of sequence artefacts from FFPE DNA as the underlying causes of the artefacts are poorly understood. As cytosine deamination to uracil is a common form of DNA damage in ancient DNA, we set out to examine whether treatment of FFPE DNA with uracil-DNA glycosylase (UDG) would lead to the reduction of C>T (and G>A) sequence artefacts. Heteroduplex formation in high resolution melting (HRM)-based assays was used for the detection of sequence variants in FFPE DNA samples. A set of samples that gave false positive HRM results for screening for the E17K mutation in exon 4 of the AKT1 gene were chosen for analysis. Sequencing of these samples showed multiple non-reproducible C:G>T:A artefacts. Treatment of the FFPE DNA with UDG prior to PCR amplification led to a very marked reduction of the sequence artefacts as indicated by both HRM and sequencing analysis, indicating that uracil lesions are the major cause of sequence artefacts. Similar results were shown for the BRAF V600 region in the same sample set and EGFR exon 19 in another sample set. UDG treatment specifically suppressed the formation of artefacts in FFPE DNA as it did not affect the detection of true KRAS codon 12 and true EGFR exon 19 and 20 mutations. We conclude that uracil in FFPE DNA leads to a significant proportion of sequence artefacts. These can be minimised by a simple UDG pretreatment which can be readily carried out, in the same tube, as the PCR immediately prior to commencing thermal cycling. HRM is a convenient way of monitoring both the degree of damage and the effectiveness of the UDG treatment. These findings have immediate and important implications for cancer diagnostics where FFPE DNA is used as the primary genetic material for mutational studies guiding personalised medicine strategies and where simple

  5. Multicomponent Synthesis of Uracil Analogues Promoted by Pd-Catalyzed Carbonylation of α-Chloroketones in the Presence of Isocyanates and Amines.

    PubMed

    Perrone, Serena; Capua, Martina; Salomone, Antonio; Troisi, Luigino

    2015-08-21

    A short and efficient one-pot synthesis of uracil derivatives with a high structural variability is described. The process is a multicomponent reaction based on a palladium-catalyzed carbonylation of α-chloroketones in the presence of primary amines and isocyanates. In most cases, when the formation of unsymmetrical N,N'-disubstituted uracil derivatives can occur, the methodology demonstrates to be highly regioselective. A mechanistic hypothesis involving β-dicarbonyl palladium intermediates and urea derivatives, generated in situ, has been discussed.

  6. Uracil content of 16S rRNA of thermophilic and psychrophilic prokaryotes correlates inversely with their optimal growth temperatures

    PubMed Central

    Khachane, Amit N.; Timmis, Kenneth N.; dos Santos, Vítor A. P. Martins

    2005-01-01

    We report here the finding of a highly significant inverse correlation of the uracil content of 16S rRNA and the optimum growth temperature (Topt) of cultured thermophilic and psychrophilic prokaryotes. This correlation was significantly different from the weaker correlations between the contents of other nucleotides and Topt. Analysis of the 16S rRNA secondary structure regions revealed a fall in the A:U base-pair content in step with the increase in Topt that was much steeper than that of mismatched base-pairs, which are thermodynamically less stable. These findings indicate that the 16S rRNA sequences of thermophiles and psychrophiles are under a strong thermo-adaptive pressure, and that structure–function constraints play a crucial role in determining their 16S rRNA nucleotide composition. The derived relationship between uracil content and Topt was used to develop an algorithm to predict the Topt values of uncultured prokaryotes lacking cultured close relatives and belonging to the phyla predominantly containing thermophiles. This algorithm may be useful in guiding the design of cultivation conditions for hitherto uncultured microbes. PMID:16030352

  7. MNDO-MOCIC evaluation of the uracil force field: Application to the interpretation of flavin vibrational spectra

    NASA Astrophysics Data System (ADS)

    Bowman, W. David; Spiro, Thomas G.

    1980-12-01

    A normal mode analysis has been carried out on the in-plane vibrations of uracil and three isotopic molecules, N,N-dideutero, C,C-dideutero, and perdeutero uracil, using a molecular orbital calculation (MNDO) to constrain the ratios of off-diagonal to diagonal elements in the compliance matrix (MOCIC). The 81 observed frequencies are calculated with a mean error of 9.7 cm-1. The force constants and normal mode patterns are discussed. The characteristic up-shift of the 1236 cm-1 mode upon N,N deuteration is reproduced by the calculation, and is ascribable to a change in mode composition from predominantly C=O deformation to predominantly C-N stretching, in agreement with the pattern observed for other cyclic imides. The analogous mode in flavin is variable in different flavoproteins and has been suggested to be sensitive to hydrogen bonding at flavin N3. This effect of hydrogen bonding is modeled in the present calculation by increasing the N-H deformation force constant. A similar perturbation of the C=O deformation constants shows that the 1236 cm-1 mode frequency may also be somewhat sensitive to hydrogen bonding at C4=0, but not at C2=0.

  8. 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.

  9. 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

  10. 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

    2016-01-01

    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 N(3)-substituted uracil derivatives containing 6-(4-bromophenoxy)hexyl or 8-(4-bromophenoxy)octyl fragment at N(1) position. PMID:27406141

  11. 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.

  12. A study of the hydration of deoxydinucleoside monophosphates containing thymine, uracil and its 5-halogen derivatives: Monte Carlo simulation.

    PubMed

    Alderfer, J L; Danilov, V I; Poltev, V I; Slyusarchuk, O N

    1999-04-01

    An extensive Monte Carlo simulation of hydration of various conformations of the dinucleoside monophosphates (DNP), containing thymine, uracil and its 5-halogen derivatives has been performed. An anti-anti conformation is the most energetically stable one for each of the DNPs. In the majority of cases the energy preference is determined by water-water interaction. For other dimers conformational energy is the most important factor, or both the factors are of nearly equal importance. The introduction of the methyl group into the 5-position of uracil ring most noticeably influences the conformational energy and leads to the decrease of its stabilizing contribution to the total interaction energy. The introduction of halogen atoms increases the relative content of anti-syn and syn-anti conformations of DNPs as compared to the parent ones due to the formation of an energetically more favorable water structure around these conformations. A correlation is observed between the Monte Carlo results for the halogenated DNPs and their experimental photoproduct distribution. The data obtained demonstrates a sequence dependence in the photochemistry of the halogenated dinucleoside monophosphates.

  13. 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

  14. A second monoclinic polymorph of {bis­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borato}{tris­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borato}cobalt(II): a structure containing a B—H⋯Co agostic inter­action

    PubMed Central

    Stibrany, Robert T.; Potenza, Joseph A.

    2011-01-01

    The title compound, [Co(C10H10BF6N4)(C15H13BF9N6)], is a polymorph of the previously reported neutral cobalt(II) complex [Stibrany & Potenza (2010 ▶). Acta Cryst. E66, m506–m507], which contains one each of the monoanionic ligands, bis­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borate (Bp) and tris­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borate (Tp). A distorted octahedral coordination geometry of the CoII atom results from ligation of an H atom, which is part of an agostic B—H⋯Co inter­action [H⋯Co = 2.12 (3) Å], and by five imine N atoms, two from a Bp ligand and three from a Tp ligand. Weak intra- and inter­molecular C—F⋯π inter­actions with F⋯centroid distances ranging from 3.025 (4) to 3.605 (4) Å are observed. PMID:21836897

  15. Protein tyrosine kinase regulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons.

    PubMed

    Wang, H; Yu, L C; Li, Y C

    2016-01-01

    This study was performed to investigate the modulation effect of protein tyrosine kinase on postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons. The cultured neurons were exposed to 1% O2 and the expression of AMPA receptor subunit GluR2 on the cell surface was significantly increased, while total GluR2 was not markedly changed. Furthermore, the hypoxia-induced increase in GluR2 expression on the cell surface was partially blocked by the protein tyrosine kinase membrane-permeable inhibitor genistein. In contrast, both the protein tyrosine kinase agonist nerve growth factor and protein tyrosine phosphatase inhibitor vanadate promoted the hypoxia-induced increase of GluR2 expression on cell surface. Moreover, GluR2 could be phosphorylated by tyrosine under normoxia and hypoxia conditions in vitro on brainstem neurons, and tyrosine phosphorylation of GluR2 was significantly stronger under hypoxia conditions. Our results indicate that acute hypoxia induces the AMPA receptor subunit GluR2 to rapidly migrate to the cell membrane to modify the strength of the synapse. This study indicates that tyrosine phosphorylation of the receptor is an important pathway regulating the rapid migration of GluR2 in the postsynaptic domain induced by hypoxia. PMID:27525851

  16. 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.

  17. 2-Amino-5-methyl­pyridinium 3-carb­oxy-4-hy­droxy­benzene­sulfonate

    PubMed Central

    Hemamalini, Madhukar; Fun, Hoong-Kun

    2010-01-01

    The asymmetric unit of the title salt, C6H9N2 +·C7H5O6S−, contains two crystallographically independent 2-amino-5-methylpyridinium cations and two sulfosalicylate anions. In the crystal structure, the sulfonate group of each 3-carb­oxy-4-hy­droxy­benzene­sulfonate anion inter­acts with the corresponding 2-amino-5-methyl­pyridinium cation via a pair of N—H⋯O hydrogen bonds, forming an R 2 2(8) ring motif. The ionic units are linked by N—H⋯O, O—H⋯O and C—H⋯O hydrogen bonds. Furthermore, the crystal structure is stabilized by π–π inter­actions between the benzene and pyridine rings [centroid–centroid distances = 3.5579 (8) and 3.8309 (8) Å]. There are also intra­molecular O—H⋯O hydrogen bonds in the anions, which generate S(6) ring motifs. PMID:21588438

  18. Spectroscopic investigation, natural bond orbital analysis, HOMO-LUMO and thermodynamic functions of 2-tert-butyl-5-methyl anisole using DFT (B3LYP) calculations.

    PubMed

    Balachandran, V; Santhi, G; Karpagam, V; Revathi, B; Karabacak, M

    2015-02-01

    The optimized molecular structure and corresponding vibrational assignments of 2-tert-butyl-5-methyl anisole (TBMA) have been investigated using density functional theory (DFT)/B3LYP with 6-31G(d,p) and 6-311++G(d,p) basis sets investigation of the relative orientation of the methoxy group has shown two conformers (O-cis) and (O-trans) exist. The vibrational analysis of the stable conformer of the title compound is performed by means of infrared absorption and Raman spectroscopy in combination with theoretical simultaneously. The natural bond orbital (NBO) analysis is useful to understand the intra-molecular hyper conjugative interaction lone pair and π(∗)(C-C), σ(∗)(C-H) bond orbital. HOMO and LUMO energies show that charge transfer occurs in the molecule, therefore; HOMO, LUMO and molecular electrostatic potential (MEP) were calculated and analyzed. (1)H and (13)C NMR spectra by using gauge including atomic orbital (GIAO) method of studied compound were compared with experimental data. The thermodynamic functions of TBMA were calculated by B3LYP/6-311+G(d,p) basis set.

  19. 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

  20. Regulation of GluA1 α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor Function by Protein Kinase C at Serine-818 and Threonine-840

    PubMed Central

    Jenkins, Meagan A.; Wells, Gordon; Bachman, Julia; Snyder, James P.; Jenkins, Andrew; Huganir, Richard L.; Oswald, Robert E.

    2014-01-01

    Three residues within the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluA1 C terminus (Ser818, Ser831, Thr840) can be phosphorylated by Ca2+/phospholipid-dependent protein kinase (PKC). Here, we show that PKC phosphorylation of GluA1 Ser818 or Thr840 enhances the weighted mean channel conductance without altering the response time course or agonist potency. These data support the idea that these residues constitute a hyper-regulatory domain for the AMPA receptor. Introduction of phosphomimetic mutations increases conductance only at these three sites within the proximal C terminus, consistent with a structural model with a flexible linker connecting the distal C-terminal domain to the more proximal domain containing a helix bracketed by Ser831 and Thr840. NMR spectra support this model and raise the possibility that phosphorylation can alter the configuration of this domain. Our findings provide insight into the structure and function of the C-terminal domain of GluA1, which controls AMPA receptor function and trafficking during synaptic plasticity in the central nervous system. PMID:24452473

  1. 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. PMID:26763002

  2. Solvent extraction of metals with 4-dinitrobenzoyl-2,4-dihydro-5-methyl-2-phenyl-3h-pyrazol-3-one (DMPP)

    SciTech Connect

    Arora, H.C.; Puram, R.K.; Rao, G.N.

    1983-01-01

    The mechanism of the extraction of copper(II), nickel(II), cobalt(II) and thorium(IV) from aqueous buffer media with 4-dinitrobenzoyl1-2,4-dihydro-5-methy1-3H-pyrazol-3-one (DMPP) in benzene has been investigated. The values of log K where K refers to the extraction equilibrium Mn/sup +/ + nHL in equilibrium MLn + nH/sup +/ are Cu(II)(+0.3), Co(II)(-6.65), Ni(II)(-5.04) and Th(IV)(+6.1). Solid complexes synthesized have the composition CuL/sub 2/ x 2H/sub 2/O and ThL/sub 4/ respectively (L=anion of the ligand). DMPP seems to be superior to the corresponding 4-benzoyl and 4-nitrobenzoyl derivatives of 2,4-dihydro-5-methyl-2-phenyl1-3H-pyrazol-3-one (MPP) and also better than thenoyltrifluoroacetone, the popularly employed fluorinated ..beta..-diketone in the systems investigated. 2 figures.

  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. Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation.

    PubMed

    Dingler, Felix A; Kemmerich, Kristin; Neuberger, Michael S; Rada, Cristina

    2014-07-01

    Excision of uracil introduced into the immunoglobulin loci by AID is central to antibody diversification. While predominantly carried out by the UNG uracil-DNA glycosylase as reflected by deficiency in immunoglobulin class switching in Ung(-/-) mice, the deficiency is incomplete, as evidenced by the emergence of switched IgG in the serum of Ung(-/-) mice. Lack of switching in mice deficient in both UNG and MSH2 suggested that mismatch repair initiated a backup pathway. We now show that most of the residual class switching in Ung(-/-) mice depends upon the endogenous SMUG1 uracil-DNA glycosylase, with in vitro switching to IgG1 as well as serum IgG3, IgG2b, and IgA greatly diminished in Ung(-/-) Smug1(-/-) mice, and that Smug1 partially compensates for Ung deficiency over time. Nonetheless, using a highly MSH2-dependent mechanism, Ung(-/-) Smug1(-/-) mice can still produce detectable levels of switched isotypes, especially IgG1. While not affecting the pattern of base substitutions, SMUG1 deficiency in an Ung(-/-) background further reduces somatic hypermutation at A:T base pairs. Our data reveal an essential requirement for uracil excision in class switching and in facilitating noncanonical mismatch repair for the A:T phase of hypermutation presumably by creating nicks near the U:G lesion recognized by MSH2.

  5. 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

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

    PubMed

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

    2016-04-01

    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.

  7. 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

  8. IR and Raman spectra, ab initio and density functional computations of the vibrational spectra, molecular geometries and atomic charges of uracil and 5-aminouracil.

    PubMed

    Singh, J S

    2014-09-15

    Infrared (IR) and Raman spectra of uracil and 5-aminouracil have been recorded and analyzed between the region 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. 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-aminouracil 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, the most of B3LYP/6-311++G** vibrational frequencies are in the excellent agreement with available experimental assignments and helped in the reassignments of some fundamental vibrational modes. On the basis of calculated results, the assignments of some missing frequencies in the experimental study are proposed. 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″) are given by 25a'+11a″, of which 30 modes (21a'+9a″) correspond to the uracil moiety and 6 modes (4a'+2a″) to the NH2 group. Kekule ring stretching mode is found to be comparatively higher frequency magnitude than the mode of uracil due to the involvement of hydrogen bonding of amino group. But, the ring breathing is found to be lower frequency magnitude compared to those for uracil which could be due to mass effect of the NH2 group in place of the hydrogen atom. All other bands have also been assigned different fundamentals/overtones/combinations.

  9. 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

  10. α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor activation protects against phencyclidine-induced caspase-3 activity by activating voltage-gated calcium channels.

    PubMed

    Timpe, Jennifer M; Wang, Cheng Z; Kim, Jisoo; Johnson, Kenneth M

    2014-12-01

    Phencyclidine (PCP) is a noncompetitive, open channel blocker of the N-methyl-D-aspartate (NMDA) receptor-ion channel complex. When administered to immature animals, it is known to cause apoptotic neurodegeneration in several regions, and this is followed by olanzapine-sensitive, schizophrenia-like behaviors in late adolescence and adulthood. Clarification of its mechanism of action could yield data that would help to inform the treatment of schizophrenia. In our initial experiments, we found that α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) inhibited PCP-induced apoptosis in organotypic neonatal rat brain slices in a concentration-dependent and 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive manner. Calcium signaling pathways are widely implicated in apoptosis, and PCP prevents calcium influx through NMDA receptor channels. We therefore hypothesized that AMPA could protect against this effect by activation of voltage-dependent calcium channels (VDCCs). In support of this hypothesis, pretreatment with the calcium channel blocker cadmium chloride eliminated AMPA-mediated protection against PCP. Furthermore, the L-type VDCC inhibitor nifedipine (10 µM) fully abrogated the effects of AMPA, suggesting that L-type VDCCs are required for AMPA-mediated protection against PCP-induced neurotoxicity. Whereas the P/Q-type inhibitor ω-agatoxin TK (200 nM) reduced AMPA protection by 51.7%, the N-type VDCC inhibitor ω-conotoxin (2 µM) had no effect. Decreased AMPA-mediated protection following cotreatment with K252a, a TrkB inhibitor, suggests that brain-derived neurotrophic factor signaling plays an important role. By analogy, these results suggest that activation of L-type, and to a lesser extent P/Q-type, VDCCs might be advantageous in treating conditions associated with diminished NMDAergic activity during early development. PMID:24995437

  11. 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.

  12. Insulin treatment restores glutamate (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor function in the hippocampus of diabetic rats.

    PubMed

    Viswaprakash, Nilmini; Vaithianathan, Thirumalini; Viswaprakash, Ajitan; Judd, Robert; Parameshwaran, Kodeeswaran; Suppiramaniam, Vishnu

    2015-09-01

    Type 1 diabetes is associated with cognitive dysfunction. Cognitive processing, particularly memory acquisition, depends on the regulated enhancement of expression and function of glutamate receptor subtypes in the hippocampus. Impairment of memory was been detected in rodent models of type 1 diabetes induced by streptozotocin (STZ). This study examines the functional properties of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the expression of synaptic molecules that regulate glutamatergic synaptic transmission in the hippocampus of STZ-diabetic rats. The AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) and single-channel properties of synaptosomal AMPA receptors were examined after 4 weeks of diabetes induction. Results show that amplitude and frequency of mEPSCs recorded from CA1 pyramidal neurons were decreased in diabetic rats. In addition, the single-channel properties of synaptic AMPA receptors from diabetic rat hippocampi were different from those of controls. These impairments in synaptic currents gated by AMPA receptors were accompanied by decreased protein levels of AMPA receptor subunit GluR1, the presynaptic protein synaptophysin, and the postsynaptic anchor protein postsynaptic density protein 95 in the hippocampus of diabetic rats. Neural cell adhesion molecule (NCAM), an extracellular matrix molecule abundantly expressed in the brain, and the polysialic acid (PSA) attached to NCAM were also downregulated in the hippocampus of diabetic rats. Insulin treatment, when initiated at the onset of diabetes induction, reduced these effects. These findings suggest that STZ-induced diabetes may result in functional deteriorations in glutamatergic synapses in the hippocampus of rats and that these effects may be reduced by insulin treatment. PMID:25807926

  13. Infrared and Electronic Spectroscopy of the Jet-Cooled 5-Methyl-2-furanylmethyl Radical Derived from the Biofuel 2,5-Dimethylfuran.

    PubMed

    Kidwell, Nathanael M; Mehta-Hurt, Deepali N; Korn, Joseph A; Zwier, Timothy S

    2016-08-18

    The electronic and infrared spectra of the 5-methyl-2-furanylmethyl (MFM) radical have been characterized under jet-cooled conditions in the gas phase. This resonance-stabilized radical is formed by H atom loss from one of the methyl groups of 2,5-dimethylfuran (DMF), a promising second-generation biofuel. As a resonance-stabilized radical, it plays an important role in the flame chemistry of DMF. The D0-D1 transition was studied using two-color resonant two-photon ionization (2C-R2PI) spectroscopy. The electronic origin is in the middle of the visible spectrum (21934 cm(-1) = 455.9 nm) and is accompanied by Franck-Condon activity involving the hindered methyl rotor. The frequencies and intensities are fit to a one-dimensional methyl rotor potential, using the calculated form of the ground state potential. The methyl rotor reports sensitively on the local electronic environment and how it changes with electronic excitation, shifting from a preferred ground state orientation with one CH in-plane and anti to the furan oxygen, to an orientation in the excited state in which one CH group is axial to the plane of the furan ring. Ground and excited state alkyl CH stretch infrared spectra are recorded using resonant ion-dip infrared (RIDIR) spectroscopy, offering a complementary view of the methyl group and its response to electronic excitation. Dramatic changes in the CH stretch transitions with electronic state reflect the changing preference for the methyl group orientation. PMID:27456434

  14. Schedule-controlled behavioral effects of the selective 2-amino-3-(5-methyl-3-hydroxyisoxazol-4-yl)propanoic acid antagonist LY293558 in pigeons.

    PubMed

    Benvenga, M J; Ornstein, P L; Leander, J D

    1995-10-01

    Behavioral effects of the selective 2-amino-3-(5-methyl-3-hydroxyisoxazol-4- yl)propanoic acid (AMPA) antagonist LY293558, along with its racemate (LY215490) and opposing enantiomer (LY293559) were evaluated in pigeons. When responding was maintained under a multiple fixed ratio 50 responses, fixed interval 5 minute (FRFI) schedule of food presentation, LY215490 completely antagonized the rate suppression induced by AMPA (10 mg/kg) and by the AMPA analog, 2-amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionic acid (ATPA; 40 mg/kg) at 1.25 and 2.5 mg/kg, respectively. In contrast, LY215490, up to 10 mg/kg, was unable to antagonize the rate suppression induced by N-methyl-D-aspartic acid. LY293558, at 0.32 mg/kg, completely blocked the rate suppression produced by AMPA in both components of the multiple schedule. Similarly, LY293558, at 0.64 mg/kg, blocked the rate suppression induced by ATPA in both components. In contrast, the opposing enantiomer, LY293559, up to 10 mg/kg, was without effect on rate suppression produced by AMPA in this model. In additional studies, behavior was maintained under a schedule in which responding was maintained by food presentation in the presence of one key color and in the presence of a second key color, responding was maintained by food and simultaneously suppressed by electric shock ("punished responding"). LY215490 significantly increased punished responding at 10 and 30 mg/kg, whereas unpunished responding was unaffected until 56 mg/kg depressed it. LY293558 significantly increased punished responding at 3 mg/kg without having an effect on unpunished responding. LY293559, on the other hand, was unable to significantly increase punished responding at doses up to 175 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. [18F]-2′-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in Prostate Cancer: Initial Preclinical Observations

    PubMed Central

    Jadvar, Hossein; Yap, Li-Peng; Park, Ryan; Li, Zibo; Chen, Kai; Hughes, Lindsey; Conti, Peter

    2011-01-01

    We hypothesized that imaging-based assessment of cellular proliferation in prostate cancer may improve tumor characterization. We therefore evaluated the biodistribution and effect of androgen on tumor uptake of the cellular proliferation imaging marker [18F]-2′-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in xenograft mouse models of human prostate cancer. Castrated and noncastrated athymic male mice were implanted with androgen-independent PC3 and androgen-sensitive CWR22 human prostate cancer cells. Dynamic microPET/CT imaging was performed for 1h followed by 10 minute static scans at 2h and 3h. Animals were sacrificed after imaging for biodistribution studies and immunohistochemical staining of tumors for androgen receptor and Ki-67/MIB expression. 18F-FMAU uptake was significantly higher in all major organs of the castrated animals in comparison to noncastrated mice with the highest uptake in liver and the lowest uptake in muscle and bone. When compared to PC3 tumors, CWR22 xenografts showed significantly higher tumor-to-muscle (2.56±0.30 vs. 1.99±0.30, p=0.008) and tumor-to-liver uptake ratios (1.72±0.12 vs. 1.26±0.17, p=0.0003) in the noncastrated animal at 3h time point. Androgen receptor and Ki-67/MIB expressions were higher in CWR22 than PC3 xenografts. Our initial preclinical observations suggest that there may be an association between androgen signaling and thymidine metabolism and that 18F-FMAU PET may be useful in prostate tumor characterization. PMID:22954187

  16. 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.

  17. Schedule-controlled behavioral effects of the selective 2-amino-3-(5-methyl-3-hydroxyisoxazol-4-yl)propanoic acid antagonist LY293558 in pigeons.

    PubMed

    Benvenga, M J; Ornstein, P L; Leander, J D

    1995-10-01

    Behavioral effects of the selective 2-amino-3-(5-methyl-3-hydroxyisoxazol-4- yl)propanoic acid (AMPA) antagonist LY293558, along with its racemate (LY215490) and opposing enantiomer (LY293559) were evaluated in pigeons. When responding was maintained under a multiple fixed ratio 50 responses, fixed interval 5 minute (FRFI) schedule of food presentation, LY215490 completely antagonized the rate suppression induced by AMPA (10 mg/kg) and by the AMPA analog, 2-amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionic acid (ATPA; 40 mg/kg) at 1.25 and 2.5 mg/kg, respectively. In contrast, LY215490, up to 10 mg/kg, was unable to antagonize the rate suppression induced by N-methyl-D-aspartic acid. LY293558, at 0.32 mg/kg, completely blocked the rate suppression produced by AMPA in both components of the multiple schedule. Similarly, LY293558, at 0.64 mg/kg, blocked the rate suppression induced by ATPA in both components. In contrast, the opposing enantiomer, LY293559, up to 10 mg/kg, was without effect on rate suppression produced by AMPA in this model. In additional studies, behavior was maintained under a schedule in which responding was maintained by food presentation in the presence of one key color and in the presence of a second key color, responding was maintained by food and simultaneously suppressed by electric shock ("punished responding"). LY215490 significantly increased punished responding at 10 and 30 mg/kg, whereas unpunished responding was unaffected until 56 mg/kg depressed it. LY293558 significantly increased punished responding at 3 mg/kg without having an effect on unpunished responding. LY293559, on the other hand, was unable to significantly increase punished responding at doses up to 175 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7562545

  18. Potent methyl oxidation of 5-methyl-2'-deoxycytidine by halogenated quinoid carcinogens and hydrogen peroxide via a metal-independent mechanism.

    PubMed

    Shao, Jie; Huang, Chun-Hua; Kalyanaraman, Balaraman; Zhu, Ben-Zhan

    2013-07-01

    Halogenated quinones are a class of carcinogenic intermediates and are newly identified chlorination disinfection by-products in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical ((•)OH) can be produced by halogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H2O2 can oxidize the nucleoside 5-methyl-2'-deoxycytidine (5mdC) to its methyl oxidation products and, if so, what the underlying molecular mechanism is. Here we show that three methyl oxidation products, 5-(hydroperoxymethyl)-, 5-(hydroxymethyl)-, and 5-formyl-2'-deoxycytidine, could be produced when 5mdC was treated with tetrachloro-1,4-benzoquinone (TCBQ) and H2O2. The formation of the oxidation products was markedly inhibited by typical (•)OH scavengers and under anaerobic conditions. Analogous effects were observed with other halogenated quinones and the classic Fenton system. Based on these data, we propose that the oxidation of 5mdC by TCBQ/H2O2 might be through the following mechanism: (•)OH produced by TCBQ/H2O2 may first abstract hydrogen from the methyl group of 5mdC, leading to the formation of 5-(2'-deoxycytidylyl)methyl radical, which may combine with O2 to form the peroxyl radical. The unstable peroxyl radical transforms into the corresponding hydroperoxide 5-(hydroperoxymethyl)-2'-deoxycytidine, which reacts with TCBQ and results in the formation of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine. This is the first report that halogenated quinoid carcinogens and H2O2 can induce potent methyl oxidation of 5mdC via a metal-independent mechanism, which may partly explain their potential carcinogenicity.

  19. 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

    2015-03-01

    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.

  20. 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.

  1. 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.

  2. 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. PMID:27189184

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

    PubMed

    Li, Quansong; Giussani, Angelo; Segarra-Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A; Mukamel, Shaul; Roca-Sanjuán, Daniel; Garavelli, Marco; Blancafort, Lluís

    2016-05-23

    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 (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb 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 (1) Lb , 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

  4. 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

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

    PubMed

    Li, Quansong; Giussani, Angelo; Segarra-Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A; Mukamel, Shaul; Roca-Sanjuán, Daniel; Garavelli, Marco; Blancafort, Lluís

    2016-05-23

    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 (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb 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 (1) Lb , 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.

  6. 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

  7. Analysis of three crystal structure determinations of a 5-methyl-6-N-methylanilino pyridopyrimidine antifolate complex with human dihydrofolate reductase.

    PubMed

    Cody, Vivian; Luft, Joseph R; Pangborn, Walter; Gangjee, Aleem

    2003-09-01

    Structural data are reported for the first example of the potent antifolate inhibitor 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxy-N-methylanilino)methyl]pyrido[2,3-d]pyrimidine (1) in complex with human dihydrofolate reductase (hDHFR) and NADPH. Small differences in crystallization conditions resulted in the growth of two different forms of a binary complex. The structure determination of an additional crystal of a ternary complex of hDHFR with NADPH and (1) grown under similar conditions is also reported. Diffraction data were collected to 2.1 A resolution for an R3 lattice from a hDHFR ternary complex with NADPH and (1) and to 2.2 A resolution from a binary complex. Data were also collected to 2.1 A resolution from a binary complex with hDHFR and (1) in the first example of a tetragonal P4(3)2(1)2 lattice. Comparison of the intermolecular contacts among these structures reveals differences in the backbone conformation (1.9-3.2 A) for flexible loop regions (residues 40-46, 77-83 and 103-107) that reflect differences in the packing environment between the rhombohedral and tetragonal space groups. Analysis of the packing environments shows that the tetragonal lattice is more tightly packed, as reflected in its smaller V(M) value and lower solvent content. The conformation of the inhibitor (1) is similar in all structures and is also similar to that observed for TMQ, the parent quinazoline compound. The activity profile for this series of 5-deaza N-substituted non-classical trimethoxybenzyl antifolates shows that the N10-CH(3) substituted (1) has the greatest potency and selectivity for Toxoplasma gondii DHFR (tgDHFR) compared with its N-H or N-CHO analogs. Models of the tgDHFR active site indicate preferential contacts with (1) that are not present in either the human or Pneumocystis carinii DHFR structures. Differences in the acidic residue (Glu30 versus Asp for tgDHFR) affect the precise positioning of the diaminopyridopyrimidine ring, while changes in other

  8. Pharmacological characterization of cGMP regulation by the biarylpropylsulfonamide class of positive, allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

    PubMed

    Ryder, John W; Falcone, Julie F; Manro, Jason R; Svensson, Kjell A; Merchant, Kalpana M

    2006-10-01

    The biarylpropylsulfonamide class of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) potentiators represented by N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide (LY404187) and (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430) are positive, allosteric AMPA receptor activators, which enhance AMPA receptor-mediated neurotransmission by reducing desensitization of the ion channel. Although these compounds have efficacy in in vivo rodent models of cognition, depression, and Parkinson's disease, little is known about biochemical pathways activated by these agents. Given the well established regulation of the nitric oxide/cGMP pathway by excitatory neurotransmission, the current study characterized AMPA receptor potentiator-mediated cGMP response in mouse cerebellum. Acute treatment by both LY404187 and LY503430 [2.0, 5.0, or 10 mg/kg subcutaneously (s.c.)] elevated basal cerebellar cGMP levels in a dose-dependent manner. Pretreatment with the noncompetitive, allosteric AMPA receptor-selective antagonist 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI) (GYKI 53655) [3.0 mg/kg intraperitoneally (i.p.)], completely blocked the effect of LY404187, demonstrating that activation of AMPA receptors induces cGMP levels. Interestingly, pretreatment with the N-methyl-d-aspartate (NMDA) open channel blocker dizocilpine (0.3 and 1.0 mg/kg i.p.) also abolished the AMPA receptor potentiator-mediated cGMP accumulation, indicating that activation of AMPA receptors leads to NMDA receptor-mediated transmission involved in cGMP regulation. Pharmacological augmentation of the endogenous glutamate tone via the alkaloid harmaline (20-60 mg/kg i.p.) synergized with AMPA potentiator activity and provided further direct evidence of in vivo allosteric activation of AMPA receptors by LY404187. The synergism between harmaline and LY404187 was

  9. 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

  10. Direct construction of 5-methyl-2-phenylisoxazol-3(2H)-ones via hypervalent iodine mediated sequential tandem oxidative cyclization of 3-oxo-N-phenylbutanamides catalyzed by zinc oxide (ZnO).

    PubMed

    Liu, Weibing; Zhou, Peng; Chen, Cui; Zhang, Qing; Zhu, Zhibo

    2013-01-28

    A sequential oxidative tandem cyclization reaction mediated by a combination of (diacetoxyiodo)benzene (DIB) with zinc oxide (ZnO) is presented for the synthesis of 5-methyl-2-phenylisoxazol-3(2H)-ones from β-ketobutylanilides. A variety of β-ketobutylanilide compounds were used in this approach, and a wide range of functionalized 5-methylisoxazol-3(2H)-ones were obtained in good to excellent yields.

  11. 1,6-Bis[(benzyloxy)methyl]uracil derivatives-Novel antivirals with activity against HIV-1 and influenza H1N1 virus.

    PubMed

    Geisman, Alexander N; Valuev-Elliston, Vladimir T; Ozerov, Alexander A; Khandazhinskaya, Anastasia L; Chizhov, Alexander O; Kochetkov, Sergey N; Pannecouque, Christophe; Naesens, Lieve; Seley-Radtke, Katherine L; Novikov, Mikhail S

    2016-06-01

    A series of 1,6-bis[(benzyloxy)methyl]uracil derivatives combining structural features of both diphenyl ether and pyridone types of NNRTIs were synthesized. Target compounds were found to inhibit HIV-1 reverse transcriptase at micro- and submicromolar levels of concentrations and exhibited anti-HIV-1 activity in MT-4 cell culture, demonstrating resistance profile similar to first generation NNRTIs. The synthesized compounds also showed profound activity against influenza virus (H1N1) in MDCK cell culture without detectable cytotoxicity. The lead compound of this assay appeared to exceed rimantadine, amantadine, ribavirin and oseltamivir carboxylate in activity. The mechanism of action of 1,6-bis[(benzyloxy)methyl]uracils against influenza virus is currently under investigation. PMID:27112451

  12. A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

    PubMed

    Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

    2016-03-01

    Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses. PMID:26899234

  13. 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%).

  14. In vitro antitumour activity of cis- and trans-5-fluoro-5,6-dihydro-6-alkoxy-uracils; effects on thymidylate synthesis.

    PubMed Central

    van der Wilt, C. L.; Visser, G. W.; Braakhuis, B. J.; Wedzinga, R.; Noordhuis, P.; Smid, K.; Peters, G. J.

    1993-01-01

    A class of new 5-fluorouracil (FU) analogues, the 5-fluoro-5,6-dihydro-6- alkoxy-uracils was synthesised with a modification at the 6-position of the pyrimidine ring. At this position the analogues have a hydroxy or alkoxy group of different chain lengths either in the cis- or trans-configuration. The antiproliferative effect of these compounds was tested on five cell lines of different origin. Generally, the analogues with a cis-configuration had a higher activity than those with a trans-configuration. The growth inhibitory effect of the compounds decreased with increasing alkoxy chain length, but the compound with a hydroxy group had the lowest growth inhibitory effect. One analogue, cis-5-F-5,6-dihydro-6-methoxy-uracil had a higher antiproliferative effect than FU in one of the cell lines. Effects on thymidylate synthase (TS), the possible target of these analogues, were evaluated by thymidine rescue of growth inhibition and incorporation of tritiated deoxyuridine (3H-UdR) into DNA. In solid tumour cell lines addition of TdR reversed the antiproliferative effect. Inhibition of TS in intact cells was determined by measuring 3H-UdR incorporation in two cell lines. The effect of cis-5-F-5,6-dihydro-6-methoxy-uracil on incorporation of 3H-UdR was 2- to 5-fold stronger than that of FU in both cell lines. All other compounds produced a higher 3H-UdR incorporation than FU both at equimolar and equi-toxic concentration. Concluding from these results we regard cis-5-F-5,6-dihydro-6-methoxy-uracil as the most promising FU analogue of this series, because of its higher antiproliferative activity than FU and marked inhibition of TS in intact cells. PMID:8398696

  15. 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%). PMID:25836367

  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. The efficacy of uracil DNA glycosylase pretreatment in amplicon-based massively parallel sequencing with DNA extracted from archived formalin-fixed paraffin-embedded esophageal cancer tissues.

    PubMed

    Serizawa, Masakuni; Yokota, Tomoya; Hosokawa, Ayumu; Kusafuka, Kimihide; Sugiyama, Toshiro; Tsubosa, Yasuhiro; Yasui, Hirofumi; Nakajima, Takashi; Koh, Yasuhiro

    2015-09-01

    Advances in mutation testing for molecular-targeted cancer therapies have led to the increased use of archived formalin-fixed paraffin-embedded (FFPE) tumors. However, DNA extracted from FFPE tumors (FFPE DNA) is problematic for mutation testing, especially for amplicon-based massively parallel sequencing (MPS), owing to DNA fragmentation and artificial C:G > T:A single nucleotide variants (SNVs) caused by deamination of cytosine to uracil. Therefore, to reduce artificial C:G > T:A SNVs in amplicon-based MPS using FFPE DNA, we evaluated the efficacy of uracil DNA glycosylase (UDG) pretreatment, which can eliminate uracil-containing DNA molecules, with 126 archived FFPE esophageal cancer specimens. We also examined the association between the frequency of C:G > T:A SNVs and DNA quality, as assessed by a quantitative PCR (qPCR)-based assay. UDG pretreatment significantly lowered the frequency of C:G > T:A SNVs in highly fragmented DNA (by approximately 60%). This effect was not observed for good- to moderate-quality DNA, suggesting that a predictive assay (i.e., DNA quality assessment) needs to be performed prior to UDG pretreatment. These results suggest that UDG pretreatment is efficacious for mutation testing by amplicon-based MPS with fragmented DNA from FFPE samples.

  18. Doubly differential distribution of electron emission in ionization of uracil in collisions with 3.5-MeV/u bare C ions

    NASA Astrophysics Data System (ADS)

    Agnihotri, A. N.; Nandi, S.; Kasthurirangan, S.; Kumar, A.; Galassi, M. E.; Rivarola, R. D.; Champion, C.; Tribedi, L. C.

    2013-03-01

    We report the energy and angular distribution of the electron emission from an RNA base molecule uracil in collisions with 3.5-MeV/u bare C ions. The absolute double differential cross sections (DDCS) are measured for emission energy between a few to 600 eV. The angular distributions are compared to those obtained for the O2 molecule in the same experiment. The single differential cross sections (SDCS) are also deduced. The energy and angular distributions of the DDCS and SDCS are compared with the state-of-the-art quantum-mechanical models based on continuum distorted wave-eikonal initial state (CDW-EIS) and correct boundary first Born (CB1) approximations which use a suitable molecular wave function for uracil. The models, however, give substantial deviations from the observed energy and angular distributions of the DDCS as well as SDCS. The CDW-EIS calculations are closer to the data compared to the CB1. In the case of uracil a large difference in the forward-backward emission of electrons was observed in comparison to that in collisions with an oxygen molecule.

  19. Uracil DNA Glycosylase Is Dispensable for Human Immunodeficiency Virus Type 1 Replication and Does Not Contribute to the Antiviral Effects of the Cytidine Deaminase Apobec3G

    PubMed Central

    Kaiser, Shari M.; Emerman, Michael

    2006-01-01

    It is well established that many host factors are involved in the replication of human immunodeficiency virus (HIV) type 1. One host protein, uracil DNA glycosylase 2 (UNG2), binds to multiple viral proteins and is packaged into HIV type 1 virions. UNG initiates the removal of uracils from DNA, and this has been proposed to be important both for reverse transcription and as a mediator to the antiviral effect of virion-incorporated Apobec3G, a cytidine deaminase that generates numerous uracils in the viral DNA during virus replication. We used a natural human UNG−/− cell line as well as cells that express a potent catalytic active-site inhibitor of UNG to assess the effects of removing UNG activity on HIV infectivity. In both cases, we find UNG2 activity and protein to be completely dispensable for virus replication. Moreover, we find that virion-associated UNG2 does not affect the loss of infectivity caused by Apobec3G. PMID:16378989

  20. 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.

  1. Fluorinated Peptide Nucleic Acids with Fluoroacetyl Side Chain Bearing 5-(F/CF3)-Uracil: Synthesis and Cell Uptake Studies.

    PubMed

    Ellipilli, Satheesh; Palvai, Sandeep; Ganesh, Krishna N

    2016-08-01

    Fluorine incorporation into organic molecules imparts favorable physicochemical properties such as lipophilicity, solubility and metabolic stability necessary for drug action. Toward such applications using peptide nucleic acids (PNA), we herein report the chemical synthesis of fluorinated PNA monomers and biophysical studies of derived PNA oligomers containing fluorine in in the acetyl side chain (-CHF-CO-) bearing nucleobase uracil (5-F/5-CF3-U). The crystal structures of fluorinated racemic PNA monomers reveal interesting base pairing of enantiomers and packing arrangements directed by the chiral F substituent. Reverse phase HPLC show higher hydrophobicity of fluorinated PNA oligomers, dependent on the number and site of the fluorine substitution: fluorine on carbon adjacent to the carbonyl group induces higher lipophilicity than fluorine on nucleobase or in the backbone. The PNA oligomers containing fluorinated bases form hybrids with cDNA/RNA with slightly lower stability compared to that of unmodified aeg PNA, perhaps due to electronic effects. The uptake of fluorinated homooligomeric PNAs by HeLa cells was as facile as that of nonfluorinated PNA. In conjunction with our previous work on PNAs fluorinated in backbone and at N-terminus, it is evident that the fluorinated PNAs have potential to emerge as a new class of PNA analogues for applications in functional inhibition of RNA. PMID:27391099

  2. [Safety and compliance with UFT (tegafur and uracil) alone and in combination with hormone therapy in patients with breast cancer].

    PubMed

    Taguchi, Tetsuya; Noguchi, Shinzaburo

    2009-09-01

    Recently, some new anticancer agents and hormonal agents can be used to treat breast cancer, and more patients are administering combinations of these drugs in clinical practice. Tegafur and Uracil (UFT) have been widely used for the postoperative chemotherapy of breast cancer, and often combined with hormonal agents. However, due consideration has not been given to safety and compliance of the combined use of UFT and aromatase inhibitor (AI). We therefore studied the safety and compliance with UFT alone and in combination with the hormonal agents tamoxifen or anastrozole as postoperative therapy in postmenopausal women with breast cancer. Our results confirmed that longterm postoperative therapy with UFT alone was feasible, provided that early adverse events are carefully monitored. Combined therapy was not associated with a significant increase in the incidence of adverse events or a decrease in compliance, even in older adult patients. Therefore, we consider UFT plus a hormonal agent (especially anastrozole) to be a treatment option for patients with HER2-negative highly or incompletely endocrine responsive disease who require relatively moderate chemotherapy or for older adult patients who require treatment with low toxicity.

  3. Novel dimeric structure of phage φ29-encoded protein p56: insights into uracil-DNA glycosylase inhibition.

    PubMed

    Asensio, Juan Luis; Pérez-Lago, Laura; Lázaro, José M; González, Carlos; Serrano-Heras, Gemma; Salas, Margarita

    2011-12-01

    Protein p56 encoded by the Bacillus subtilis phage φ29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions. Each polypeptide chain contains three stretches of anti-parallel β-sheets and a helical region linked by three short loops. In addition, microcalorimetry titration experiments showed that it forms a tight 2:1 complex with UDG, strongly suggesting that the dimer represents the functional form of the inhibitor. This was further confirmed by the functional analysis of p56 mutants unable to assemble into dimers. We have also shown that the highly anionic region of the inhibitor plays a significant role in the inhibition of UDG. Thus, based on these findings and taking into account previous results that revealed similarities between the association mode of p56 and the phage PBS-1/PBS-2-encoded inhibitor Ugi with UDG, we propose that protein p56 might inhibit the enzyme by mimicking its DNA substrate.

  4. 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.

  5. Nano- and microstructuration of supramolecular materials driven by H-bonded uracil.2,6-diamidopyridine complexes

    NASA Astrophysics Data System (ADS)

    Marangoni, Tomas; Bonifazi, Davide

    2013-09-01

    In the last few decades, multiple H-bonded arrays have been shown to be versatile tools to prepare functional supramolecular materials. Supramolecular complexes formed by uracil (Ur) and 2,6-diamidopyridine (DAP) developed by Lehn are the first examples of multiple H-bonded systems governing the formation of supramolecular polymers in solution. Although a large variety of complementary multiple H-bonded complexes has been prepared, the use of the heteromolecular Ur.DAP complex still remains very promising due to its ease of preparation and its intermediate association strength that ensures a dynamical character to the self-assembly and self-organisation processes. In this feature article, we report a detailed account on the results that our group has obtained in this field by designing and engineering a novel library of shape persistent molecular modules able to transfer their geometrical information to the final supramolecular architectures through the formation of Ur.DAP complexes both at the nanoscopic and microscopic levels.

  6. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase.

    PubMed

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

    2014-03-18

    Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR.

  7. 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.

  8. 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.

  9. 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.

  10. Classical trajectory Monte Carlo model calculations for ionization of the uracil molecule by impact of heavy ions

    NASA Astrophysics Data System (ADS)

    Sarkadi, L.

    2016-09-01

    The ionization of the uracil molecule induced by heavy-ion impact has been investigated using the classical trajectory Monte Carlo (CTMC) method. Assuming the validity of the independent-particle model approximation, the collision problem is solved by considering the three-body dynamics of the projectile, an active electron and the molecule core. The interaction of the molecule core with the other two particles is described by a multi-center potential built from screened atomic potentials. The cross section differential with respect to the energy and angle of the electrons ejected in the ionization process has been calculated for an impact of 3.5 MeV u-1 {{{C}}}6+ ions. Total electron emission cross sections (TCS) are presented for {{{C}}}q+ (q=0-6) and {{{O}}}6+ projectiles as a function of the impact energy in the range from 10 keV u-1 to 10 MeV u-1. The dependence of the TCS on the charge state of the projectile has been investigated for 2.5 MeV u-1 {{{O}}}q+ (q=4-8) and {{{F}}}q+ (q=5-9) ions. The results of the calculations are compared with available experimental data and the predictions of other theoretical models: the first Born approximation with correct boundary conditions (CB1), the continuum-distorted-wave-eikonal-initial-state approach (CDW-EIS), and the combined classical-trajectory Monte Carlo-classical over-the-barrier model (CTMC-COB).

  11. Cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in normal human cells.

    PubMed Central

    Mansur, N R; Meyer-Siegler, K; Wurzer, J C; Sirover, M A

    1993-01-01

    The cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene was examined in normal human cells. Steady state RNA levels were monitored by Northern blot analysis using a plasmid (pChug 20.1) which contained the 1.3 kb GAPDH/UDG cDNA. The biosynthesis of the 37 kDa GAPDH/UDG protein was determined using an anti-human placental GAPDH/UDG monoclonal antibody to immunoprecipitate the radiolabeled protein. Increases in steady state GAPDH/UDG mRNA levels were cell cycle specific. A biphasic pattern was observed resulting in a 19-fold increase in the amount of GAPDH/UDG mRNA. The biosynthesis of the 37 kDa GAPDH/UDG protein displayed a similar biphasic regulation with a 7-fold increase. Pulse-chase experiments revealed a remarkably short half life of less than 1 hr. for the newly synthesized 37 kDa protein, comparable to that previously documented for a number of oncogenes. GAPDH/UDG mRNA levels were markedly reduced at 24 hr. when DNA synthesis was maximal. These results define the GAPDH/UDG gene as cell cycle regulated with a characteristic temporal sequence of expression in relation to DNA synthesis. The cell cycle synthesis of a labile 37 kDa monomer suggests a possible regulatory function for this multidimensional protein. Further, modulation of the GAPDH/UDG gene in the cell cycle may preclude its use as a reporter gene when the proliferative state of the cell is not kept constant. Images PMID:8451199

  12. Classical trajectory Monte Carlo model calculations for ionization of the uracil molecule by impact of heavy ions

    NASA Astrophysics Data System (ADS)

    Sarkadi, L.

    2016-09-01

    The ionization of the uracil molecule induced by heavy-ion impact has been investigated using the classical trajectory Monte Carlo (CTMC) method. Assuming the validity of the independent-particle model approximation, the collision problem is solved by considering the three-body dynamics of the projectile, an active electron and the molecule core. The interaction of the molecule core with the other two particles is described by a multi-center potential built from screened atomic potentials. The cross section differential with respect to the energy and angle of the electrons ejected in the ionization process has been calculated for an impact of 3.5 MeV u‑1 {{{C}}}6+ ions. Total electron emission cross sections (TCS) are presented for {{{C}}}q+ (q=0-6) and {{{O}}}6+ projectiles as a function of the impact energy in the range from 10 keV u‑1 to 10 MeV u‑1. The dependence of the TCS on the charge state of the projectile has been investigated for 2.5 MeV u‑1 {{{O}}}q+ (q=4-8) and {{{F}}}q+ (q=5-9) ions. The results of the calculations are compared with available experimental data and the predictions of other theoretical models: the first Born approximation with correct boundary conditions (CB1), the continuum-distorted-wave–eikonal-initial-state approach (CDW-EIS), and the combined classical-trajectory Monte Carlo–classical over-the-barrier model (CTMC-COB).

  13. Mammalian 5-formyluracil-DNA glycosylase. 2. Role of SMUG1 uracil-DNA glycosylase in repair of 5-formyluracil and other oxidized and deaminated base lesions.

    PubMed

    Masaoka, Aya; Matsubara, Mayumi; Hasegawa, Rei; Tanaka, Tamon; Kurisu, Satofumi; Terato, Hiroaki; Ohyama, Yoshihiko; Karino, Naoko; Matsuda, Akira; Ide, Hiroshi

    2003-05-01

    In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.

  14. Thermochemistry of uracils. Experimental and computational enthalpies of formation of 5,6-dimethyl-, 1,3,5-trimethyl-, and 1,3,5,6-tetramethyluracils.

    PubMed

    Notario, Rafael; Emel'yanenko, Vladimir N; Roux, María Victoria; Ros, Francisco; Verevkin, Sergey P; Chickos, James S; Liebman, Joel F

    2013-01-10

    We describe in the current paper an experimental and computational study of three methylated uracils, in particular, the 5,6-dimethyl-, 1,3,5-trimethyl-, and 1,3,5,6-tetramethyl derivatives. The values of the standard (p(0) = 0.1 MPa) molar enthalpies of formation in the gas phase at T = 298.15 K have been determined. The energies of combustion were measured by static bomb combustion calorimetry, and from the results obtained, the standard molar enthalpies of formation in the crystalline state at T = 298.15 K were calculated. The enthalpies of sublimation were determined using the transpiration method in a saturated N(2) stream. Values of -(376.2 ± 2.6), -(355.9 ± 3.0), and -(381.7 ± 2.8) kJ·mol(-1) for the gas-phase enthalpies of formation at T = 298.15 K of 5,6-dimethyluracil, 1,3,5-trimethyluracil, and 1,3,5,6-tetramethyluracil, respectively, were obtained from the experimental thermochemical study. An extended theoretical study with the G3 and the G4 quantum-chemical methods has been carried out for all the possible methylated uracils. There is a very good agreement between experimental and calculated enthalpies of formation for the three derivatives studied. A Free-Wilson analysis on G4-calculated enthalpies of formation has been carried out, and the contribution of methylation in the different positions of the uracil ring has been estimated. PMID:23215007

  15. 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.

  16. Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

    PubMed Central

    De Silva, Frank S; Moss, Bernard

    2008-01-01

    Background Low levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV), the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase) hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG) cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed. Results VACV mutants lacking the gene encoding dUTPase or with catalytic site mutations in UNG and double UNG/dUTPase mutants were constructed. Replication of UNG and UNG/dUTPase mutants were slightly reduced compared to wild type or the dUTPase mutant in actively dividing cells. Viral DNA replication was reduced about one-third under these conditions. After high multiplicity infection of quiescent fibroblasts, yields of wild type and mutant viruses were decreased by 2-logs with relative differences similar to those observed in active fibroblasts. However, under low multiplicity multi-step growth conditions in quiescent fibroblasts, replication of the dUTPase/UNG mutant was delayed and 5-fold lower than that of either single mutant or parental virus. This difference was exacerbated by 1-day serial passages on quiescent fibroblasts, resulting in 2- to 3-logs lower titer of the double mutant compared to the parental and single mutant viruses. Each mutant was more attenuated than a revertant

  17. 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.

  18. Interaction of cyclic cytosine-, guanine-, thymine-, uracil- and mixed guanine-cytosine base tetrads with K+, Na+ and Li+ ions -- a density functional study.

    PubMed

    Meyer, Michael; Sühnel, Jürgen

    2003-02-01

    We have carried out B3LYP hybrid density functional studies of complexes formed by cyclic cytosine-, guanine-, thymine-, uracil- and mixed guanine cytosine-tetrads with Li+, Na+ and K+ ions to determine their structures and interaction energies. The conformations studied have been restricted to a hydrogen bond pattern closely related to the tetrads observed in experimental nucleic acid structures. A comparison of the alkali metal ion/tetrad complexes with the tetrads without cations indicates that alkali metal ions modulate the tetrad structures significantly and that even the hydrogen bond pattern may change. Guanine-tetrad cation complexes show the strongest interaction energy compared to other tetrads that occur less frequently in experimental structures. The most stable G-tetrad/metal ion structure adopts a nearly planar geometry that is especially suitable for tetraplex formation, which requires approximately parallel tetrad planes. In the cytosine-tetrad there is a very large central cavity suitable for cation recognition, but the complexes adopt a non-planar structure unsuitable for stacking, except possibly for ions with very large radii. Uracil and thymine tetrads show a significant different characteristics which may contribute to the differences between DNA and RNA PMID:12529150

  19. Deletion of the Uracil Permease Gene Confers Cross-Resistance to 5-Fluorouracil and Azoles in Candida lusitaniae and Highlights Antagonistic Interaction between Fluorinated Nucleotides and Fluconazole

    PubMed Central

    Gabriel, Frédéric; Sabra, Ayman; El-Kirat-Chatel, Sofiane; Pujol, Sophie; Fitton-Ouhabi, Valérie; Brèthes, Daniel; Dementhon, Karine; Accoceberry, Isabelle

    2014-01-01

    We characterized two additional membrane transporters (Fur4p and Dal4p) of the nucleobase cation symporter 1 (NCS1) family involved in the uptake transport of pyrimidines and related molecules in the opportunistic pathogenic yeast Candida lusitaniae. Simple and multiple null mutants were constructed by gene deletion and genetic crosses. The function of each transporter was characterized by supplementation experiments, and the kinetic parameters of the uptake transport of uracil were measured using radiolabeled substrate. Fur4p specifically transports uracil and 5-fluorouracil. Dal4p is very close to Fur4p and transports allantoin (glyoxyldiureide). Deletion of the FUR4 gene confers resistance to 5-fluorouracil as well as cross-resistance to triazoles and imidazole antifungals when they are used simultaneously with 5-fluorouracil. However, the nucleobase transporters are not involved in azole uptake. Only fluorinated pyrimidines, not pyrimidines themselves, are able to promote cross-resistance to azoles by both the salvage and the de novo pathway of pyrimidine synthesis. A reinterpretation of the data previously obtained led us to show that subinhibitory doses of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine also were able to trigger resistance to fluconazole in susceptible wild-type strains of C. lusitaniae and of different Candida species. Our results suggest that intracellular fluorinated nucleotides play a key role in azole resistance, either by preventing azoles from targeting the lanosterol 14-alpha-demethylase or its catalytic site or by acting as a molecular switch for the triggering of efflux transport. PMID:24867971

  20. Synthesis and Characterization of bis[(2-ethyl-5-methyl-imidazo-4-yl)methyl]Sulfide and Its Coordination Behavior toward Cu(II) as a Possible Approach of a Copper Site Type I

    PubMed Central

    Barrón-Garcés, Juan D.; Mendoza-Díaz, Guillermo; Vilchez-Aguado, Florina; Bernès, Sylvain

    2009-01-01

    The synthesis and characterization of a novel ligand, bis[(2-ethyl-5-methyl-imidazo-4-yl)methyl]sulfide (bemims), as well as a bemims-containing copper(II) coordination complex are described. In this complex, [Cu(bemims)X2] with X = NO3−, bemims acts as a tridentate ligand and two monodentate nitrate ions complete the coordination sphere. Both imidazole N atoms and the thioether S atom of bemims participate in coordination. The Cu(II) ion is five-coordinated with a slightly distorted square-pyramidal geometry (τ = .09). Electrochemical studies and spectroscopic data for this complex are compared with some blue copper proteins in order to assess its ability to mimic the copper center of type I copper proteins. PMID:19587830

  1. Contactin-associated Protein 1 (Caspr1) Regulates the Traffic and Synaptic Content of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)-type Glutamate Receptors*

    PubMed Central

    Santos, Sandra D.; Iuliano, Olga; Ribeiro, Luís; Veran, Julien; Ferreira, Joana S.; Rio, Pedro; Mulle, Christophe; Duarte, Carlos B.; Carvalho, Ana Luísa

    2012-01-01

    Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type mediate fast excitatory synaptic transmission in the CNS. Synaptic strength is modulated by AMPA receptor binding partners, which regulate receptor synaptic targeting and functional properties. We identify Contactin-associated protein 1 (Caspr1) as an AMPA receptor interactor. Caspr1 is present in synapses and interacts with AMPA receptors in brain synaptic fractions. Coexpression of Caspr1 with GluA1 increases the amplitude of glutamate-evoked currents. Caspr1 overexpression in hippocampal neurons increases the number and size of synaptic GluA1 clusters, whereas knockdown of Caspr1 decreases the intensity of synaptic GluA1 clusters. Hence, Caspr1 is a regulator of the trafficking of AMPA receptors to synapses. PMID:22223644

  2. Contactin-associated protein 1 (Caspr1) regulates the traffic and synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors.

    PubMed

    Santos, Sandra D; Iuliano, Olga; Ribeiro, Luís; Veran, Julien; Ferreira, Joana S; Rio, Pedro; Mulle, Christophe; Duarte, Carlos B; Carvalho, Ana Luísa

    2012-02-24

    Glutamate receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type mediate fast excitatory synaptic transmission in the CNS. Synaptic strength is modulated by AMPA receptor binding partners, which regulate receptor synaptic targeting and functional properties. We identify Contactin-associated protein 1 (Caspr1) as an AMPA receptor interactor. Caspr1 is present in synapses and interacts with AMPA receptors in brain synaptic fractions. Coexpression of Caspr1 with GluA1 increases the amplitude of glutamate-evoked currents. Caspr1 overexpression in hippocampal neurons increases the number and size of synaptic GluA1 clusters, whereas knockdown of Caspr1 decreases the intensity of synaptic GluA1 clusters. Hence, Caspr1 is a regulator of the trafficking of AMPA receptors to synapses. PMID:22223644

  3. A DFT/TD DFT study of the structure and spectroscopic properties of 5-methyl-2-(8-quinolinyl)benzoxazole and its complexes with Zn(II) ion.

    PubMed

    Guzow, Katarzyna; Milewska, Magda; Czaplewski, Cezary; Wiczk, Wiesław

    2010-02-01

    The structure and spectroscopic properties of 5-methyl-2-(8-quinolinyl)benzoxazole and its complexes with Zn(II) ion were studied using a DFT and TD DFT methods with def2-TZVP basis set. It was shown that the type of functional used (B3-LYP or pbe0) implemented in TURBOMOLE package does not have essential influence on the geometry (small differences in bond length, valence and dihedral angles) of studied compounds in both ground and excited states. However, significant differences were obtained for the position of vertical absorption and emission transition but not for the oscillator strength of transition. Application of pbe0 functional seems to reproduce better the experimental spectrum.

  4. 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

  5. 5-Methyl-Tetrahydrofolate and the S-Adenosylmethionine Cycle in C57BL/6J Mouse Tissues: Gender Differences and Effects of Arylamine N-Acetyltransferase-1 Deletion

    PubMed Central

    Witham, Katey L.; Butcher, Neville J.; Sugamori, Kim S.; Brenneman, Debbie; Grant, Denis M.; Minchin, Rodney F.

    2013-01-01

    Folate catabolism involves cleavage of the C9-N10 bond to form p-aminobenzoylgluamate (PABG) and pterin. PABG is then acetylated by human arylamine N-acetyltransferase 1 (NAT1) before excretion in the urine. Mice null for the murine NAT1 homolog (Nat2) show several phenotypes consistent with altered folate homeostasis. However, the exact role of Nat2 in the folate pathway in vivo has not been reported. Here, we examined the effects of Nat2 deletion in male and female mice on the tissue levels of 5-methyl-tetrahydrofolate and the methionine-S-adenosylmethionine cycle. We found significant gender differences in hepatic and renal homocysteine, S-adenosylmethionine and methionine levels consistent with a more active methionine-S-adenosylmethionine cycle in female tissues. In addition, methionine levels were significantly higher in female liver and kidney. PABG was higher in female liver tissue but lower in kidney compared to male tissues. In addition, qPCR of mRNA extracted from liver tissue suggested a significantly lower level of Nat2 expression in female animals. Deletion of Nat2 affected liver 5- methyl-tetrahydrofolate in female mice but had little effect on other components of the methionine-S-adenosylmethionine cycle. No N-acetyl-PABG was observed in any tissues in Nat2 null mice, consistent with the role of Nat2 in PABG acetylation. Surprisingly, tissue PABG levels were similar between wild type and Nat2 null mice. These results show that Nat2 is not required to maintain tissue PABG homeostasis in vivo under normal conditions. PMID:24205029

  6. 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.

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

    PubMed

    Corsi, Steven R; Geis, Steven W; Loyo-Rosales, Jorge E; Rice, Clifford P

    2006-12-01

    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.

  8. 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

  9. Synergistic inhibition of human immunodeficiency virus type 1 replication by 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU) and azidothymidine in vitro.

    PubMed Central

    Baba, M; Ito, M; Shigeta, S; Tanaka, H; Miyasaka, T; Ubasawa, M; Umezu, K; Walker, R T; De Clercq, E

    1991-01-01

    A novel 6-substituted acyclouridine derivative, 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU), has recently proved to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. Combinations of 3'-azido-2',3'-dideoxythymidine (AZT) and E-EPU synergistically inhibit the replication of HIV-1 in MT-4 cells, whereas the cytotoxic effects of AZT and E-EPU on mock-infected MT-4 cells are not enhanced by the drug combination. Synergistic inhibition of HIV-1 replication has also been observed in peripheral blood lymphocytes. These results indicate that the combination of AZT and E-EPU should be further pursued in the treatment of AIDS. PMID:1929304

  10. Phosphate-triggered self-assembly of N-[(Uracil-5-yl)methyl]urea: a minimalistic urea-derived hydrogelator.

    PubMed

    Kleinsmann, Alexander J; Weckenmann, Nicole M; Nachtsheim, Boris J

    2014-07-28

    N-[(Uracil-5-yl)methyl]urea is reported as a minimalistic low-molecular-weight hydrogelator (LMWHG). The unusual phosphate-induced assembly of this compound has been thoroughly investigated by IR, UV/Vis, and NMR spectroscopy, electron microscopy, and rheological experiments. This rare example of an anion-triggered urea-based LMWHG is the first example of a pyrimidine- and urea-containing molecule that can be forced into self-assembly in aqueous solution without additional aromatic or lipophilic groups. The gelator/phosphate ratio within the hydrogel was successfully determined by (31) P MAS NMR spectroscopy. The hydrogel exhibits a very fast and repeatable self-healing property, and remarkable G' values. The viscoelastic properties of the hydrogel can easily be tuned by variation of the phosphate ratio.

  11. Photoisomerization of fluorinated 1,3-dimethyl-5-propenyl uracils as a cycle of sequential reactions: Electrocyclization in its use in light-fueled nanomotors

    NASA Astrophysics Data System (ADS)

    Siodla, Tomasz; Hoffmann, Marcin; Karwatka, Piotr; Koroniak, Henryk

    2011-01-01

    Research in the last ten years has demonstrated that light energy can be used to power artificial nanomotors by exploiting photochemical processes in appropriately designed systems. The photochemical properties of fluorinated derivatives of 1,3-dimethyl-5-propenyl uracils were studied assuming that the electrocyclization of the diene would be a dominant reaction. The primarily formed intermediate, a cyclobutene derivative was expected to undergo further electrocyclic conrotatory ring-opening leading to corresponding E or Z isomer. Keeping in mind the Woodward-Hoffmann and torquoselectivity rules, this photoisomerization is the stereospecific reaction, where the isomerization of each of isomers processes through a different trans-cis cyclobutene-intermediates. This is a cycle of a sequent reactions capable to provide the selective 360° clockwork rotation, which can be used as a model to design a light-fueled nanomotor.

  12. Cisplatin/Tegafur/Uracil/Irinotecan Triple Combination Therapy for Recurrent/Metastatic Head and Neck Squamous Cell Carcinoma: A Phase I/II Clinical Study

    PubMed Central

    Chen, San-Chi; Chang, Peter Mu-Hsin

    2016-01-01

    Lessons Learned Cisplatin/tegafur/uracil/irinotecan triple combination therapy shows moderate response, especially in patients without previous chemoradiotherapy within the 6 months before this combination therapy. Toxicity is tolerable, and quality of life is improved in responders. Background. The prognosis is poor in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). Triple combination therapy may increase tumor response. Methods. This phase I/II prospective trial first determined the dose-limiting toxicity and recommended dose of irinotecan with cisplatin and tegafur/uracil (UFUR) in phase I. Irinotecan was supplied at doses of 40, 50, 60, and 70 mg/m2 by using a standard 3+3 design. Doses of cisplatin and UFUR were held stable. In phase II, the recommended dose of irinotecan was administered intravenously (i.v.) over 90 min on day 1, with cisplatin 50 mg/m2 i.v. over 60 min also on day 1, and oral UFUR 200 mg twice a day for 5 days every 2 weeks a cycle. Results. In the phase I portion, 14 patients were enrolled, and the dose level of irinotecan at 60 mg/m2 was defined as the recommended dose for the phase II portion of the study. Among 43 patients enrolled in the phase II portion, complete response was seen in 2 patients (4.7%) and partial response in 10 patients (23.3%), and the disease control rate was 39.5%. In a subgroup analysis of patients whose prior chemoradiotherapy was more than 6 months earlier, a response rate of 40.7% and disease control rate of 59.3% were observed. Conclusion. Cisplatin/UFUR/irinotecan triple combination therapy is tolerated and effective for selected patients. Individualized choice of treatment will influence prognosis and quality of life in R/M HNSCC patients. PMID:27091418

  13. 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

  14. In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.

    PubMed

    Erbs, P; Regulier, E; Kintz, J; Leroy, P; Poitevin, Y; Exinger, F; Jund, R; Mehtali, M

    2000-07-15

    Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies. PMID:10919655

  15. 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.

  16. 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.

  17. Synthesis, spectral characterization and antioxidant activity studies of a bidentate Schiff base, 5-methyl thiophene-2-carboxaldehyde-carbohydrazone and its Cd(II), Cu(II), Ni(II) and Zn(II) complexes

    NASA Astrophysics Data System (ADS)

    Harinath, Y.; Harikishore Kumar Reddy, D.; Naresh Kumar, B.; Apparao, Ch.; Seshaiah, K.

    2013-01-01

    A new Schiff base bidentate ligand (L), 5-methyl thiophene-2-carboxaldehyde-carbohydrazone and its metal (Cu(II), Cd(II), Ni(II) and Zn(II)) complexes with general stoichiometry [M(L)2X2] (where X = Cl) were synthesized. The ligand and its metal complexes were characterized by elemental analyses, IR, 1H NMR, ESR spectral analyses, and molar conductance studies. The molar conductance data revealed that all the metal chelates are non-electrolytes. IR spectra showed that ligand (L) is coordinated to the metal ions in a bidentate manner with N and O donor sites of the azomethine-N, and carbonyl-O. ESR and UV-Vis spectral data showed that the geometrical structure of the complexes are Orthorhombic. Furthermore, the antioxidant activity of the ligand and its complexes was determined by hydroxyl radical scavenging, DPPH, NO, reducing power methods in vitro. The obtained IC50 value of the DPPH activity for the copper complex (IC50 = 66.4 μm) was higher than other compounds. Microbial assay of the above complexes against Staphylococcus aureus, Escherichia coli, Rhizocotonia bataticola and Alternaria alternata showed that copper complex exhibited higher activity than the other complexes.

  18. Design, Synthesis, and Preclinical Evaluation of 4-Substituted-5-methyl-furo[2,3-d]pyrimidines as Microtubule Targeting Agents That Are Effective against Multidrug Resistant Cancer Cells.

    PubMed

    Devambatla, Ravi Kumar Vyas; Namjoshi, Ojas A; Choudhary, Shruti; Hamel, Ernest; Shaffer, Corena V; Rohena, Cristina C; Mooberry, Susan L; Gangjee, Aleem

    2016-06-23

    The design, synthesis, and biological evaluations of eight 4-substituted 5-methyl-furo[2,3-d]pyrimidines are reported. Synthesis involved N(4)-alkylation of N-aryl-5-methylfuro[2,3-d]pyrimidin-4-amines, obtained from Ullmann coupling of 4-amino-5-methylfuro[2,3-d]pyrimidine and appropriate aryl iodides. Compounds 3, 4, and 9 showed potent microtubule depolymerizing activities, while compounds 6-8 had slightly lower potency. Compounds 4, 6, 7, and 9 inhibited tubulin assembly with IC50 values comparable to that of combretastatin A-4 (CA-4). Compounds 3, 4, and 6-9 circumvented Pgp and βIII-tubulin mediated drug resistance, mechanisms that can limit the efficacy of paclitaxel, docetaxel, and the vinca alkaloids. In the NCI 60-cell line panel, compound 3 exhibited GI50 values less than 10 nM in 47 of the cell lines. In an MDA-MB-435 xenograft model, compound 3 had statistically significant antitumor effects. The biological effects of 3 identify it as a novel, potent microtubule depolymerizing agent with antitumor activity. PMID:27213719

  19. 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.

  20. 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.

  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. 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.

  3. Combined ESR and thermodynamic studies of the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO): hindered rotation around the O-O bond evidenced by two-dimensional simulation of temperature-dependent spectra.

    PubMed

    Rockenbauer, Antal; Clément, Jean-Louis; Culcasi, Marcel; Mercier, Anne; Tordo, Paul; Pietri, Sylvia

    2007-06-14

    Experiments were performed to elucidate the origin of the superhyperfine structure and line width alternation (LWA) seen in the ESR spectrum of the major diastereoisomer (1) of DEPMPO-OOH, the remarkably persistent superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). Using selectively deuterated DEPMPO derivatives, we demonstrated that the superhyperfine pattern can be unambiguously attributed to long-range couplings. The recording in pyridine of highly resolved spectra in a wide temperature range, combined with two-dimensional simulation, allowed us to characterize an inverted LWA in 1 and revealed a uniform line broadening in the spectrum of the minor DEPMPO-OOH diastereoisomer (2), with both effects originating from a chemical exchange between conformers. When the individual spectra of 1 presenting LWA in the fast-exchange regime were simulated, four equally good fits were obtained and this ambiguity could be resolved by using a two-dimensional simulation technique. The thermodynamic and kinetic constants of this exchange were consistent with a rotation around the O-O bond. We propose that line broadening effects in 1 and 2 result from this O-O rotation concerted with the pseudo-rotation of the pyrrolidine ring. PMID:17518450

  4. 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.

  5. Crystal structure of (2-hy-droxy-5-methyl-phen-yl)(3-methyl-1-phenyl-1H-pyrazolo-[3,4-b]pyridin-5-yl)methanone.

    PubMed

    Raja, Rajamani; Poomathi, Nataraj; Perumal, Paramasivam T; SubbiahPandi, A

    2015-07-01

    In the title compound, C21H17N3O2, the 2-hy-droxy-5-methyl-phenyl ring and the phenyl ring are inclined to the mean plane of the pyrazolo-pyridine moiety (r.m.s. deviation = 0.013 Å) by 52.89 (9) and 19.63 (8)°, respectively, and to each other by 42.83 (11)°. In the mol-ecule, there are intra-molecular O-H⋯O and C-H⋯N hydrogen bonds, both enclosing an S(6) ring motif. In the crystal, mol-ecules stack along the c-axis direction, forming columns within which there are a number of π-π inter-actions [the inter-centroid distances vary from 3.5278 (10) to 3.8625 (10) Å]. The columns are linked by C-H⋯π inter-actions, forming slabs parallel to (100). PMID:26279931

  6. Germline ablation of SMUG1 DNA glycosylase causes loss of 5-hydroxymethyluracil- and UNG-backup uracil-excision activities and increases cancer predisposition of Ung-/-Msh2-/- mice.

    PubMed

    Kemmerich, Kristin; Dingler, Felix A; Rada, Cristina; Neuberger, Michael S

    2012-07-01

    Deamination of cytosine (C), 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) occurs spontaneously in mammalian DNA with several hundred deaminations occurring in each cell every day. The resulting potentially mutagenic mispairs of uracil (U), thymine (T) or 5-hydroxymethyluracil (hmU) with guanine (G) are substrates for repair by various DNA glycosylases. Here, we show that targeted inactivation of the mouse Smug1 DNA glycosylase gene is sufficient to ablate nearly all hmU-DNA excision activity as judged by assay of tissue extracts from knockout mice as well as by the resistance of their embryo fibroblasts to 5-hydroxymethyldeoxyuridine toxicity. Inactivation of Smug1 when combined with inactivation of the Ung uracil-DNA glycosylase gene leads to a loss of nearly all detectable uracil excision activity. Thus, SMUG1 is the dominant glycosylase responsible for hmU-excision in mice as well as the major UNG-backup for U-excision. Both Smug1-knockout and Smug1/Ung-double knockout mice breed normally and remain apparently healthy beyond 1 year of age. However, combined deficiency in SMUG1 and UNG exacerbates the cancer predisposition of Msh2(-/-) mice suggesting that when both base excision and mismatch repair pathways are defective, the mutagenic effects of spontaneous cytosine deamination are sufficient to increase cancer incidence but do not preclude mouse development.

  7. Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. I. Rodent cerebral white matter and cortex.

    PubMed

    Talos, Delia M; Fishman, Rachel E; Park, Hyunkyung; Folkerth, Rebecca D; Follett, Pamela L; Volpe, Joseph J; Jensen, Frances E

    2006-07-01

    This is the first part of a two-part study to investigate the cellular distribution and temporal regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunits in the developing white matter and cortex in rat (part I) and human (part II). Western blot and immunocytochemistry were used to evaluate the differential expression of AMPAR subunits on glial and neuronal subtypes during the first 3 postnatal weeks in the Long Evans and Sprague Dawley rat strains. In Long Evans rats during the first postnatal week, GluR2-lacking AMPARs were expressed predominantly on white matter cells, including radial glia, premyelinating oligodendrocytes, and subplate neurons, whereas, during the second postnatal week, these AMPARs were highly expressed on cortical neurons, coincident with decreased expression on white matter cells. Immunocytochemical analysis revealed that cell-specific developmental changes in AMPAR expression occurred 2-3 days earlier by chronological age in Sprague Dawley rats compared with Long Evans rats, despite overall similar temporal sequencing. In both white and gray matter, the periods of high GluR2 deficiency correspond to those of regional susceptibility to hypoxic/ischemic injury in each of the two rat strains, supporting prior studies suggesting a critical role for Ca2+-permeable AMPARs in excitotoxic cellular injury and epileptogenesis. The developmental regulation of these receptor subunits strongly suggests that Ca2+ influx through GluR2-lacking AMPARs may play an important role in neuronal and glial development and injury in the immature brain. Moreover, as demonstrated in part II, there are striking similarities between rat and human in the regional and temporal maturational regulation of neuronal and glial AMPAR expression.

  8. Developmental regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit expression in forebrain and relationship to regional susceptibility to hypoxic/ischemic injury. II. Human cerebral white matter and cortex.

    PubMed

    Talos, Delia M; Follett, Pamela L; Folkerth, Rebecca D; Fishman, Rachel E; Trachtenberg, Felicia L; Volpe, Joseph J; Jensen, Frances E

    2006-07-01

    This report is the second of a two-part evaluation of developmental differences in alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunit expression in cell populations within white matter and cortex. In part I, we reported that, in rat, developmental expression of Ca2+-permeable (GluR2-lacking) AMPARs correlated at the regional and cellular level with increased susceptibility to hypoxia/ischemia (H/I), suggesting an age-specific role of these receptors in the pathogenesis of brain injury. Part II examines the regional and cellular progression of AMPAR subunits in human white matter and cortex from midgestation through early childhood. Similarly to the case in the rodent, there is a direct correlation between selective vulnerability to H/I and expression of GluR2-lacking AMPARs in human brain. For midgestational cases aged 20-24 postconceptional weeks (PCW) and for premature infants (25-37 PCW), we found that radial glia, premyelinating oligodendrocytes, and subplate neurons transiently expressed GluR2-lacking AMPARs. Notably, prematurity represents a developmental window of selective vulnerability for white matter injury, such as periventricular leukomalacia (PVL). During term (38-42 PCW) and postterm neonatal (43-46 PCW) periods, age windows characterized by increased susceptibility to cortical injury and seizures, GluR2 expression was low in the neocortex, specifically on cortical pyramidal and nonpyramidal neurons. This study indicates that Ca2+-permeable AMPAR blockade may represent an age-specific therapeutic strategy for potential use in humans. Furthermore, these data help to validate specific rodent maturational stages as appropriate models for evaluation of H/I pathophysiology.

  9. 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

  10. Solvation Effects on the Static and Dynamic First-Order Electronic and Vibrational Hyperpolarizabilities of Uracil: A Polarized Continuum Model Investigation

    PubMed Central

    Alparone, Andrea

    2013-01-01

    Electronic (βe) and vibrational (βv) first-order hyperpolarizabilities of uracil were determined in gas and water solution using the Coulomb-attenuating Density Functional Theory level with the Dunning's correlation-consistent aug-cc-pVDZ basis set. Frequency-dependent βe values were computed for the Second Harmonic Generation (SHG) and Electric Optical Pockels Effect (EOPE) nonlinear optical phenomena. The Polarized Continuum Model was employed to study the solvent effects on the electronic and vibrational properties. The introduction of solvation contributions increases the βe(static) value by ca. 110%. In comparison, smaller enhancements are found for the βe(EOPE) and βe(SHG) data evaluated at the typical wavelength of 694 nm (by 40–50%). The gas-water hyperpolarizability difference was rationalised through a density analysis study. The magnitudes of the vibrational first-order hyperpolarizabilities are comparable to their electronic counterparts and noticeably increase in solution: βv(EOPE) ~ βe(EOPE) in aqueous phase at λ = 694 nm. Analysis of the IR and Raman spectra is useful to elucidate the most important contributing modes to the vibrational first-order hyperpolarizabilities. PMID:24453886

  11. Structural and Energetic Impact of Non-Natural 7-Deaza-8-Azaadenine and Its 7-Substituted Derivatives on H-Bonding Potential with Uracil in RNA Molecules.

    PubMed

    Chawla, Mohit; Credendino, Raffaele; Oliva, Romina; Cavallo, Luigi

    2015-10-15

    Non-natural (synthetic) nucleobases, including 7-ethynyl- and 7-triazolyl-8-aza-7-deazaadenine, have been introduced in RNA molecules for targeted applications, and have been characterized experimentally. However, no theoretical characterization of the impact of these modifications on the structure and energetics of the corresponding H-bonded base pair is available. To fill this gap, we performed quantum mechanics calculations, starting with the analysis of the impact of the 8-aza-7-deaza modification of the adenine skeleton, and we moved then to analyze the impact of the specific substituents on the modified 8-aza-7-deazaadenine. Our analysis indicates that, despite of these severe structural modifications, the H-bonding properties of the modified base pair gratifyingly replicate those of the unmodified base pair. Similar behavior is predicted when the same skeleton modifications are applied to guanine when paired to cytosine. To stress further the H-bonding pairing in the modified adenine-uracil base pair, we explored the impact of strong electron donor and electron withdrawing substituents on the C7 position. Also in this case we found minimal impact on the base pair geometry and energy, confirming the validity of this modification strategy to functionalize RNAs without perturbing its stability and biological functionality.

  12. Structural and Energetic Impact of Non-Natural 7-Deaza-8-Azaadenine and Its 7-Substituted Derivatives on H-Bonding Potential with Uracil in RNA Molecules.

    PubMed

    Chawla, Mohit; Credendino, Raffaele; Oliva, Romina; Cavallo, Luigi

    2015-10-15

    Non-natural (synthetic) nucleobases, including 7-ethynyl- and 7-triazolyl-8-aza-7-deazaadenine, have been introduced in RNA molecules for targeted applications, and have been characterized experimentally. However, no theoretical characterization of the impact of these modifications on the structure and energetics of the corresponding H-bonded base pair is available. To fill this gap, we performed quantum mechanics calculations, starting with the analysis of the impact of the 8-aza-7-deaza modification of the adenine skeleton, and we moved then to analyze the impact of the specific substituents on the modified 8-aza-7-deazaadenine. Our analysis indicates that, despite of these severe structural modifications, the H-bonding properties of the modified base pair gratifyingly replicate those of the unmodified base pair. Similar behavior is predicted when the same skeleton modifications are applied to guanine when paired to cytosine. To stress further the H-bonding pairing in the modified adenine-uracil base pair, we explored the impact of strong electron donor and electron withdrawing substituents on the C7 position. Also in this case we found minimal impact on the base pair geometry and energy, confirming the validity of this modification strategy to functionalize RNAs without perturbing its stability and biological functionality. PMID:26389789

  13. Resonance Raman Intensities Demonstrate that C5 Substituents Affect the Initial Excited-State Structural Dynamics of Uracil More than C6 Substituents.

    PubMed

    Teimoory, Faranak; Loppnow, Glen R

    2016-05-01

    Resonance Raman derived initial excited-state structural dynamics provide insight into the photochemical mechanisms of pyrimidine nucleobases, in which the photochemistry appears to be dictated by the C5 and C6 substituents. The absorption and resonance Raman spectra and excitation profiles of 5,6-dideuterouracil were measured to further test this photochemical dependence on the C5 and C6 substituents. The resulting set of excited-state reorganization energies of the observed internal coordinates were calculated and compared to those of other 5- and 6-substituted uracils. The results show that the initial excited-state dynamics along the C5C6 stretch responds to changes in mass at C5 and C6 in the same manner but that the in-plane bends at C5 and C6 are more sensitive to substituents at the C5 position than at the C6 position. In addition, the presence of two deuterium substituents at C5 and C6 decreases the initial excited-state structural dynamics along these in-plane bends, in contrast to what is observed in the presence of two CH3 groups on C5 and C6. The results are discussed in the context of DNA nucleobase photochemistry.

  14. Crystal structures of (E)-N'-(2-hy-droxy-5-methyl-benzyl-idene)isonicotinohydrazide and (E)-N'-(5-fluoro-2-hy-droxy-benzyl-idene)isonicotinohydrazide.

    PubMed

    Chainok, Kittipong; Makmuang, Sureerat; Kielar, Filip

    2016-07-01

    Two derivatives of the well-known iron chelator, (E)-N'-(2-hy-droxy-benzyl-idene)isonicotinohydrazide (SIH), substituted in the 5-position of the 2-hy-droxy-benzene ring by a methyl and a fluorine group viz. (E)-N'-(2-hy-droxy-5-methyl-benzyl-idene)isonicotinohydrazide, C14H13N3O2, (I), and (E)-N'-(5-fluoro-2-hy-droxy-benzyl-idene)isonicotinohydrazide, C13H10FN3O2, (II), have been prepared and characterized by single-crystal X-ray diffraction, (1)H NMR and mass spectrometry. The mol-ecules of both compounds deviate slightly from planarity [r.m.s. deviations are 0.145 and 0.110 Å for (I) and (II), respectively] and adopt an E conformation with respect to the double bond of the hydrazone bridge. In each mol-ecule, there is an intra-molecular O-H⋯N hydrogen bond forming an S(6) ring motif. The dihedral angles between the mean planes of the isonicotinoyl ring and the cresol ring in (I) or the fluoro-phenol ring in (II) are 10.49 (6) and 9.43 (6)°, respectively. In the crystals of both compounds, zigzag chains are formed via N-H⋯N hydrogen bonds, in the [10-1] direction for (I) and [010] for (II). In (I), the chains are linked by weak C-H⋯π and π-π stacking inter-actions [centroid-to-centroid distances = 3.6783 (8) Å; inter-planar angle = 10.94 (5)°], leading to the formation of a three-dimensional supra-molecular architecture. In (II), adjacent chains are connected through C-H⋯O hydrogen bonds to form sheets parallel to (100), which enclose R 4 (4)(30) ring motifs. The sheets are linked by weak C-H⋯π and π-π [centroid-to-centroid distance = 3.7147 (8) Å; inter-planar angle = 10.94 (5)°] inter-actions, forming a three-dimensional supra-molecular architecture. PMID:27555945

  15. Crystal structures of (E)-N′-(2-hy­droxy-5-methyl­benzyl­idene)isonicotinohydrazide and (E)-N′-(5-fluoro-2-hy­droxy­benzyl­idene)isonicotinohydrazide

    PubMed Central

    Chainok, Kittipong; Makmuang, Sureerat; Kielar, Filip

    2016-01-01

    Two derivatives of the well-known iron chelator, (E)-N′-(2-hy­droxy­benzyl­idene)isonicotinohydrazide (SIH), substituted in the 5-position of the 2-hy­droxy­benzene ring by a methyl and a fluorine group viz. (E)-N′-(2-hy­droxy-5-methyl­benzyl­idene)isonicotinohydrazide, C14H13N3O2, (I), and (E)-N′-(5-fluoro-2-hy­droxy­benzyl­idene)isonicotinohydrazide, C13H10FN3O2, (II), have been prepared and characterized by single-crystal X-ray diffraction, 1H NMR and mass spectrometry. The mol­ecules of both compounds deviate slightly from planarity [r.m.s. deviations are 0.145 and 0.110 Å for (I) and (II), respectively] and adopt an E conformation with respect to the double bond of the hydrazone bridge. In each mol­ecule, there is an intra­molecular O—H⋯N hydrogen bond forming an S(6) ring motif. The dihedral angles between the mean planes of the isonicotinoyl ring and the cresol ring in (I) or the fluoro­phenol ring in (II) are 10.49 (6) and 9.43 (6)°, respectively. In the crystals of both compounds, zigzag chains are formed via N—H⋯N hydrogen bonds, in the [10-1] direction for (I) and [010] for (II). In (I), the chains are linked by weak C—H⋯π and π–π stacking inter­actions [centroid-to-centroid distances = 3.6783 (8) Å; inter-planar angle = 10.94 (5)°], leading to the formation of a three-dimensional supra­molecular architecture. In (II), adjacent chains are connected through C—H⋯O hydrogen bonds to form sheets parallel to (100), which enclose R 4 4(30) ring motifs. The sheets are linked by weak C—H⋯π and π–π [centroid-to-centroid distance = 3.7147 (8) Å; inter-planar angle = 10.94 (5)°] inter­actions, forming a three-dimensional supra­molecular architecture. PMID:27555945

  16. Downregulation of stargazin inhibits the enhanced surface delivery of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor GluR1 subunit in rat dorsal horn and ameliorates postoperative pain

    PubMed Central

    Guo, Ruijuan; Zhao, Yujie; Zhang, Meijuan; Wang, Yue; Shi, Rong; Liu, Yang; Xu, Jie; Wu, Anshi; Yue, Yun; Wu, Jing; Guan, Yun; Wang, Yun

    2014-01-01

    Background Stargazin is the first transmembrane protein known to regulate synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. Yet, it is unclear whether regulation of the surface delivery of spinal AMPA receptor subunits by stargazin contributes to postoperative pain development. Methods Western blot analysis was used to examine changes in the surface delivery of AMPA receptor subunits GluR1 and GluR2 in rat dorsal horn. The interaction between stargazin and GluR1 and GluR2 was examined by coimmunoprecipitation. Expression of stargazin was suppressed by intrathecal administration of small interfering ribonucleic acid (siRNA)311. Results Membrane-bound GluR1, but not GluR2, in ipsilateral dorsal horn was increased at 3 h (1.49±0.15-fold of β-tubulin, mean±SEM) and 1 day (1.03±0.25) after incision, as compared to that in control rats (naïve, 0.63±0.23, P < 0.05, n=6/group). The amount of GluR1 coimmunoprecipitated with stargazin was greater at 3 h after incision (1.48±0.31-fold of input) than in control animals (0.45±0.24, P < 0.05, n=6/group). Importantly, the increase in membrane GluR1 at 3 h after incision was normalized to near control level (0.72±0.20-fold of β-tubulin) by pretreatment with intrathecal stargazin siRNA311 (0.87±0.09), but not scrambled siRNA (1.48±0.24) or vehicle (1.25±0.13, P < 0.05, n=6/group). Stargazin siRNA311 pretreatment prevented the increase in stargazin–GluR1 interaction and decreased postoperative pain after incision. Conclusions This study suggests a critical role of stargazin-mediated surface delivery of GluR1 subunit in the development of postoperative pain. A better therapeutic strategy for postoperative pain may involve selectively downregulating spinal stargazin to inhibit synaptic targeting of GluR1 subunit. PMID:25093662

  17. Positive allosteric modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors differentially modulates the behavioural effects of citalopram in mouse models of antidepressant and anxiolytic action.

    PubMed

    Fitzpatrick, Ciarán M; Larsen, Maria; Madsen, Louise H; Caballero-Puntiverio, Maitane; Pickering, Darryl S; Clausen, Rasmus P; Andreasen, Jesper T

    2016-09-01

    Drugs that increase monoamine neurotransmission are effective in both anxiety and depression. The therapeutic effects of monoamine-based antidepressant drugs may involve indirect effects on neurotransmission through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR). Thus, chronic antidepressant treatment increases AMPAR-mediated neurotransmission and AMPAR-positive allosteric modulators have shown antidepressant-like efficacy in rodents. Here, the effect of enhanced AMPAR neurotransmission on the antidepressant-like and anxiolytic-like actions of the selective serotonin reuptake inhibitor citalopram (0-10 mg/kg) was investigated in mice using the AMPAR-positive allosteric modulator LY451646 (0-3 mg/kg). Antidepressant-like effects were assessed using the forced-swim test (FST), whereas anxiolytic-like effects were tested using the elevated zero maze (EZM) and the marble burying test. LY451646 (3 mg/kg) increased swim distance in the FST and a subactive dose of LY451646 (1 mg/kg) enhanced the effect of citalopram in the FST. In the EZM, LY451646 (3 mg/kg) did not show anxiogenic effects alone, but blocked the anxiolytic-like action of citalopram in the EZM, as reflected by an increase in the latency to enter the open areas and a decrease in the number of entries and time spent in the open areas in citalopram-treated mice. In the marble burying test, LY451646 (3 mg/kg) showed no effect alone, but significantly attenuated the anxiolytic-like effect of citalopram (1.25-2.5 mg/kg) by increasing the number of marbles buried in citalopram-treated mice. These results suggest that AMPAR neurotransmission plays opposite roles in anxiety and depression as AMPAR potentiation facilitated the antidepressant-like effects of citalopram while attenuating its anxiolytic-like effect. These findings have ramifications in the search for AMPAR-based novel anxiolytic and antidepressant treatments. PMID:27341500

  18. 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.

  19. Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer specific cytotoxicity and tumor growth delay

    PubMed Central

    Christensen, Camilla L.; Gjetting, Torben; Poulsen, Thomas T.; Cramer, Frederik; Roth, Jack A.; Poulsen, Hans S.

    2012-01-01

    Purpose Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment and novel therapies are therefore in high demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug Experimental design The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter Insulinoma-associated 1 (INSM1). Therapeutic effect was evaluated in vitro in SCLC cell lines and in vivo in SCLC xenografted nude mice using the non-viral nanoparticle, DOTAP:Cholesterol for transgene delivery. Results INSM1-YCD/5-FC and INSM1-YCD-YUPRT/5-FC therapy induced high cytotoxicity in a range of SCLC cell lines. The highest therapeutic effect was obtained from the YCD-YUPRT fusion gene strategy. No cytotoxicity was induced after treatment of cell lines of other origin than SCLC. In addition the INSM1-YCD-YUPRT/5-FC therapy was superior to an established suicide gene system consisting of the Herpes Simplex Virus Thymidine Kinase (HSVTK) gene and prodrug Ganciclovir (GCV). The superior effect was in part due to massive bystander cytotoxicity of YCD-YUPRT-produced toxins. Finally, INSM1-YCD-YUPRT/5-FC therapy induced significant tumor growth delay in SCLC xenografts compared to control treated xenografts. Conclusions The current study is the first to test cytosine deaminase-based suicide gene therapy for SCLC and the first to demonstrate an anti-tumor effect from the delivery of suicide gene therapeutics for SCLC in vivo. PMID:20371678

  20. Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to Toxoplasma gondii ▿ †

    PubMed Central

    Fox, Barbara A.; Bzik, David J.

    2010-01-01

    The orotidine-5′-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting ΔOMPDC, ΔUP, and ΔOMPDC ΔUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The ΔUP knockout strain was replication competent and virulent. In contrast, the ΔOMPDC and ΔOMPDC ΔUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the ΔOMPDC strain but not the ΔOMPDC ΔUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the ΔOMPDC and ΔOMPDC ΔUP knockout strains exhibited extreme attenuation in murine virulence (∼8 logs). Genetic complementation of the ΔOMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated ΔOMPDC strain or the ΔOMPDC ΔUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting ΔOMPDC and ΔOMPDC ΔUP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered. PMID:20605980

  1. X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

    PubMed

    Jones, Kate L; Roche, Michael; Gantier, Michael P; Begum, Nasim A; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R G; Mak, Johnson

    2010-06-11

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  2. X4 and R5 HIV-1 Have Distinct Post-entry Requirements for Uracil DNA Glycosylase during Infection of Primary Cells

    PubMed Central

    Jones, Kate L.; Roche, Michael; Gantier, Michael P.; Begum, Nasim A.; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R. G.; Mak, Johnson

    2010-01-01

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

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

    PubMed

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

    2014-08-01

    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 (Kb) 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. PMID:25084329

  4. 5-Thiocyanato-2′-deoxyuridine as a Possible Radiosensitizer: Electron-Induced Formation of Uracil-C5-Thiyl Radical and Its Dimerization

    PubMed Central

    Zdrowowicz, Magdalena; Chomicz, Lidia; Żyndul, Michał; Wityk, Paweł; Wiegand, Tyler J.; Hanson, Cameron G.; Adhikary, Amitava

    2015-01-01

    In this work, we have synthesized 5-thiocyanato-2′-deoxyuridine (SCNdU) along with the C6-deuterated nucleobase 5-thiocyanatouracil (6-D-SCNU) and studied their reactions with radiation-produced electrons. ESR spectra in γ-irradiated nitrogen-saturated frozen homogeneous solutions (7.5 M LiCl in H2O or D2O) of these compounds show that electron-induced S-CN bond cleavage occurs to form a thiyl radical (dU-5-S• or 6-D-U-5-S•) and CN− via the initial π-anion radical (SCNdU•−) intermediate in which the excess electron is on the uracil base. HPLC and LC-MS/MS studies of γ-irradiated N2-saturated aqueous solutions of SCNdU in the presence of sodium formate as a OH-radical scavenger at ambient temperature show the formation of the dU-5S-5S-dU dimer in preference to dU by about 10 to 1 ratio. This shows that both possible routes of electron-induced bond cleavage (dUC5-SCN and S-CN) in SCNdU•− and dU-5-S• formation are preferred for the production of the σ-type uracilyl radical (dU•) by 10 fold. DFT/M06-2x/6-31++G(d,p) calculations employing the polarizable continuum model (PCM) for aqueous solutions show that dU-5-S• and CN− formation was thermodynamically favored by over 15 kcal/mol (ΔG) compared to dU• and SCN− production. The activation barriers for C5-S and S-CN bond cleavage in SCNdU•− amount to 8.7 and 4.0 kcal/mol, respectively, favoring dU-5-S• and CN− formation. These results support the experimental observation of S-CN bond cleavage by electron addition to SCNdU that results in the formation of dU-5-S• and the subsequent dU-5S-5S-dU dimer. This establishes SCNdU as a potential radiosensitizer that could cause intra- and inter-strand crosslinking as well as DNA-protein crosslinking via S-S dimer formation. PMID:26059609

  5. 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

  6. 5-Thiocyanato-2'-deoxyuridine as a possible radiosensitizer: electron-induced formation of uracil-C5-thiyl radical and its dimerization.

    PubMed

    Zdrowowicz, Magdalena; Chomicz, Lidia; Żyndul, Michał; Wityk, Paweł; Rak, Janusz; Wiegand, Tyler J; Hanson, Cameron G; Adhikary, Amitava; Sevilla, Michael D

    2015-07-14

    In this work, we have synthesized 5-thiocyanato-2'-deoxyuridine (SCNdU) along with the C6-deuterated nucleobase 5-thiocyanatouracil (6-D-SCNU) and studied their reactions with radiation-produced electrons. ESR spectra in γ-irradiated nitrogen-saturated frozen homogeneous solutions (7.5 M LiCl in H2O or D2O) of these compounds show that electron-induced S-CN bond cleavage occurs to form a thiyl radical (dU-5-S˙ or 6-D-U-5-S˙) and CN(-)via the initial π-anion radical (SCNdU˙(-)) intermediate in which the excess electron is on the uracil base. HPLC and LC-MS/MS studies of γ-irradiated N2-saturated aqueous solutions of SCNdU in the presence of sodium formate as a OH-radical scavenger at ambient temperature show the formation of the dU-5S-5S-dU dimer in preference to dU by about 10 to 1 ratio. This shows that both possible routes of electron-induced bond cleavage (dUC5-SCN and S-CN) in SCNdU˙(-) and dU-5-S˙ formation are preferred for the production of the σ-type uracilyl radical (dU˙) by 10 fold. DFT/M06-2x/6-31++G(d,p) calculations employing the polarizable continuum model (PCM) for aqueous solutions show that dU-5-S˙ and CN(-) formation was thermodynamically favored by over 15 kcal mol(-1) (ΔG) compared to dU˙ and SCN(-) production. The activation barriers for C5-S and S-CN bond cleavage in SCNdU˙(-) amount to 8.7 and 4.0 kcal mol(-1), respectively, favoring dU-5-S˙ and CN(-) formation. These results support the experimental observation of S-CN bond cleavage by electron addition to SCNdU that results in the formation of dU-5-S˙ and the subsequent dU-5S-5S-dU dimer. This establishes SCNdU as a potential radiosensitizer that could cause intra- and inter-strand crosslinking as well as DNA-protein crosslinking via S-S dimer formation.

  7. 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

  8. 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

  9. 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. PMID:27146565

  10. Potent anti-viral 5-(2-bromovinyl) uracil nucleosides are inactive at inducing gene mutations in Salmonella typhimurium and V79 Chinese hamster cells and unscheduled DNA synthesis in primary rat hepatocytes.

    PubMed

    Marquardt, H; Westendorf, J; De Clercq, E; Marquardt, H

    1985-08-01

    (E)-5-(2-bromovinyl)-2'-deoxyuridine (BDVU), one of the most potent and selective anti-herpes agents described to date, and its close congeners (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) and (E)-5-(2-bromovinyl)uracil (BVU), as well as the reference compounds 5-iodo-2'-deoxyuridine (IDU) and 5-trifluoro-2'-deoxythymidine (TFT) were examined for their genotoxic potential. With the exception of a weak activity of TFT in the newly developed strain TA 102, none of the compounds was active in a bacterial cell mutagenesis (Salmonella/microsome) assay. Nor did they induce DNA repair (unscheduled DNA synthesis) in primary rat hepatocytes. In a mammalian cell mutagenesis assay using V79 Chinese hamster cells, the reference compounds IDU and TFT proved highly cytotoxic and mutagenic, whereas BVDU, BVaraU and BVU were neither cytotoxic nor mutagenic.

  11. New 2,6-bis-[uracil-imino] ethylpyridine complexes containing the CdN(6) core: Synthesis, crystal structures, luminescent properties and antiproliferative activity against C6 glioma cells.

    PubMed

    Illán-Cabeza, Nuria A; Jiménez-Pulido, Sonia B; Martínez-Martos, José M; Ramírez-Expósito, María J; Moreno-Carretero, Miguel N

    2009-08-01

    Three new Cd(II) complexes with the Schiff base ligand derived from the condensation 1+2 of 2,6-diacetylpyridine and 5,6-diamino-1,3-dimethyluracil have been "in template" synthesized. The molecular structures of complexes were determined by single-crystal X-ray diffraction. The metal center shows a very distorted mer-bis-tridentate CdN(6) octahedral geometry as consequence of the reduced bite angles of the ligand and the existence of long-distanced interactions with donor atoms in the neighbourhood. The luminescent properties of complexes in CH(3)CN solution were investigated showing the emission energies depend on the uracil part of the ligand. The evaluation of their biological properties against C6 glioma cell line indicates that cadmium(II) complexes could be an interesting tools to treat drug-resistant brain tumors. PMID:19616303

  12. 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.

  13. 2-Amino-5-methyl-pyridinium 4-chloro-benzoate.

    PubMed

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

    2013-01-01

    The 4-chloro-benzoate anion of the title salt, C6H9N2(+)·C7H4ClO2(-), is nearly planar with a dihedral angle of 5.14 (16)° between the benzene ring and the carboxyl-ate group. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl-ate O atoms of the anion via a pair of N-H⋯O hydrogen bonds with an R2(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 inter-action between the pyridinium and benzene rings with a centroid-centroid distance of 3.7948 (9) Å. PMID:23476392

  14. 2-Amino-5-methyl-pyridinium 4-methyl-benzoate.

    PubMed

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

    2013-01-01

    The 4-methyl-benzoate anion of the title salt, C6H9N2(+)·C8H7O2(-), is nearly planar, with a dihedral angle of 6.26 (10)° between the benzene ring and the carboxyl-ate group. In the crystal, the protonated N atom and the 2-amino group of the cation are hydrogen bonded to the carboxyl-ate O atoms of the anion via a pair of N-H⋯O hydrogen bonds with an R2(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

  15. Methyl 4-(butyrylamino)-5-methyl-2-nitro-benzoate.

    PubMed

    Yuan, Lian-Shan; Li, Xiang; Yao, Cheng

    2008-03-29

    The title compound, C(13)H(16)N(2)O(5), is useful as an inter-mediate in the field of agrochemicals. Intra-molecular C-H⋯O hydrogen bonds result in the formation of one six- and one five-membered nearly planar ring; the six-membered ring is also nearly coplanar with the adjacent benzene ring. In the crystal structure, inter-molecular C-H⋯O hydrogen bonds link the mol-ecules.

  16. 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.

  17. 7-Chloro-5-(furan-3-yl)-3-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-Dioxide as Positive Allosteric Modulator of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor. The End of the Unsaturated-Inactive Paradigm?

    PubMed

    Citti, Cinzia; Battisti, Umberto M; Cannazza, Giuseppe; Jozwiak, Krzysztof; Stasiak, Natalia; Puja, Giulia; Ravazzini, Federica; Ciccarella, Giuseppe; Braghiroli, Daniela; Parenti, Carlo; Troisi, Luigino; Zoli, Michele

    2016-02-17

    5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1. PMID:26580317

  18. Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus.

    PubMed

    Tang, Yi; Chen, Hao; Diao, Youxiang

    2016-06-07

    Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 10(0.89) - 1 × 10(1.55) tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22-3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the "gold standard" in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10(-16) g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV.

  19. Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

    PubMed Central

    Tang, Yi; Chen, Hao; Diao, Youxiang

    2016-01-01

    Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 100.89 − 1 × 101.55 tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22–3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the “gold standard” in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10−16 g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV. PMID:27270462

  20. 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

  1. Redetermination of 3-deaza­uracil

    PubMed Central

    Portalone, Gustavo

    2008-01-01

    The crystal structure of the title compound, 4-hydr­oxy-2-pyridone, C5H5NO2, which has been the subject of several determinations using X-rays and neutron diffraction, was first reported by Low & Wilson [Acta Cryst. (1983). C39, 1688–1690]. It has been redetermined, providing a significant increase in the precision of the derived geometric parameters. The asymmetric unit comprises a planar 4-enol tautomer having some degree of delocalization of π-electron density through the mol­ecule. In the crystal structure, the mol­ecules are connected into chains by two strong O—H⋯O and N—H⋯O hydrogen bonds between the OH and NH groups and the carbonyl O atom. PMID:21202619

  2. Synthesis, spectroscopic and structural characterization of cobalt(II) complex with uracil-containing 2,6-diformylpyridine ligand: Theoretical studies on the ligand and pentagonal-bipyramidal [Co(L)(H 2O) 2] 2+ and [Zn(L)(H 2O) 2] 2+ cations

    NASA Astrophysics Data System (ADS)

    Koz, Gamze; Özdemir, Namık; Astley, Demet; Dinçer, Muharrem; Astley, Stephen T.

    2010-03-01

    The title complex, trans-diaqua{5,5'-[( E, E)-pyridine-2,6-diylbis(methylidynenitrilo)]bis-[pyrimidine-2,4(1 H,3 H)-dione]}cobalt(II) bis(hexafluorophosphate) dihydrate [Co(C 15H 11N 7O 4) (H 2O) 2]·2(PF 6)·2(H 2O), has been synthesized, and characterized by IR spectroscopy and single crystal X-ray diffraction. The compound crystallizes in the monoclinic space group P2 1/ c with a = 10.7301(4) Å, b = 12.0537(3) Å, c = 21.6030(9) Å and β = 109.392 (3)°. In the title complex, the Co 2+ centre is seven-coordinated in a slightly distorted pentagonal-bipyramidal geometry, with the two water O atoms located in the apical positions, and the pyridine N atom, the two imine N atoms and two carbonyl O atoms of the uracil groups located in the equatorial plane. The positions of fluorine atoms in the hexafluorophosphate groups were disordered. The charge is balanced by two PF 62- anions. In addition to the molecular geometry from X-ray experiment, theoretical studies have been carried out on the structures of the pentagonal-bipyramidal [Co(L)(H 2O) 2] 2+ and [Zn(L)(H 2O) 2] 2+ cations using the Hartree-Fock (HF) and density functional theory (DFT-B3LYP) methods in conjunction with effective core potential basis set (LANL2DZ) to clarify the solid state behaviour of these cations. Besides, frontier molecular orbitals (FMO) analysis and natural bond orbital (NBO) analysis of [Co(L)(H 2O) 2] 2+ cation are presented here together with vibrational frequencies and gauge including atomic orbital (GIAO) 1H and 13C NMR chemical shift values of the pentadentate ligand calculated at HF and DFT (B3LYP) levels with 6-31G(d) basis set.

  3. β-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU) prevents varicella-zoster virus replication in a SCID-Hu mouse model and does not interfere with 5-fluorouracil catabolism.

    PubMed

    De, Chandrav; Liu, Dongmei; Zheng, Bo; Singh, Uma S; Chavre, Satish; White, Catherine; Arnold, Robert D; Hagen, Fred K; Chu, Chung K; Moffat, Jennifer F

    2014-10-01

    The alphaherpesvirus varicella-zoster virus (VZV) causes chickenpox and shingles. Current treatments are acyclovir (ACV) and its derivatives, foscarnet and brivudine (BVdU). Additional antiviral compounds with increased potency and specificity are needed to treat VZV, especially to treat post-herpetic neuralgia. We evaluated β-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU, 1) and 5'-O-valyl-l-BHDU (2) in three models of VZV replication: primary human foreskin fibroblasts (HFFs), skin organ culture (SOC) and in SCID-Hu mice with skin xenografts. The efficacy of l-BHDU in vivo and its drug-drug interactions were previously not known. In HFFs, 200μM l-BHDU was noncytotoxic over 3days, and l-BHDU treatment reduced VZV genome copy number and cell to cell spread. The EC50 in HFFs for l-BHDU and valyl-l-BHDU were 0.22 and 0.03μM, respectively. However, l-BHDU antagonized the activity of ACV, BVdU and foscarnet in cultured cells. Given its similar structure to BVdU, we asked if l-BHDU, like BVdU, inhibits 5-fluorouracil catabolism. BALB/c mice were treated with 5-FU alone or in combination with l-BHDU or BVdU. l-BHDU did not interfere with 5-FU catabolism. In SCID-Hu mice implanted with human skin xenografts, l-BHDU and valyl-l-BHDU were superior to ACV and valacyclovir. The maximum concentration (Cmax) levels of l-BHDU were determined in mouse and human tissues at 2h after dosing, and comparison of concentration ratios of tissue to plasma indicated saturation of uptake at the highest dose. For the first time, an l-nucleoside analog, l-BHDU, was found to be effective and well tolerated in mice.

  4. 2-(2-Chloro-phen-yl)-5-methyl-1,3-dioxane-5-carboxylic acid.

    PubMed

    Jia, Guo-Kai; Yuan, Lin; Zhang, Min; Yuan, Xian-You

    2012-07-01

    In the title compound, C(12)H(13)ClO(4), the 1,3-dioxane ring adopts a chair conformation and the 2-chloro-benzene and methyl substituents occupy equatorial sites. The carboxyl group is in an axial inclination. In the crystal, carb-oxy-lic acid inversion dimers linked by pairs of O-H⋯O hydrogen bonds generate R(2) (2)(8) loops. PMID:22807863

  5. 2-Amino-5-methyl-pyridinium 2-hy-droxy-5-chloro-benzoate.

    PubMed

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

    2013-01-01

    In the 5-chloro-salicylate anion of the title salt, C6H9N2(+)·C7H4ClO3(-), an intra-molecular 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 carboxyl-ate O atoms via a pair of N-H⋯O hydrogen bonds, forming an R2(2)(8) ring motif. The crystal structure also features N-H⋯O and weak C-H⋯O inter-actions, resulting in a layer parallel to (10-1). PMID:23476391

  6. 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

  7. 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

  8. 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

  9. 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

  10. Detection and quantification of flavivirus NS5 methyl-transferase activities.

    PubMed

    Lim, Siew Pheng; Bodenreider, Christophe; Shi, Pei-Yong

    2013-01-01

    Flavivirus NS5 is the most conserved protein amongst the flavivirus proteins and is an essential enzyme for viral mRNA capping and replication. It encodes a methyl-transferase (MTase) domain at its N-terminal region which carries out sequential N7 and 2'-O methylation, resulting in the formation of the cap1 structure on its viral RNA genome. Two key methods have been established to measure these activities in vitro: thin-layer chromatography (TLC) and scintillation proximity assays (SPA). TLC offers the advantage of direct visualization of the amounts and types of cap structures formed whilst the SPA assay is more sensitive and quantitative. It is also amenable to high-throughput compound screening. The drawback of both assays is the need for radioisotope usage. We further describe the adaptation of a nonradioactive immune-competitive fluorescence polarization assay for detection of dengue virus MTase activity. PMID:23821274

  11. Molecular beacon probes of photodamage in thymine and uracil oligonucleotides.

    PubMed

    Yarasi, Soujanya; McConachie, Cheryl; Loppnow, Glen R

    2005-01-01

    Molecular beacons (MB) are becoming more common as sequence-selective detectors of nucleic acids. Although they can easily detect single-base mismatches, they have never been used to directly detect DNA or RNA damage. To measure the degree of ultraviolet (UV) light damage in oligonucleotides, we report a novel MB approach for general detection of photoproducts in UV-irradiated rU17 and dT17 oligonucleotides. With monochromatic UV light irradiation at ca 280 nm under anoxic conditions, the oligonucleotide absorption decays with a single-exponential time constant of 123+/-1 min for rU17 and with double-exponential time constants of 78+/-0.5 min (99%) and 180+/-5 min (0.05%) for dT17 oligonucleotides. Under the same conditions, the MB fluorescence decays more quickly, with single-exponential time constants of 19+/-2 and 26+/-3 min for rU17 and dT17, respectively. Similar kinetics were observed with broadband UV light irradiation of oligonucleotides. The differences in the UV damage kinetics of dT17 and rU17 and their detection by absorption and fluorescence techniques will be discussed in the context of differential instabilities introduced in the nucleic acid-MB duplex by the different photoproducts formed.

  12. 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.

  13. Crystal structure of N-(2-hy­droxy-5-methyl­phen­yl)benzamide

    PubMed Central

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

    2015-01-01

    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 hy­droxy­phenyl rings, respectively. A short intra­molecular N—H⋯O contact is present. In the crystal, the mol­ecules 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 inter­actions link the mol­ecules into inversion dimers. PMID:26870541

  14. N-tert-Butyl O-2-isopropyl-5-methyl­cyclo­hexyl phenyl­phospho­namidate

    PubMed Central

    Liu, Li-Juan; Meng, Fan-Jie; Xu, Hao; Wang, Daqi; Zhao, Chang-Qiu

    2011-01-01

    In the title compound, C20H34NO2P, the P atom has an irregular tetra­hedral environment and exhibits S p chirality. In the crystal, weak inter­molecular N—H⋯O and C—H⋯O hydrogen bonds link the mol­ecules into chains extending in [010]. PMID:21754537

  15. 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.

  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. 3-Ethyl-2-methyl-5-methyl-ene-6,7-di-hydroindol-4(5H)-one.

    PubMed

    Sonar, Vijayakumar N; Parkin, Sean; Crooks, Peter A

    2007-01-01

    The title compound, C(12)H(15)NO, a degradation product of molindone hydro-chloride, was prepared by the reaction of molindone with methyl iodide and subsequent reaction of the resulting quaternary ammonium salt with 2N aqueous sodium hydroxide. The newly formed double bond is exocyclic in nature and the carbonyl group is conjugated with the π-electrons of the pyrrole ring. The six-membered ring is in the half-chair conformation. The H atom attached to the N atom is involved in an inter-molecular hydrogen bond with the O atom of a screw-related mol-ecule, thus forming a continuous chain.

  19. Crystal structure of (5-methyl­imidazo[1,2-a]pyridin-2-yl)methanol

    PubMed Central

    Elaatiaoui, Abdelmalik; Koudad, Mohammed; Saddik, Rafik; Benchat, Noureddine; El Ammari, Lahcen

    2014-01-01

    In the title compound, C9H10N2O, the imidazo[1,2-a]pyridine moiety is approximately planar (r.m.s. deviation = 0.024 Å). The methanol group is nearly perpendicular to its mean plane as indicated by the C—C—C—O and N—C—C—O torsion angles of 80.04 (16) and −96.30 (17)°, respectively. In the crystal, mol­ecules are linked by O—H⋯N hydrogen bonds, forming inversion dimers with an R 2 2(10) ring motif. The dimers are liked via C—H⋯O hydrogen bonds, enclosing R 2 2(10) ring motifs and forming ribbons along [201]. The ribbons are linked via a number of π–π inter­actions [centroid–centroid distances vary from 3.4819 (8) to 3.7212 (8) Å], forming a three-dimensional structure. PMID:25484822

  20. Synthesis and Some Reactions of 1-aryl-4-acetyl-5-methyl-1,2,3-triazole Derivatives with Anticonvulsant Activity.

    PubMed

    Nassar, Ekhlass M; Abdelrazek, Fathy M; Ayyad, Rezk R; El-Farargy, Ahmed F

    2016-01-01

    The triazoles 3a-d underwent condensation reactions with 4-(piperidin-1-yl)-benzaldehyde to afford the chalcones 5a-d. Chalcone derivatives 5a-d were reacted with 2,3-diaminomaleonitrile, thiourea and hydrazine hydrate to afford the novel diazepine-dicarbonitrile derivatives 7a-d, the pyrimidine-2-thiol derivatives 9a-d and hydrazino-pyrimidines 10a-d respectively. Structures of the prepared compounds were elucidated by physical and spectral data like FT-IR, (1)H NMR, (13)C NMR, and mass spectroscopy. Some of the synthesized compounds were screened for their anticonvulsant activity and SAR. PMID:26776225

  1. Natural folates from biofortified tomato and synthetic 5-methyl-tetrahydrofolate display equivalent bioavailability in a murine model.

    PubMed

    Castorena-Torres, Fabiola; Ramos-Parra, Perla A; Hernández-Méndez, Rogelio V; Vargas-García, Andrés; García-Rivas, Gerardo; de la Garza, Rocío I Díaz

    2014-03-01

    Folate deficiency is a global health problem related to neural tube defects, cardiovascular disease, dementia, and cancer. Considering that folic acid (FA) supply through industrialized foods is the most successful intervention, limitations exist for its complete implementation worldwide. Biofortification of plant foods, on the other hand, could be implemented in poor areas as a complementary alternative. A biofortified tomato fruit that accumulates high levels of folates was previously developed. In this study, we evaluated short-term folate bioavailability in rats infused with this folate-biofortified fruit. Fruit from tomato segregants hyperaccumulated folates during an extended ripening period, ultimately containing 3.7-fold the recommended dietary allowance in a 100-g portion. Folate-depleted Wistar rats separated in three groups received a single dose of 1 nmol of folate/g body weight in the form of lyophilized biofortified tomato fruit, FA, or synthetic 5-CH3-THF. Folate bioavailability from the biofortified tomato was comparable to that of synthetic 5-CH3-THF, with areas under the curve (AUC(0-∞)) of 2,080 ± 420 and 2,700 ± 220 pmol · h/mL, respectively (P = 0.12). Whereas, FA was less bioavailable with an AUC(0-∞) of 750 ± 10 pmol · h/mL. Fruit-supplemented animals reached maximum levels of circulating folate in plasma at 2 h after administration with a subsequent steady decline, while animals treated with FA and synthetic 5-CH3-THF reached maximum levels at 1 h. Pharmacokinetic parameters revealed that biofortified tomato had slower intestinal absorption than synthetic folate forms. This is the first study that demonstrates the bioavailability of folates from a biofortified plant food, showing its potential to improve folate deficiency. PMID:24445671

  2. 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.

  3. Interaction of sodium and potassium ions with sandwiched cytosine-, guanine-, thymine-, and uracil-base tetrads.

    PubMed

    Meyer, Michael; Hocquet, Alexandre; Sühnel, Jürgen

    2005-03-01

    Nucleic acid tetraplexes and lipophilic self-assembling G-quadruplexes contain stacked base tetrads with intercalated metal ions as basic building blocks. Thus far, quantum-chemical studies have been used to explore the geometric and energetic properties of base tetrads with and without metal ions. Recently, for the first time, work on a sandwiched G-tetrad complex has been studied. We report here results of a systematic B3LYP density functional study on sandwiched G-, C-, U-, and T-tetrads with Na+ and K+ at different symmetries that substantially extend the recent work. The results include detailed information on total energies as well as on metal ion tetrad and base-base interaction energies. The geometrical parameters of the sandwiched metal ion complexes are compared to both experimental structures and to calculated geometries of complexes of single tetrads with metal ions. A microsolvation model explains the ion selectivity preference of K+ over Na+ in a qualitative sense. PMID:15648098

  4. 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...

  5. Dynamic Regulation of N-Methyl-d-aspartate (NMDA) and α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by Posttranslational Modifications.

    PubMed

    Lussier, Marc P; Sanz-Clemente, Antonio; Roche, Katherine W

    2015-11-27

    Many molecular mechanisms underlie the changes in synaptic glutamate receptor content that are required by neuronal networks to generate cellular correlates of learning and memory. During the last decade, posttranslational modifications have emerged as critical regulators of synaptic transmission and plasticity. Notably, phosphorylation, ubiquitination, and palmitoylation control the stability, trafficking, and synaptic expression of glutamate receptors in the central nervous system. In the current review, we will summarize some of the progress made by the neuroscience community regarding our understanding of phosphorylation, ubiquitination, and palmitoylation of the NMDA and AMPA subtypes of glutamate receptors. PMID:26453298

  6. 5-methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni

    PubMed Central

    2013-01-01

    Background Biomphalaria glabrata is the mollusc intermediate host for Schistosoma mansoni, a digenean flatworm parasite that causes human intestinal schistosomiasis. An estimated 200 million people in 74 countries suffer from schistosomiasis, in terms of morbidity this is the most severe tropical disease after malaria. Epigenetic information informs on the status of gene activity that is heritable, for which changes are reversible and that is not based on the DNA sequence. Epigenetic mechanisms generate variability that provides a source for potentially heritable phenotypic variation and therefore could be involved in the adaptation to environmental constraint. Phenotypic variations are particularly important in host-parasite interactions in which both selective pressure and rate of evolution are high. In this context, epigenetic changes are expected to be major drivers of phenotypic plasticity and co-adaptation between host and parasite. Consequently, with characterization of the genomes of invertebrates that are parasite vectors or intermediate hosts, it is also essential to understand how the epigenetic machinery functions to better decipher the interplay between host and parasite. Methods The CpGo/e ratios were used as a proxy to investigate the occurrence of CpG methylation in B. glabrata coding regions. The presence of DNA methylation in B. glabrata was also confirmed by several experimental approaches: restriction enzymatic digestion with isoschizomers, bisulfite conversion based techniques and LC-MS/MS analysis. Results In this work, we report that DNA methylation, which is one of the carriers of epigenetic information, occurs in B. glabrata; approximately 2% of cytosine nucleotides are methylated. We describe the methylation machinery of B. glabrata. Methylation occurs predominantly at CpG sites, present at high ratios in coding regions of genes associated with housekeeping functions. We also demonstrate by bisulfite treatment that methylation occurs in multiple copies of Nimbus, a transposable element. Conclusions This study details DNA methylation for the first time, one of the carriers of epigenetic information in B. glabrata. The general characteristics of DNA methylation that we observed in the B. glabrata genome conform to what epigenetic studies have reported from other invertebrate species. PMID:23742053

  7. 2,2-Dimethyl-5-[(5-methyl-furan-2-yl)methyl-idene]-1,3-dioxane-4,6-dione.

    PubMed

    Zeng, Wu-Lan

    2011-01-01

    The asymmetric unit of the title compound, C(12)H(12)O(5), contains two independent mol-ecules. In each, the 1,3-dioxane ring adopts an envelope conformation with the dimethyl-substituted C atom forming the flap. The crystal structure is stabilized by weak inter-molecular C-H⋯O hydrogen bonds. PMID:21523135

  8. Crystal structure of 2,6-bis-(2-hy-droxy-5-methyl-phen-yl)-4-phenyl-pyridinium bromide di-chloro-methane hemisolvate hemihydrate.

    PubMed

    Mankaev, Badma N; Zaitsev, Kirill V; Karlov, Sergey S; Egorov, Mikhail P; Churakov, Andrei V

    2015-12-01

    The asymmetric unit in the structure of the title compound, C25H22NO2 (+)·Br (-)·0.5CH2Cl2·0.5H2O, comprises two pseudosymmetry-related cations, two bromide anions, a di-chloro-methane molecule and a water mol-ecule of solvation. The two independent cations are conformationally similar with the comparative dihedral angles between the central pyridine ring and the three benzene substituent rings being 3.0 (2), 36.4 (1) and 24.2 (1)°, and 3.7 (2), 36.5 (1) and 24.8 (1)°, respectively. In the crystal, the cations, anions and water mol-ecules are linked through O-H⋯O and O-H⋯Br hydrogen bonds, forming an insular unit. Within the cations there are also intra-molecular N-H⋯O hydrogen bonds. Adjacent centrosymmetrically related aggregates are linked by π-π stacking inter-actions between the pyridine ring and a benzene ring in both cations [ring-centroid separations = 3.525 (3) and 3.668 (3) Å], forming chains extending across the ac diagonal. Voids between these chains are filled by dichloromethane molecules. PMID:26870547

  9. 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

  10. 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.

  11. Crystal structure of 4-[(5-methyl-isoxazol-3-yl)amino-sulfon-yl]anilinium 3,5-di-nitro-salicylate.

    PubMed

    Malathy, Sevaiyan; Nirmalram, Jeyaraman Selvaraj; Muthiah, Packianathan Thomas

    2015-06-01

    The title mol-ecular salt, C10H12N3O3S(+)·C7H3N2O7 (-), protonation occurs at the amino N atom attached to the benzene ring of sulfamethoxazole. In the anion, there is an intra-molecular O-H⋯O hydrogen bond and the cation is linked to the anion by an N-H⋯O hydrogen bond. In the extended structure, the cations and anions are linked via N-H⋯O, N-H⋯N and C-H⋯O hydrogen bonds, forming a three-dimensional framework. PMID:26090134

  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, 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...

  13. Di-aqua-bis-(2-ethyl-5-methyl-imidazole-4-sulfonato-κ(2) N (3),O)nickel(II) dihydrate.

    PubMed

    Purdy, Andrew P; Butcher, Ray J

    2014-01-01

    In the title complex, [Ni(C6H9N2O3S)2(H2O)2]·2H2O, the Ni(II) atom lies on an inversion center and is chelated by N and O atoms of two symmetry-equivalent imidazole-sulfonate ligands in the basal plane, and two water O atoms in axial positions in an overall octa-hedral configuration. The crystal structure displays O-H⋯O and N-H⋯O hydrogen bonds, which connect the components into an extended three-dimensional network. PMID:24526946

  14. 2-Amino-5-methyl­pyridinium 6-oxo-1,6-dihydro­pyridine-2-carboxyl­ate

    PubMed Central

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

    2012-01-01

    The anion of the title salt, C6H9N2 +·C6H4NO3 −, undergoes an enol-to-keto tautomerism during the crystallization. In the crystal structure, the cation and anion are held together by a relatively short N—H⋯O hydrogen bond, and the two anions are further connected to each other by a pair of N—H⋯O hydrogen bonds with an R 2 2(8) ring motif, thus forming a centrosymmetric 2 + 2 aggregate. The aggregates are further linked through weak N—H⋯O and C—H⋯O hydrogen bonds, resulting a three-dimensional network. PMID:23284469

  15. 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

  16. 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. PMID:27067148

  17. 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

  18. Crystal structure of (2-{[3,5-bis­(1,1-di­methyl­eth­yl)-4-hy­droxy­phen­yl](5-methyl-2H-pyrrol-2-yl­idene)meth­yl}-5-methyl-1H-pyrrolido-κ2 N,N′)di­fluoridoboron

    PubMed Central

    Morimoto, Yukio; Ogawa, Keizo; Uto, Yoshihiro; Nagasawa, Hideko; Hori, Hitoshi

    2015-01-01

    The title compound, C25H31BF2N2O, is a potential boron tracedrug in boron neutron capture therapy (BNCT), in which the B atom adopts a distorted BN2F2 tetra­hedral geometry: it is soluble in dimethyl sulfoxide, di­methyl­formamide and methanol. The pyrrolyl­idene­methyl­pyrrole triple fused ring system is almost planar (r.m.s. deviation = 0.031 Å) and subtends a dihedral angle of 47.09 (5)° with the plane of the pendant phenol ring. The phenol –OH group is blocked from forming hydrogen bonds by the adjacent bulky tert-butyl groups. In the crystal, inversion dimers linked by pairs of very weak C—H⋯F inter­actions generate R 2 2(22) loops. PMID:26396909

  19. Characterization of the three-dimensional free energy manifold for the uracil ribonucleoside from asynchronous replica exchange simulations.

    PubMed

    Radak, Brian K; Romanus, Melissa; Lee, Tai-Sung; Chen, Haoyuan; Huang, Ming; Treikalis, Antons; Balasubramanian, Vivekanandan; Jha, Shantenu; York, Darrin M

    2015-02-10

    Replica exchange molecular dynamics has emerged as a powerful tool for efficiently sampling free energy landscapes for conformational and chemical transitions. However, daunting challenges remain in efficiently getting such simulations to scale to the very large number of replicas required to address problems in state spaces beyond two dimensions. The development of enabling technology to carry out such simulations is in its infancy, and thus it remains an open question as to which applications demand extension into higher dimensions. In the present work, we explore this problem space by applying asynchronous Hamiltonian replica exchange molecular dynamics with a combined quantum mechanical/molecular mechanical potential to explore the conformational space for a simple ribonucleoside. This is done using a newly developed software framework capable of executing >3,000 replicas with only enough resources to run 2,000 simultaneously. This may not be possible with traditional synchronous replica exchange approaches. Our results demonstrate 1.) the necessity of high dimensional sampling simulations for biological systems, even as simple as a single ribonucleoside, and 2.) the utility of asynchronous exchange protocols in managing simultaneous resource requirements expected in high dimensional sampling simulations. It is expected that more complicated systems will only increase in computational demand and complexity, and thus the reported asynchronous approach may be increasingly beneficial in order to make such applications available to a broad range of computational scientists. PMID:26580900

  20. 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...

  1. 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

    2013-06-01

    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.

  2. Blood folate status and expression of proteins involved in immune function, inflammation, and coagulation: biochemical and proteomic changes in the plasma of humans in response to long-term synthetic folic acid supplementation.

    PubMed

    Duthie, Susan J; Horgan, Graham; de Roos, Baukje; Rucklidge, Garry; Reid, Martin; Duncan, Gary; Pirie, Lynn; Basten, Graham P; Powers, Hilary J

    2010-04-01

    We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

  3. Crystal structure of 2,6-bis­(2-hy­droxy-5-methyl­phen­yl)-4-phenyl­pyridinium bromide di­chloro­methane hemisolvate hemihydrate

    PubMed Central

    Mankaev, Badma N.; Zaitsev, Kirill V.; Karlov, Sergey S.; Egorov, Mikhail P.; Churakov, Andrei V.

    2015-01-01

    The asymmetric unit in the structure of the title compound, C25H22NO2 +·Br −·0.5CH2Cl2·0.5H2O, comprises two pseudosymmetry-related cations, two bromide anions, a di­chloro­methane molecule and a water mol­ecule of solvation. The two independent cations are conformationally similar with the comparative dihedral angles between the central pyridine ring and the three benzene substituent rings being 3.0 (2), 36.4 (1) and 24.2 (1)°, and 3.7 (2), 36.5 (1) and 24.8 (1)°, respectively. In the crystal, the cations, anions and water mol­ecules are linked through O—H⋯O and O—H⋯Br hydrogen bonds, forming an insular unit. Within the cations there are also intra­molecular N—H⋯O hydrogen bonds. Adjacent centrosymmetrically related aggregates are linked by π–π stacking inter­actions between the pyridine ring and a benzene ring in both cations [ring-centroid separations = 3.525 (3) and 3.668 (3) Å], forming chains extending across the ac diagonal. Voids between these chains are filled by dichloromethane molecules. PMID:26870547

  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. Crystal structure of 5-{3-[2,6-dimethyl-4-(5-methyl-1,2,4-oxa-diazol-3-yl)phen-oxy]prop-yl}-N-(11-hy-droxy-undec-yl)isoxazole-3-carboxamide hemihydrate.

    PubMed

    Salorinne, K; Lahtinen, T

    2015-05-01

    The title compound, C29H42N4O5·0.5H2O, comprises four structural units. A flexible prop-yloxy unit in a gauche conformation, with a -C(H2)-C(H2)-C(H2)-O- torsion angle of -64.32 (18)°, connects an isoxazole ring and an approximately planar phenyl-oxa-diazole ring system [with a maxixmum devation of 0.061 (2) Å], which are oriented almost parallel to one another with a dihedral angle of 10.75 (7)°. Furthermore, a C11-alkyl chain with a terminal hy-droxy group links to the 3-position of the isoxazole ring via an amide bond. In the crystal, a half-occupancy solvent water mol-ecule connects to a neighbouring mol-ecule via an inter-molecular O-H⋯O(water) hydrogen bond to the C11-alkyl chain hy-droxy group.

  6. 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.

  7. (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

  8. Synthesis, structure and catalase-like activity of dimanganese(III) complexes of 1,5-bis(X-salicylidenamino)pentan-3-ol (X = 3- and 5-methyl). Influence of phenyl-ring substituents on catalytic activity.

    PubMed

    Moreno, Diego; Palopoli, Claudia; Daier, Verónica; Shova, Sergiu; Vendier, Laure; Sierra, Manuel González; Tuchagues, Jean-Pierre; Signorella, Sandra

    2006-11-21

    The diMn(III) complexes [Mn2(5-Me-salpentO)(mu-MeO)(mu-AcO)(H2O)Br] (1) and [Mn2(3-Me-salpentO)(mu-MeO)(mu-AcO)(MeOH)2]Br (2), where salpentOH = 1,5-bis(salicylidenamino)pentan-3-ol, were synthesised and structurally characterized. The two complexes include a bis(micro-alkoxo)(micro-acetato) triply-bridged diMn(III) core with an Mn...Mn separation of 2.93-2.94 A, the structure of which is retained upon dissolution. Complexes 1 and 2 show catalytic activity toward disproportionation of H2O2, with first-order dependence on the catalyst, and saturation kinetics on [H2O2], in methanol and DMF. In DMF, the two complexes are able to disproportionate at least 1500 eq. of H2O2 without significant decomposition, while in methanol, they rapidly lose activity with formation of a non-coupled Mn(II) species. Electrospray ionisation mass spectrometry, EPR and UV/vis spectroscopy used to monitor the reaction suggest that the major active form of the catalyst occurs in the Mn2(III) oxidation state during cycling. The correlation between log(k(cat)) and the redox potentials of 1, 2 and analogous complexes of other X-salpentOH derivatives indicates that, in this series, the oxidation of the catalyst is probably the rate-limiting step in the catalytic cycle. It is also noted that formation of the catalyst-peroxide adduct is more sensitive to steric effects in DMF than in methanol. Overall, kinetics and spectroscopic studies of H2O2 dismutation by these complexes converge at a catalytic cycle that involves the Mn2(III) and Mn2(IV) oxidation states. PMID:17077889

  9. 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

    Qin, Xike; Jiang, Yongjun; Tse, Yiu Chung; Wang, Yunling; Wong, Tak Pan; Paudel, Hemant K

    2015-12-01

    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

  10. A Compact Approach to an Isomeric Iheyamine A System and X-ray Crystal Structure of 5-Methyl-5H-azepino[2,3-b:4,5-b']diindole

    SciTech Connect

    Bremner, John B.; Sengpracha, Waya; Skelton, Brian W.

    2008-11-03

    A compact three-step synthesis of a new fused bisindole system isomeric with the heterocyclic skeleton present in the marine natural product iheyamine A has been achieved. The structure of the synthetic product was confirmed by a single-crystal X-ray structure.

  11. 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

    2014-08-01

    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.

  12. 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

    Hsu, Wei-Lun; Chung, Hui-Wen; Wu, Chih-Yueh; Wu, Huei-Ing; Lee, Yu-Tao; Chen, En-Chan; Fang, Weilun; Chang, Yen-Chung

    2015-08-21

    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 Ca(2+), resulting from Ca(2+) influxes through calcium-permeable AMPA receptors, voltage-gated Ca(2+) 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 Ca(2+) 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, Ca(2+) 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.

  13. Inhibitory Interactions between Phosphorylation Sites in the C Terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor GluA1 Subunits*

    PubMed Central

    Gray, Erin E.; Guglietta, Ryan; Khakh, Baljit S.; O'Dell, Thomas J.

    2014-01-01

    The C terminus of AMPA-type glutamate receptor (AMPAR) GluA1 subunits contains several phosphorylation sites that regulate AMPAR activity and trafficking at excitatory synapses. Although many of these sites have been extensively studied, little is known about the signaling mechanisms regulating GluA1 phosphorylation at Thr-840. Here, we report that neuronal depolarization in hippocampal slices induces a calcium and protein phosphatase 1/2A-dependent dephosphorylation of GluA1 at Thr-840 and a nearby site at Ser-845. Despite these similarities, inhibitors of NMDA-type glutamate receptors and protein phosphatase 2B prevented depolarization-induced Ser-845 dephosphorylation but had no effect on Thr-840 dephosphorylation. Instead, depolarization-induced Thr-840 dephosphorylation was prevented by blocking voltage-gated calcium channels, indicating that distinct Ca2+ sources converge to regulate GluA1 dephosphorylation at Thr-840 and Ser-845 in separable ways. Results from immunoprecipitation/depletion assays indicate that Thr-840 phosphorylation inhibits protein kinase A (PKA)-mediated increases in Ser-845 phosphorylation. Consistent with this, PKA-mediated increases in AMPAR currents, which are dependent on Ser-845 phosphorylation, were inhibited in HEK-293 cells expressing a Thr-840 phosphomimetic version of GluA1. Conversely, mimicking Ser-845 phosphorylation inhibited protein kinase C phosphorylation of Thr-840 in vitro, and PKA activation inhibited Thr-840 phosphorylation in hippocampal slices. Together, the regulation of Thr-840 and Ser-845 phosphorylation by distinct sources of Ca2+ influx and the presence of inhibitory interactions between these sites highlight a novel mechanism for conditional regulation of AMPAR phosphorylation and function. PMID:24706758

  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. Novel photodynamic effect of a psoralen-conjugated oligonucleotide for the discrimination of the methylation of cytosine in DNA.

    PubMed

    Yamayoshi, Asako; Matsuyama, Yohei; Kushida, Mikihiko; Kobori, Akio; Murakami, Akira

    2014-01-01

    DNA methylation and demethylation significantly affect the deactivation and activation processes of gene expression significantly. In particular, C-5-methylation of cytosine in the CpG islands is important for the epigenetic modification in genes, which plays a key role in regulating gene expression. The determination of the location and frequency of DNA methylation is important for the elucidation of the mechanisms of cell differentiation and carcinogenesis. Here we designed a psoralen-conjugated oligonucleotide (PS-oligo) for the discrimination of 5-methylcytosine (5-mC) in DNA. The cross-linking behavior of psoralen derivatives with pyrimidine bases, such as thymine, uracil and cytosine has been well discussed, but there are no reports which have examined whether cross-linking efficiency of psoralen with cytosine would be changed with or without C-5 methylation. We found that the cross-linking efficiency of PS-oligo with target-DNA containing 5-mC was greatly increased compared to the case of target-DNA without 5-mC, approximately seven-fold higher. Here we report a new aspect of the photocross-linking behavior of psoralen with 5-mC that is applicable to a simple, sequence-specific and quantitative analysis for the discrimination of 5-mC in DNA, which can be applicable to study the epigenetic behavior of gene expressions.

  16. Thiophene aldehyde-diamino uracil Schiff base: A novel fluorescent probe for detection and quantification of cupric, silver and ferric ions.

    PubMed

    Hammud, Hassan H; El Shazly, Shawky; Sonji, Ghassan; Sonji, Nada; Bouhadir, Kamal H

    2015-01-01

    A new Schiff base from the condensation of 5,6-diamino-1,3-dimethyluracil with 5-methylthiophene-2-carboxaldehyde was synthesized. The compound was characterized by spectral data (UV-Vis, IR, (1)H NMR, fluorescence, MS). Ethanolic solutions of the Schiff base exhibit a strong fluorescence emission at 385 nm (λex=341 nm), and have been employed as a "turn-off" fluorescent probe for selective detection of Ag(+), Cu(2+) and Fe(3+) ions in presence of other cations such as Na(+), K(+), Ca(2+) and Mg(2+) ions abundant in natural water. The interaction between the tested compound and copper, silver or iron ions is associated with a significant fluorescence decrease, showing detection limits of 2.1-14.2 ppb. Under optimal conditions, the developed sensor was successfully employed to determine Ag(+), Cu(2+) and Fe(3+) ions in real samples and proved to be selective and sensitive.

  17. Randomized phase II trial of TEGAFIRI (tegafur/uracil, oral leucovorin, irinotecan) compared with FOLFIRI (folinic acid, 5-fluorouracil, irinotecan) in patients with unresectable/recurrent colorectal cancer.

    PubMed

    Shigeta, Kohei; Hasegawa, Hirotoshi; Okabayashi, Koji; Tsuruta, Masashi; Ishii, Yoshiyuki; Endo, Takashi; Ochiai, Hiroki; Kondo, Takayuki; Kitagawa, Yuko

    2016-08-15

    Irinotecan-based chemotherapy with bevacizumab is one of the first-line standard therapies for metastatic colorectal cancer (mCRC). TEGAFIRI (UFT/LV + irinotecan) is an irinotecan-based chemotherapy regimen. Currently, few clinical data regarding TEGAFIRI are available. This study evaluated the efficacy and safety of TEGAFIRI in Japanese patients with mCRC. This is a multicenter, randomized, phase II study. The major inclusion criteria were previously untreated patients with mCRC (age: 20-75 years, Eastern Cooperative Oncology Group performance status: 0-1). Eligible patients were randomly assigned (1:1) to receive either FOLFIRI ± bevacizumab or TEGAFIRI ± bevacizumab. The primary endpoint was progression-free survival (PFS). The secondary endpoints were response rate, overall survival, dose intensity and toxicity. From November 2007 to October 2011, 36 and 35 patients assigned to the FOLFIRI and TEGAFIRI groups were included in the primary analysis. No significant difference in PFS was observed between the groups {median PFS: TEGAFIRI 9.9 months [95% confidence interval (CI), 6.5-14.7], FOLFIRI 10.6 months [95% CI, 7.7-16.5]; Hazard ratio, 0.98, 95% CI, 0.57-1.66, p = 0.930}. The response rates in the FOLFIRI and TEGAFIRI groups were 56% and 66%, respectively. Relative dose intensity was similar between the groups. The most common Grade 3/4 adverse event was diarrhea (26%) in TEGAFIRI group and neutropenia (39%) in the FOLFIRI group. The results of the present study indicate that TEGAFIRI ± bevacizumab is an effective and tolerable first-line treatment regimen for mCRC. PMID:27061810

  18. 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.

  19. Thiophene aldehyde-diamino uracil Schiff base: A novel fluorescent probe for detection and quantification of cupric, silver and ferric ions.

    PubMed

    Hammud, Hassan H; El Shazly, Shawky; Sonji, Ghassan; Sonji, Nada; Bouhadir, Kamal H

    2015-01-01

    A new Schiff base from the condensation of 5,6-diamino-1,3-dimethyluracil with 5-methylthiophene-2-carboxaldehyde was synthesized. The compound was characterized by spectral data (UV-Vis, IR, (1)H NMR, fluorescence, MS). Ethanolic solutions of the Schiff base exhibit a strong fluorescence emission at 385 nm (λex=341 nm), and have been employed as a "turn-off" fluorescent probe for selective detection of Ag(+), Cu(2+) and Fe(3+) ions in presence of other cations such as Na(+), K(+), Ca(2+) and Mg(2+) ions abundant in natural water. The interaction between the tested compound and copper, silver or iron ions is associated with a significant fluorescence decrease, showing detection limits of 2.1-14.2 ppb. Under optimal conditions, the developed sensor was successfully employed to determine Ag(+), Cu(2+) and Fe(3+) ions in real samples and proved to be selective and sensitive. PMID:26026307

  20. Cationic lipids containing protonated cyclen and different hydrophobic groups linked by uracil-PNA monomer: synthesis and application for gene delivery.

    PubMed

    Liu, Jun-Liang; Ma, Qing-Ping; Huang, Qing-Dong; Yang, Wei-Han; Zhang, Ji; Wang, Jing-Yi; Zhu, Wen; Yu, Xiao-Qi

    2011-09-01

    In this report, as candidates for non-viral gene vectors, cationic lipids L1, L2 and L3 based on protonated cyclen and different hydrophobic groups (cholesterol, dodecanol or diosgenin) linked by PNA monomer were designed and synthesized. Their liposomes were easily prepared by mixing the synthesized lipids with dioleoylphosphatidyl ethanolamine (DOPE) under appropriate mole ratios. Agarose gel retardation and fluorescent titration by ethidium bromide (EB) showed the strong DNA-binding ability with the K(sv) values of 1.21 × 10(7), 3.76 × 10(6) and 2.90 × 10(6) M(-1) for the liposomes formed from L1-L3, respectively. These liposomes could retard pDNA at an N/P ratio of 3 and form lipoplexes with sizes around 200-300 nm and zeta-potential values of +20-50 mV at N/P ratio from 4 to 10. Besides, the cytotoxicity of the three lipoplexes assayed by MTT is quite different. The results from in vitro transfection in HEK 293T and A549 cell lines showed that the transfection efficiency of L3/DOPE/DNA lipoplex at an N/P ratio of 6 and lipid/DOPE mole ratio of 1:2 is slightly higher than that of Lipofectamine 2000™, indicating that the title PNA monomer-based cationic lipids have great potential to be efficient non-viral gene vector.

  1. 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.

  2. Structural Simplification of Bioactive Natural Products with Multicomponent Synthesis. 3. Fused Uracil-Containing Heterocycles as Novel Topoisomerase-Targeting Agents

    PubMed Central

    Evdokimov, Nikolai M.; Van slambrouck, Severine; Heffeter, Petra; Tu, Lee; Le Calvé, Benjamin; Lamoral-Theys, Delphine; Hooten, Carla J.; Uglinskii, Pavel Y.; Rogelj, Snezna; Kiss, Robert; Steelant, Wim F.A.; Berger, Walter; Yang, Jeremy J.; Bologa, Cristian G.; Kornienko, Alexander; Magedov, Igor V.

    2011-01-01

    After the initial discovery of antiproliferative and apoptosis-inducing properties of a camptothecin-inspired pentacycle based on 1H-indeno[2’,1’:5,6]dihydropyrido[2,3-d]pyrimidine scaffold, a library of its analogues as well as their oxidized planar counterparts were prepared utilizing a practical multicomponent synthetic protocol. The synthesized compounds exhibited submicromolar to low micromolar antiproliferative potencies toward a panel of human cancer cell lines. Biochemical experiments are consistent with the dihydropyridine library members undergoing intracellular oxidation to the corresponding planar pyridines, which then inhibit topoisomerase II activity leading to inhibition of proliferation and cell death. Because of facile synthetic preparation and promising antitopoisomerase activity, both the dihydropyridine and planar pyridine-based compounds represent a convenient starting point for anticancer drug discovery. PMID:21388138

  3. Synthesis, structure and study of azo-hydrazone tautomeric equilibrium of 1,3-dimethyl-5-(arylazo)-6-amino-uracil derivatives.

    PubMed

    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.

  4. Ab initio quantum mechanical study of the structures and energies for the pseudorotation of 5{prime}-dehydroxy analogues of 2{prime}-deoxyribose and ribose sugars

    SciTech Connect

    Brameld, K.A.; Goddard, W.A. III

    1999-02-10

    The authors have used ab initio quantum mechanical (QM) methods to determine the potential energy of pseudorotation for 3,4-dihydroxy-5-methyl-2-(1-pyrollyl)tetrahydrofuran and 4-hydroxy-5-methyl-2-(1-pyrollyl)-tetrahydrofuran, close analogues of 2{prime}-deoxyribose and ribose sugars. The pyrrole is a substitute for the naturally occurring nucleic acid bases: adenine, thymine, guanine, cytosine, and uracil. At the highest calculation level (LMP2/cc-pVTZ(-f)//HF/6-31G**) for 2{prime}-deoxyribose, they find the C2{prime}-endo conformation is the global minimum. The C3{prime}-endo conformation is a local minimum 0.6 kcal/mol higher in energy, and an eastern barrier of 1.6 kcal/mol separates the two minima. Pseudorotation energies of ribose are quite complex and are strongly affected by local orientations of the 2{prime} and 3{prime} hydroxyl groups. When the hydroxyl groups are allowed to assume any conformation, the global minimum remains the C2{prime}-endo conformation. The eastern barrier increases slightly to 1.8 kcal/mol, and the C3{prime}-endo local minimum lies 0.6 kcal/mol above the global minimum. Constraining the torsion angle of the C3{prime} hydroxyl group to {minus}146{degree}, as is found in RNA polymers, results in the C3{prime}-endo conformation becoming the only energy minimum with a C2{prime}-endo conformation 1.9 kcal/mol higher in energy. Bond angles within the pentofuranose ring are correlated to the pseudorotational phase, as is observed by X-ray crystallography and is predicted by pseudorotation theory. Finally, a force field for use in molecular mechanics and dynamics simulations is presented which reproduces the QM potential energies for the 2{prime}-deoxyribose and ribose sugars.

  5. Thermodynamics and mechanism of the interaction of willardiine partial agonists with a glutamate receptor: implications for drug development.

    PubMed

    Martinez, Madeline; Ahmed, Ahmed H; Loh, Adrienne P; Oswald, Robert E

    2014-06-17

    Understanding the thermodynamics of binding of a lead compound to a receptor can provide valuable information for drug design. The binding of compounds, particularly partial agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy. Using a series of partial agonists based on the structure of the natural product, willardiine, we show that the charged state of the ligand determines the enthalpic contribution to binding. Willardiines have uracil rings with pKa values ranging from 5.5 to 10. The binding of the charged form is largely driven by enthalpy, while that of the uncharged form is largely driven by entropy. This is due at least in part to changes in the hydrogen bonding network within the binding site involving one water molecule. This work illustrates the importance of charge to the thermodynamics of binding of agonists and antagonists to AMPA receptors and provides clues for further drug discovery. PMID:24850223

  6. Thermodynamics and mechanism of the interaction of willardiine partial agonists with a glutamate receptor: implications for drug development.

    PubMed

    Martinez, Madeline; Ahmed, Ahmed H; Loh, Adrienne P; Oswald, Robert E

    2014-06-17

    Understanding the thermodynamics of binding of a lead compound to a receptor can provide valuable information for drug design. The binding of compounds, particularly partial agonists, to subtypes of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor is, in some cases, driven by increases in entropy. Using a series of partial agonists based on the structure of the natural product, willardiine, we show that the charged state of the ligand determines the enthalpic contribution to binding. Willardiines have uracil rings with pKa values ranging from 5.5 to 10. The binding of the charged form is largely driven by enthalpy, while that of the uncharged form is largely driven by entropy. This is due at least in part to changes in the hydrogen bonding network within the binding site involving one water molecule. This work illustrates the importance of charge to the thermodynamics of binding of agonists and antagonists to AMPA receptors and provides clues for further drug discovery.

  7. Preclinical investigation of L-FMAU as an anti-hepatitis B virus agent.

    PubMed

    Chu, C K; Boudinot, F D; Peek, S F; Hong, J H; Choi, Y; Korba, B E; Gerin, J L; Cote, P J; Tennant, B C; Cheng, Y C

    1998-01-01

    Preclinical aspects of a potent anti-hepatitis B virus (HBV) L-nucleoside, 1-(2-fluoro-5-methyl-beta-L-arabino-furanosyl)uracil (L-FMAU) are described. L-FMAU was prepared from L-ribose derivatives via either L-xylose or L-arabinose. L-FMAU shows potent antiviral activity against hepatitis B virus (EC50 5.0 microM in H1 cells) with high selectivity in vitro. L-FMAU is not incorporated into mitochondrial DNA and no significant lactic acid production was observed in vitro. L-FMAU is phosphorylated by thymidine kinase as well as deoxycytidine kinase, ultimately to the triphosphate, which inhibits HBV DNA polymerase as the mechanism of antiviral action. Preliminary in vivo toxological studies suggest no apparent toxicity for 30 days at 50 mg/kg/day in mice and for 3 months in woodchucks (10 mg/kg/day). L-FMAU also has respectable bioavailability in rats. L-FMAU shows potent anti-HBV activity in vivo against woodchuck hepatitis virus in chronically infected woodchucks and there is no significant virus rebound after cessation of the drug treatment.

  8. (1SR,3RS,3aSR,6aRS)-Methyl 5-methyl-4,6-dioxo-3-[2-(trifluoro­meth­yl)phen­yl]octa­hydro­pyrrolo­[3,4-c]pyrrole-1-carboxyl­ate

    PubMed Central

    Kudryavtsev, Konstantin V.; Ivantcova, Polina M.; Churakov, Andrei V.

    2013-01-01

    In the title compound, C16H15F3N2O4, the relative stereochemistry of the four stereogenic C atoms has been determined. The carb­oxy­methyl and 2-(trifluoro­meth­yl)­phenyl substituents of the pyrrolidine cycle have a cis mutual arrangement. The five-membered saturated aza­cycle adopts an envelope conformation with the N atom occupying the flap position. In the crystal, adjacent mol­ecules are combined in centrosymmetric dimers by two weak N—H⋯O hydrogen bonds. PMID:23424450

  9. (5-Methyl-pyrazine-2-carboxyl-ato-κ(2) N (1),O)bis-[2-(4-methyl-pyridin-2-yl-κN)-3,5-bis-(tri-fluoro-meth-yl)phenyl-κC (1)]iridium(III) chloro-form hemisolvate.

    PubMed

    Kim, Young-Inn; Song, Young-Kwang; Kang, Sung Kwon

    2014-02-01

    In the title complex, [Ir(C14H8F6N)2(C6H5N2O2)]·0.5CHCl3, the Ir(III) atom adopts a distorted octa-hedral geometry, being coordinated by three N atoms (arranged meridionally), two C atoms and one O atom of three bidentate ligands. The complex mol-ecules pack with no specific inter-molecular inter-actions between them. The SQUEEZE procedure in PLATON [Spek (2009 ▶). Acta Cryst. D65, 148-155] was used to model a disordered chloro-form solvent mol-ecule; the calculated unit-cell data allow for the presence of half of this mol-ecule in the asymmetric unit. PMID:24764808

  10. Discovery of [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl]methanone (MK-6096): a dual orexin receptor antagonist with potent sleep-promoting properties.

    PubMed

    Coleman, Paul J; Schreier, John D; Cox, Christopher D; Breslin, Michael J; Whitman, David B; Bogusky, Michael J; McGaughey, Georgia B; Bednar, Rodney A; Lemaire, Wei; Doran, Scott M; Fox, Steven V; Garson, Susan L; Gotter, Anthony L; Harrell, C Meacham; Reiss, Duane R; Cabalu, Tamara D; Cui, Donghui; Prueksaritanont, Thomayant; Stevens, Joanne; Tannenbaum, Pamela L; Ball, Richard G; Stellabott, Joyce; Young, Steven D; Hartman, George D; Winrow, Christopher J; Renger, John J

    2012-03-01

    Insomnia is a common disorder that can be comorbid with other physical and psychological illnesses. Traditional management of insomnia relies on general central nervous system (CNS) suppression using GABA modulators. Many of these agents fail to meet patient needs with respect to sleep onset, maintenance, and next-day residual effects and have issues related to tolerance, memory disturbances, and balance. Orexin neuropeptides are central regulators of wakefulness, and orexin antagonism has been identified as a novel mechanism for treating insomnia with clinical proof of concept. Herein we describe the discovery of a series of α-methylpiperidine carboxamide dual orexin 1 and orexin 2 receptor (OX(1) R/OX(2) R) antagonists (DORAs). The design of these molecules was inspired by earlier work from this laboratory in understanding preferred conformational properties for potent orexin receptor binding. Minimization of 1,3-allylic strain interactions was used as a design principle to synthesize 2,5-disubstituted piperidine carboxamides with axially oriented substituents including DORA 28. DORA 28 (MK-6096) has exceptional in vivo activity in preclinical sleep models, and has advanced into phase II clinical trials for the treatment of insomnia.

  11. 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.

  12. Resistance of human immunodeficiency virus type 1 to acyclic 6-phenylselenenyl- and 6-phenylthiopyrimidines.

    PubMed Central

    Nguyen, M H; Schinazi, R F; Shi, C; Goudgaon, N M; McKenna, P M; Mellors, J W

    1994-01-01

    Acyclic 6-phenylselenenyl- and 6-phenylthiopyrimidine derivatives are potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1). The development of in vitro resistance to two derivatives, 5-ethyl-1-(ethoxymethyl)-(6-phenylthio)-uracil (E-EPU), was evaluated by serial passage of HIV-1 in increasing concentrations of inhibitor. HIV-1 variants exhibiting > 500-fold resistance to E-EPSeU and E-EPU were isolated after sequential passage in 1, 5, and 10 microM inhibitor. The resistant variants exhibited coresistance to related acyclic 6-substituted pyrimidines and the HIV-1-specific inhibitors (+)-(5S)-4,5,6,7-tetrahydro-5- pyrimidines and the HIV-1-specific inhibitors (+)-(5S)-4,5,6,7-tetrahydro-5- methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk]benzodiazepin-2(1H)- thione (TIBO R82150) and nevirapine, but remained susceptible to 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and phosphonoformic acid. DNA sequence analysis of reverse transcriptase (RT) derived from E-EPSeU-resistant virus identified a Tyr (TAT)-to-Cys (TGT) mutation at either codon 188 (Cys-188; 9 of 15 clones) or codon 181 (Cys-181; 5 of 15 clones). The same amino acid changes were found in RT from E-EPU-resistant virus, but the Cys-181 mutation was more common (9 of 10 clones) than the Cys-188 mutation (1 of 10 clones). Site-specific mutagenesis and production of mutant recombinant viruses demonstrated that both the Cys-181 and Cys-188 mutations cause resistance to E-EPSeU and E-EPU. Of the two mutations, the Cys-188 substitution produced greater E-EPSeU and E-EPU resistance. The predominance of the Cys-188 mutation in E-EPSeU-resistant variants has not been noted for other classes of HIV-1 specific RT inhibitors. HIV-1 resistance is likely to limit the therapeutic efficacy of acyclic 6-substituted pyrimidines if they are used as monotherapy. PMID:7840579

  13. Mimicking Damaged DNA with a Small Moleclue Inhibitor of Human UNG2

    SciTech Connect

    Krosky,D.; Bianchet, M.; Seiple, L.; Chung, S.; Amzel, L.; Stivers, J.

    2006-01-01

    Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases.

  14. The Synthesis of Ribosomes in E. coli

    PubMed Central

    Mccarthy, B. J.; Britten, R. J.

    1962-01-01

    C14-uracil is rapidly incorporated by E. coli at low concentrations. Approximately half the radioactivity passes directly into RNA with very little delay. The remaining half enters a large metabolic pool and later is incorporated into RNA. The total rate of uptake (growing cells) is not greater than the requirement for uracil and cytosine for RNA synthesis. The size of the metabolic pool is not influenced measurably by the external uracil concentration. No evidence is found for the existence of a fraction of RNA which is rapidly synthesized and degraded. PMID:19431314

  15. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA.

    PubMed

    Briggs, Adrian W; Stenzel, Udo; Meyer, Matthias; Krause, Johannes; Kircher, Martin; Pääbo, Svante

    2010-04-01

    DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil-DNA-glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms.

  16. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA.

    PubMed

    Briggs, Adrian W; Stenzel, Udo; Meyer, Matthias; Krause, Johannes; Kircher, Martin; Pääbo, Svante

    2010-04-01

    DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil-DNA-glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms. PMID:20028723

  17. 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.

  18. Counterselection System for Geobacillus kaustophilus HTA426 through Disruption of pyrF and pyrR

    PubMed Central

    Murakami, Ayano; Yoshida, Ken-ichi

    2012-01-01

    Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems. PMID:22885745

  19. Heteroadamantanes and their derivatives. V. Synthesis of 5-monosubstituted 6-oxo- and 6-hydroxy-1,3-diazaadamantanes

    SciTech Connect

    Kuznetsov, A.I.; Basargin, E.B.; Mamadu Hadi Ba; Yakushev, P.F.; Unkovskii, B.V.

    1986-05-20

    The difficulty obtainable 5-methyl- and 5-phenyl-6-oxo-1,3-diazaadamantanes are formed when methyl ethyl ketone and methyl benzyl ketone are heated with hexamethylenetetraamine and glacial acetic acid in 1-butanol by a modified Mannich reaction. Their reduction gave 5-methyl- and 5-phenyl-6-hydroxy-1,3-diazaadamantanes.

  20. Photoelectron spectroscopy and density functional theory studies on the uridine homodimer radical anions

    NASA Astrophysics Data System (ADS)

    Jae Ko, Yeon; Storoniak, Piotr; Wang, Haopeng; Bowen, Kit H.; Rak, Janusz

    2012-11-01

    We report the photoelectron spectrum (PES) of the homogeneous dimer anion radical of uridine, (rU)2•-. It features a broad band consisting of an onset of ˜1.2 eV and a maximum at the electron binding energy (EBE) ranging from 2.0 to 2.5 eV. Calculations performed at the B3LYP/6-31++G** level of theory suggest that the PES is dominated by dimeric radical anions in which one uridine nucleoside, hosting the excess charge on the base moiety, forms hydrogen bonds via its O8 atom with hydroxyl of the other neutral nucleoside's ribose. The calculated adiabatic electron affinities (AEAGs) and vertical detachment energies (VDEs) of the most stable homodimers show an excellent agreement with the experimental values. The anionic complexes consisting of two intermolecular uracil-uracil hydrogen bonds appeared to be substantially less stable than the uracil-ribose dimers. Despite the fact that uracil-uracil anionic homodimers are additionally stabilized by barrier-free electron-induced proton transfer, their relative thermodynamic stabilities and the calculated VDEs suggest that they do not contribute to the experimental PES spectrum of (rU)2•-.

  1. APOBEC3B: pathological consequences of an innate immune DNA mutator.

    PubMed

    Burns, Michael B; Leonard, Brandon; Harris, Reuben S

    2015-01-01

    Cancer is a disease that results from alterations in the cellular genome. Several recent studies have identified mutational signatures that implicate a variety of mutagenic processes in cancer, a major one of which is explained by the enzymatic activity of the DNA cytosine deaminase, APOBEC3B. As a deaminase, APOBEC3B converts cytosines to uracils in single-stranded DNA. Failure to properly repair these uracil lesions can result in a diverse array of mutations. For instance, DNA uracils can template the insertion of complementary adenines leading to C-to-T transition mutations. DNA uracils can also be converted into abasic sites that, depending upon the DNA polymerase recruited to bypass this lesion in the template strand, can lead to adenine insertion and C-to-T mutations as well as cytosine insertion and C-to-G transversion mutations. Finally, DNA uracils can also be converted into DNA breaks that may precipitate some types of larger chromosomal aberrations observed in cancer. These studies cumulatively demonstrate that APOBEC3B is a major source of genetic heterogeneity in several human cancers and, as such, this enzyme may prove to be a critical diagnostic and therapeutic target.

  2. 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. PMID:24621654

  3. 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.

  4. Cloning and transcriptional control of a eucaryotic permease gene.

    PubMed Central

    Chevallier, M R

    1982-01-01

    The uracil permease gene of the yeast Saccharomyces cerevisiae was cloned on a hybrid plasmid which replicates autonomously in both yeast and Escherichia coli. Cloning was carried out by complementation in yeast. The smallest DNA fragment found to complement the uracil permease deficiency in recipient yeast cells measured approximately 2.3 kilobases. In strains transformed by the plasmid with the uracil permease gene inserted, initial rates of uracil uptake increased up to 25 times more than the rates found in the wild type. Using DNA probes carrying several regions of the cloned gene, I showed that a strain carrying the dhul-I mutation, which is not linked to the permease structural gene and is responsible for enhanced uptake velocity of uracil, had enhanced transcription of the permease gene. By using DNA probes recloned in phage M13 mp7, the direction of transcription of the permease gene relative to the restriction map was deduced. A half-life of 2 min was found for the permease mRNA in labeling kinetics experiments. PMID:6290876

  5. 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. PMID:24143868

  6. Biosynthesis of the pyrimidinyl amino acid lathyrine by Lathyrus tingitanus L.

    PubMed Central

    Brown, E G; Al-Baldowi, N F

    1977-01-01

    The biosynthesis of the pyrimidinyl amino acid lathyrine by seedlings of Lathyrus tingitanus L. was shown to be stimulated by uracil. [6(-14)C]Orotate, [2(-14)C]uracil and [3(-14)C]serine were incorporated into lathyrine; the incorporation of [6(-14)C]orotate was substantially decreased in the presence of uracil. Chemical degradation to locate the 14C incorporated from labelled precursors showed that 90% of the radioactivity incorporated into lathyrine from [3(-14)C]serine could be recovered in the alanine side chain. Over 80% of the radioactivity incorporated from [2(-14)C]uracil was shown to be located in C-2 of lathyrine. It is concluded that under the conditions studied, lathyrine arises from a preformed pyrimidine arising via the orotate pathway. Paradoxically, it was also possible to confirm previous reports that radioactivity from L-[guanidino-14C]homoarginine is incorporated into lathyrine and gamma-hydroxyhomoarginine. However, as homoarginine and gamma-hydroxyhomoarginine are also both labelled by [2(-14)C]uracil, it is suggested that they are products of the ring-opening of lathyrine and that reversibility of this process accounts, at least in part, for their observed experimental incorporation into lathyrine. PMID:883953

  7. Attempted prebiotic synthesis of pseudouridine

    NASA Technical Reports Server (NTRS)

    Dworkin, J. P.; Miller, S. L. (Principal Investigator)

    1997-01-01

    Pseudouridine is a modified base found in all tRNA and rRNA. Hence, it is reasonable to think that pseudouridine was important in the early evolution, if not the origin, of life. Since uracil reacts rapidly with formaldehyde and other aldehydes at the C-5 position, it is plausible that pseudouridine could be synthesized in a similar way by the reaction of the C-5 of uracil with the C-1 of ribose. The determining factor is whether the ribose could react with the uracil faster than ribose decomposes. However, both rates are determined by the amount of free aldehyde in the ribose. Various plausible prebiotic reactions were investigated and none showed pseudouridine above the detection limit (<0.01%). Only unreacted uracil and ribose decomposition products could be observed. Thus the rate of addition of ribose to uracil is much slower than the decomposition of ribose under any reasonable prebiotic conditions. Unless efficient non-biological catalysts for any of these reactions exist, pseudouridine would not have been synthesized to any significant extent without the use of biologically produced enzymes.

  8. Characterization of carbamoyl phosphate synthetase of Streptomyces spp.

    PubMed

    Vaishnav, P; Randev, S; Jatiani, S; Aggarwal, S; Keharia, H; Vyas, P R; Nareshkumar, G; Archana, G

    2000-09-01

    Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc). PMID:12561954

  9. 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.

  10. Mutational analysis of the damage-recognition and catalytic mechanism of human SMUG1 DNA glycosylase.

    PubMed

    Matsubara, Mayumi; Tanaka, Tamon; Terato, Hiroaki; Ohmae, Eiji; Izumi, Shunsuke; Katayanagi, Katsuo; Ide, Hiroshi

    2004-01-01

    Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.

  11. 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.

  12. 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. PMID:23082747

  13. 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

  14. Biological UV dosimeters in simulated space irradiation conditions

    NASA Astrophysics Data System (ADS)

    Rontó, G.; Bérces, A.; Fekete, A.; Kovács, G.; Lammer, H.

    For the measurement of the harmful biological effect of solar UV radiation bacteriophage T7 and polycrystalline uracil dosimeters were used. For terrestrial dosimetric purposes bacteriophage T7 has been applied in solution, while uracil in the form of thin layers. For space irradiation dosimetry the uracil, phage T7-DNA and bacteriophage T7 thin layer samples were prepared in vacuum tightly closed sandwich forms covered either by calciumfluoride or quartz windows. The experimental conditions tested correspond to the conditions planned in the EXPOSE facility: the samples were surrounded by nitrogen atmosphere at various humidities, their vacuum stability was tested in the vacuum chamber of the Institute of Space Research,, Graz. All kinds of the thin film samples have been stored in an atmosphere containing Nitrogen and Hidrogen, in quality control no change in the structure of them has been found. To attenuate the high extraterrestrial irradiance neutral filters of 0.5 and 1.0 optical densities have been tested. Irradiation of the samples has been performed with various UV sources: solar simulator, low pressure Mercury lamp, Deuterium lamp. Dose-effect functions have been determined using for the evaluation spectrophotometry in the characteristic UV range, HPLC of photoproducts, PCR of two different primer sequences of phage T7-DNA. Photoproduct formation kinetics was followed by the saturation level of uracil thin layer. Attenuation ability of the neutral filters was controlled with low pressure Mercury lamp by the exposure necessary for saturation of uracil dosimeters. A three and tenfold increase in the exposure was found respectively, while the influence of spectral composition of the irradiation source was tested using Deuterium lamp supplied with Ca F2 and quartz filters respectively. A doubling of the irradiance was necessary for the saturation of uracil with quartz filter.

  15. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA

    PubMed Central

    Briggs, Adrian W.; Stenzel, Udo; Meyer, Matthias; Krause, Johannes; Kircher, Martin; Pääbo, Svante

    2010-01-01

    DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil–DNA–glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms. PMID:20028723

  16. 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.

    2016-03-01

    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.

  17. 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...

  18. Anti-wrinkle and anti-inflammatory effects of active garlic components and the inhibition of MMPs via NF-κB signaling.

    PubMed

    Kim, So Ra; Jung, Yu Ri; An, Hye Jin; Kim, Dae Hyun; Jang, Eun Ji; Choi, Yeon Ja; Moon, Kyoung Mi; Park, Min Hi; Park, Chan Hum; Chung, Ki Wung; Bae, Ha Ram; Choi, Yung Whan; Kim, Nam Deuk; Chung, Hae Young

    2013-01-01

    Skin aging is a multisystem degenerative process caused by several factors, such as, UV irradiation, stress, and smoke. Furthermore, wrinkle formation is a striking feature of photoaging and is associated with oxidative stress and inflammatory response. In the present study, we investigated whether caffeic acid, S-allyl cysteine, and uracil, which were isolated from garlic, modulate UVB-induced wrinkle formation and effect the expression of matrix-metalloproteinase (MMP) and NF-κB signaling. The results obtained showed that all three compounds significantly inhibited the degradation of type І procollagen and the expressions of MMPs in vivo and attenuated the histological collagen fiber disorder and oxidative stress in vivo. Furthermore, caffeic acid and S-allyl cysteine were found to decrease oxidative stress and inflammation by modulating the activities of NF-κB and AP-1, and uracil exhibited an indirect anti-oxidant effect by suppressing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions levels and downregulating transcriptional factors. These results suggest that the anti-wrinkle effects of caffeic acid, S-allyl cysteine, and uracil are due to anti-oxidant and/or anti-inflammatory effects. Summarizing, caffeic acid, S-allyl cysteine, and uracil inhibited UVB-induced wrinkle formation by modulating MMP via NF-κB signaling.

  19. Structures of [M(Ura-H)(H2 O)n ](+) (M = Mg, Ca, Sr, Ba; n = 1-3) complexes in the gas phase by IRMPD spectroscopy and theoretical studies.

    PubMed

    Power, Barry; Haldys, Violette; Salpin, Jean-Yves; Fridgen, Travis D

    2016-03-01

    The structures of singly and doubly (and for Mg, triply) hydrated group 2 metal dications bound to deprotonated uracil were explored in the gas phase using infrared multiple photon dissociation spectroscopy in the mid-infrared region (1000-1900 cm(-1) ) and the O-H/N-H stretching region (2700-3800 cm(-1) ) in a Fourier transform ion cyclotron resonance mass spectrometer. The infrared multiple photon dissociation spectra were then compared with the computed IR spectra for various isomers. Calculations were performed using B3LYP with the 6-31 + G(d,p) basis set for all atoms except Ba(2+) and Sr(2+) , for which the LANL2DZ or the def2-TZVPP basis sets with relativistic core potentials were used. Atoms-in-molecules analysis was conducted for all lowest energy structures. The lowest energy isomers in all cases are those in which the one uracil is deprotonated at the N3 position, and the metal is coordinated to the N3 and O4 of uracil. Regardless of the degree of solvation, all water molecules are bound to the metal ion and participate in a hydrogen bond with a carbonyl of the uracil moiety.

  20. Structures of [M(Ura-H)(H2 O)n ](+) (M = Mg, Ca, Sr, Ba; n = 1-3) complexes in the gas phase by IRMPD spectroscopy and theoretical studies.

    PubMed

    Power, Barry; Haldys, Violette; Salpin, Jean-Yves; Fridgen, Travis D

    2016-03-01

    The structures of singly and doubly (and for Mg, triply) hydrated group 2 metal dications bound to deprotonated uracil were explored in the gas phase using infrared multiple photon dissociation spectroscopy in the mid-infrared region (1000-1900 cm(-1) ) and the O-H/N-H stretching region (2700-3800 cm(-1) ) in a Fourier transform ion cyclotron resonance mass spectrometer. The infrared multiple photon dissociation spectra were then compared with the computed IR spectra for various isomers. Calculations were performed using B3LYP with the 6-31 + G(d,p) basis set for all atoms except Ba(2+) and Sr(2+) , for which the LANL2DZ or the def2-TZVPP basis sets with relativistic core potentials were used. Atoms-in-molecules analysis was conducted for all lowest energy structures. The lowest energy isomers in all cases are those in which the one uracil is deprotonated at the N3 position, and the metal is coordinated to the N3 and O4 of uracil. Regardless of the degree of solvation, all water molecules are bound to the metal ion and participate in a hydrogen bond with a carbonyl of the uracil moiety. PMID:26956390

  1. 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.

  2. 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.

  3. High-resolution photoelectron spectra of the pyrimidine-type nucleobases

    SciTech Connect

    Fulfer, K. D.; Hardy, D.; Poliakoff, E. D.; Aguilar, A. A.

    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.

  4. The metabolism of histamine in the Drosophila optic lobe involves an ommatidial pathway: β-alanine recycles through the retina.

    PubMed

    Borycz, Janusz; Borycz, Jolanta A; Edwards, Tara N; Boulianne, Gabrielle L; Meinertzhagen, Ian A

    2012-04-15

    Flies recycle the photoreceptor neurotransmitter histamine by conjugating it to β-alanine to form β-alanyl-histamine (carcinine). The conjugation is regulated by Ebony, while Tan hydrolyses carcinine, releasing histamine and β-alanine. In Drosophila, β-alanine synthesis occurs either from uracil or from the decarboxylation of aspartate but detailed roles for the enzymes responsible remain unclear. Immunohistochemically detected β-alanine is present throughout the fly's entire brain, and is enhanced in the retina especially in the pseudocone, pigment and photoreceptor cells of the ommatidia. HPLC determinations reveal 10.7 ng of β-alanine in the wild-type head, roughly five times more than histamine. When wild-type flies drink uracil their head β-alanine increases more than after drinking l-aspartic acid, indicating the effectiveness of the uracil pathway. Mutants of black, which lack aspartate decarboxylase, cannot synthesize β-alanine from l-aspartate but can still synthesize it efficiently from uracil. Our findings demonstrate a novel function for pigment cells, which not only screen ommatidia from stray light but also store and transport β-alanine and carcinine. This role is consistent with a β-alanine-dependent histamine recycling pathway occurring not only in the photoreceptor terminals in the lamina neuropile, where carcinine occurs in marginal glia, but vertically via a long pathway that involves the retina. The lamina's marginal glia are also a hub involved in the storage and/or disposal of carcinine and β-alanine.

  5. 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.

  6. 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.

  7. Apurinic/apyrimidinic endonuclease 1 is the essential nuclease during immunoglobulin class switch recombination.

    PubMed

    Masani, Shahnaz; Han, Li; Yu, Kefei

    2013-04-01

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) that catalyzes numerous DNA cytosine deaminations within switch regions. The resulting uracils are processed by uracil base excision and/or mismatch repair enzymes that ultimately generate switch region DNA double-strand breaks (DSBs). Uracil glycosylase 2 (UNG2) is required for CSR, most likely by removing uracils to generate abasic sites. Although it is presumed that the apurinic/apyrimidinic endonuclease 1 (APE1) generates DNA strand incisions (a prerequisite for CSR) at these abasic sites, a direct test of the requirement for APE1 in CSR has been difficult because of the embryonic lethality of APE1 ablation in mice. Here, we report the successful deletion of the APE1 gene in a mouse B cell line (CH12F3) capable of robust CSR in vitro. In contrast to the general assumption that APE1 is essential for cellular viability, deletion of APE1 in CH12F3 cells has no apparent effect on cell viability or growth. Moreover, CSR in APE1-null CH12F3 cells is drastically reduced, providing direct evidence for an essential role for APE1 in switch region cleavage and CSR. Finally, deletion of AP endonuclease 2 (APE2) has no effect on CSR in either APE1-proficient or -deficient cells.

  8. 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. PMID:27411074

  9. Alternative bases in the RNA world: the prebiotic synthesis of urazole and its ribosides.

    PubMed

    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.

  10. Aging of Escherichia coli

    PubMed Central

    Clifton, C. E.

    1966-01-01

    Clifton, C. E. (Stanford University, Stanford, Calif.). Aging of Escherichia coli. J. Bacteriol. 92:905–912. 1966.—The rates of endogenous and exogenous (glucose) respiration decreased much more rapidly than did the viable count during the first 24 hr of aging of washed, C14-labeled cells of Escherichia coli K-12 suspended in a basal salt medium devoid of ammonium salts. The rates of decrease of respiration and of death approached each other as the age of the cells increased, but death was not the only factor involved in decreased respiratory activity of the suspensions. The greatest decrease in cellular contents with aging was noted in the ribonucleic acid fraction, of which the ribose appeared to be oxidized, while uracil accumulated in the suspension medium. The viable count and respiratory activities remained higher in glucose-fed than in nonfed suspensions. Proline-labeled cells fed glucose tended to lose more of their proline and to convert more proline into C14O2 than in unfed controls. On the other hand, uracil-labeled cells fed glucose retained more of the uracil than did nonfed cells, but glucose elicited some oxidation of uracil. An exogenous energy source such as glucose aided in the maintenance of a population, but it was not the only factor needed for such maintenance. PMID:5332874

  11. Immunoglobulin genes: generating diversity with AID and UNG.

    PubMed

    Storb, Ursula; Stavnezer, Janet

    2002-10-29

    Somatic hypermutation and switch recombination of immunoglobulin genes require the activity of the activation-induced deaminase, AID. Recent studies of mice deficient for the uracil-DNA glycosylase UNG, which removes U from DNA, suggest that AID catalyses the deamination of dC to dU during antibody diversification.

  12. [Isolation and identification of the secondary metabolites from Hydroclathrus clathratus].

    PubMed

    Xiaoling, Li; Shihai, Xu; Kai, Yang; Shuhao, Guo; Xiangchao, Zeng

    2004-04-01

    Four compounds have been isolated from the algae Hydroclathrus clathratus collected from the South China Sea. Their structures were established as 1-glyceryl hexadecanoate (1), eicosanamide, N-[2'-hydroxy-1'-(hydroxymethyl) pentadecyl] (2), uracil (3), mannitol (4) by MS, 1HNMR and 13CNMR. Compound 1, 2 and 3 were isolated from this algae for the first time.

  13. Acetylene as an essential building block for prebiotic formation of pyrimidine bases on Titan.

    PubMed

    Jeilani, Yassin A; Fearce, Chelesa; Nguyen, Minh Tho

    2015-10-01

    Prebiotic building blocks for the formation of biomolecules are important in understanding the abiotic origin of biomolecules. However, there is a limited choice of the building blocks as precursors for the biomolecules. Acetylene (HCCH) is found in Titan's atmosphere and is an abiotic-precursor of pyrimidine bases. HCCH reacts with urea to form both cytosine and uracil. The mechanisms for the formation of both cytosine and uracil were studied by density functional theory at B3LYP/6-311G(d,p) level. Ethynyl radicals (˙CCH) are relevant for the chemistry of Titan's atmosphere therefore both HCCH and ˙CCH were evaluated as carbon sources. The pathways, for both HCCH and ˙CCH, lead to intermediates with an unsaturated-group that facilitate the formation of the six-membered ring of the pyrimidine bases. The predicted structures for cytosine and uracil were compared with labeled cytosine and uracil that were formed from the reaction of DCCD with urea. The results suggest that cytosine is formed from HCCH while uracil is formed from ˙CCH. The mechanisms are energetically feasible and there is no conclusive evidence for the preferred pathway (HCCH or ˙CCH). The pathways were further extended for the formation of both uric acid and 8-oxoguanine from HCCH and urea, and demonstrate the utility of HCCH as a carbon source for diverse biomolecules. Biuret is identified as a precursor for the pyridimine bases, and it unifies the free radical pathways for the pyrimidine bases with those of triazines. The pathways are appropriate for the reducing atmosphere that creates both radicals and electrons due to ionizing radiation on Titan. The mechanisms are feasible for the extraterrestrial formation of the pyrimidine bases. PMID:26325173

  14. 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

  15. 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. PMID:26393397

  16. 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.

  17. 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.

  18. Adjuvant Chemotherapy for the Completely Resected Stage IB Nonsmall Cell Lung Cancer

    PubMed Central

    He, Jiaxi; Shen, Jianfei; Yang, Chenglin; Jiang, Long; Liang, Wenhua; Shi, Xiaoshun; Xu, Xin; He, Jianxing

    2015-01-01

    Abstract Adjuvant chemotherapy is recommended for postoperative stage II-IIIB nonsmall cell lung cancer patients. However, its effect remains controversial in stage IB patients. We, therefore, performed a meta-analysis to compare the efficacy of adjuvant chemotherapy versus surgery alone in stage IB patients. Six electronic databases were searched for relevant articles. The primary and secondary outcomes were overall survival (OS) and disease-free survival (DFS). The time-to-event outcomes were compared by hazard ratio using log-rank test. Sixteen eligible trials were identified. A total of 4656 patients were included and divided into 2 groups: 2338 in the chemotherapy group and 2318 in the control group (surgery only). Patients received platinum-based therapy, uracil-tegafur, or a combination of them. Our results demonstrated that patients can benefit from the adjuvant chemotherapy in terms of OS (HR 0.74 95% CI 0.63–0.88) and DFS (HR 0.64 95% CI 0.46–0.89). Patients who received 6-cycle platinum-based therapy (HR 0.45 95% CI 0.29–0.69), uracil-tegafur (HR 0.71 95% CI 0.56–0.90), or a combination of them (HR 0.51 95% CI 0.36–0.74) had better OS, but patients who received 4 or fewer cycles platinum-based therapy (HR 0.97 95% CI 0.85–1.11) did not. Moreover, 6-cycle platinum-based therapy (HR 0.29 95% CI 0.13–0.63) alone or in combination with uracil-tegafur (HR 0.44 95% CI 0.30–0.66) had advantages in DFS. However, 4 or fewer cycles of platinum-based therapy (HR 0.89 95% CI 0.76–1.04) or uracil-tegafur alone (HR 1.19 95% CI 0.79–1.80) were not beneficial. Six-cycle platinum-based chemotherapy can improve OS and DFS in stage IB NSCLC patients. Uracil-tegafur alone or in combination with platinum-based therapy is beneficial to the patients in terms of OS, but uracil-tegafur seems to have no advantage in prolonging DFS, unless it is administered with platinum-based therapy. PMID:26039122

  19. [Development of Eimeria tenella in MDBK cell culture with a note on enhancing effect of preincubation with chicken spleen cells].

    PubMed

    Chai, J Y; Lee, S H; Kim, W H; Yun, C K

    1989-06-01

    Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella oöcysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of

  20. Three oxygenated cyclohexenone derivatives produced by an endophytic fungus.

    PubMed

    Shiono, Yoshihito; Murayama, Tetsuya; Takahashi, Koetsu; Okada, Katsuhide; Katohda, Shigeyoshi; Ikeda, Michimasa

    2005-02-01

    Three cyclohexenone derivatives, (4S,5S,6S)-5,6-epoxy-4-hydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (1), (4R,5R)-4,5-dihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (2), and (4R,5S,6R)-4,5,6-trihydroxy-3-methoxy-5-methyl-cyclohex-2-en-1-one (3), were isolated from unpolished rice fermented with an xylariaceous endophytic fungus (strain YUA-026). The structures of three compounds were established on the basis of spectroscopic analyses and chemical conversion. The minimum inhibitory concentrations of 1 and 3 were 100 microg/ml and 400 microg/ml against Staphylococcus aureus, 100 microg/ml and 200 microg/ml against Pseudomonas aeruginosa, and 200 microg/ml and >400 microg/ml against Candida albicans, respectively. In addition, 1 and 3 exhibited phytotoxic activity against lettuce. PMID:15725652

  1. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K 12. Involvement of the glnLG and purR genes in the regulation of codA expression.

    PubMed

    Andersen, L; Kilstrup, M; Neuhard, J

    1989-01-01

    Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.

  2. Catalysis of a Flavoenzyme-Mediated Amide Hydrolysis

    SciTech Connect

    Mukherjee, Tathagata; Zhang, Yang; Abdelwahed, Sameh; Ealick, Steven E.; Begley, Tadhg P.

    2010-09-13

    A new pyrimidine catabolic pathway (the Rut pathway) was recently discovered in Escherichia coli K12. In this pathway, uracil is converted to 3-hydroxypropionate, ammonia, and carbon dioxide. The seven-gene Rut operon is required for this conversion. Here we demonstrate that the flavoenzyme RutA catalyzes the initial uracil ring-opening reaction to give 3-ureidoacrylate. This reaction, while formally a hydrolysis reaction, proceeds by an oxidative mechanism initiated by the addition of a flavin hydroperoxide to the C4 carbonyl. While peroxide-catalyzed amide hydrolysis has chemical precedent, we are not aware of a prior example of analogous chemistry catalyzed by flavin hydroperoxides. This study further illustrates the extraordinary catalytic versatility of the flavin cofactor.

  3. 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.

  4. Synthesis of new, base-modified PNA monomers.

    PubMed

    Sipos, Ferenc; Sági, Gyula

    2007-01-01

    A number of N-Boc-protected peptide nucleic acids (PNA) monomers containing 5-aryl- and 5-alkynyl-uracil bases have been synthesized using different palladium-catalyzed cross-coupling reactions. Starting from the base-unprotected 5-iodo-uracil PNA monomer, only the Stille couplings were accomplished successfully, while Suzuki couplings with boronic acids containing the same aryl groups failed. During Sonogashira couplings with terminal alkynes, significant amounts of unrequired furano[2,3-d]pyrimidine by-products were formed. Protection of the lactam function by p-methoxybenzylation prevented the opportunity for intramolecular cyclization as well as formation of a negative charge on the 4-O atom, making it possible to reach almost quantitative yields at Sonogashira couplings and acceptable conversions in Suzuki reactions. PMID:18066879

  5. Bond- and Site-Selective Loss of H{sup -} from Pyrimidine Bases

    SciTech Connect

    Ptasinska, Sylwia; Denifl, Stephan; Grill, Verena; Maerk, Tilmann D.; Illenberger, Eugen; Scheier, Paul

    2005-08-26

    Electron attachment to gas phase thymine and uracil leads to H{sup -} loss within a broad and structured feature in the energy range between about 5 and 12 eV consisting of 4 overlapping resonances. By using thymine and uracil methylated at the N1 and N3 positions, respectively, and taking into account recent results from partly deuterated thymine, we find that by tuning the electron energy, H{sup -} loss turns out to be not only bond selective, i.e., (C-H) versus (N-H) bonds, but also site selective (N1 versus N3 site). Such a bond and site selectivity by energy has not been observed before in dissociative electron attachment. Implications for the mechanism of strand breaks observed in plasmid DNA are considered.

  6. Discovery of sodium R-(+)-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyrate (elagolix), a potent and orally available nonpeptide antagonist of the human gonadotropin-releasing hormone receptor.

    PubMed

    Chen, Chen; Wu, Dongpei; Guo, Zhiqiang; Xie, Qiu; Reinhart, Greg J; Madan, Ajay; Wen, Jenny; Chen, Takung; Huang, Charles Q; Chen, Mi; Chen, Yongsheng; Tucci, Fabio C; Rowbottom, Martin; Pontillo, Joseph; Zhu, Yun-Fei; Wade, Warren; Saunders, John; Bozigian, Haig; Struthers, R Scott

    2008-12-11

    The discovery of novel uracil phenylethylamines bearing a butyric acid as potent human gonadotropin-releasing hormone receptor (hGnRH-R) antagonists is described. A major focus of this optimization was to improve the CYP3A4 inhibition liability of these uracils while maintaining their GnRH-R potency. R-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyric acid sodium salt, 10b (elagolix), was identified as a potent and selective hGnRH-R antagonist. Oral administration of 10b suppressed luteinizing hormone in castrated macaques. These efforts led to the identification of 10b as a clinical compound for the treatment of endometriosis.

  7. Assignment of the human dihydropyrimidine dehydrogenase gene (DPYD) to chromosome region 1p22 by fluorescence in situ hybridization

    SciTech Connect

    Takai, Setsuo; Fernandez-Salguero, Pedro; Kimura, Shioko

    1994-12-01

    Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the three-step pathway of uracil and thymine catabolism leading to the formation of {beta}-alanine and {beta}-aminobutyric acid, respectively. Several studies have demonstrated the importance of DPD in cancer patients, particularly in those lacking or having only low levels of activity. Patients exhibiting severe toxicity when administered 5-fluorouracil were shown to have low DPD activity. Studies of affected families demonstrated that the deficiency was inherited in an autosomal recessive pattern. DPD deficiency is one of several inherited disorders of pyrimidine metabolism, clinically termed thymine-uracil-uria. 14 refs., 1 fig.

  8. 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.

  9. Uptake and incorporation of pyrimidines in Euglena gracilis.

    PubMed

    Wasternack, C H

    1976-08-01

    In photoorganotrophically grown cells of Euglena gracilis the uptake and incorporation degree of 12 different pyrimidines were tested. The rate of uptake of pyrimidines has distinct maxima in the late log phase and in the stationary phase of cell multiplication. The kinetics of uptake are linear in the first 2 h, do not show saturation at various concentrations and increase with the concetrations. No accumulation of the pyrimidines at various concentrations could be observed in the first 2 h of incubation. Membrane inhibitors as uranyl acetate inhibit the uptake of the reference substance alpha-AIB, which is wellknown transported by an active transport mechanism, but have no effect on uptake rate of uracil and cytosine. It could not be observed an energy requirement tested in temperature dependence and with electron transport inhibitors. Uptake of uridine, uracil, barbituric acid and alpha-AIB is inhibited by cycloheximide in a different manner after 5 - 10 min.

  10. Interactions of model biomolecules. Benchmark CC calculations within MOLCAS

    NASA Astrophysics Data System (ADS)

    Urban, Miroslav; PitoÅák, Michal; Neogrády, Pavel; Dedíková, Pavlína; Hobza, Pavel

    2015-01-01

    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.

  11. Impairment of cobalt-induced riboflavin biosynthesis in a Debaryomyces hansenii mutant.

    PubMed

    Seda-Miró, Jasmine M; Arroyo-González, Nancy; Pérez-Matos, Ana; Govind, Nadathur S

    2007-11-01

    Flavinogenic yeasts such as Debaryomyces hansenii overproduce riboflavin (RF) in the presence of heavy metals. Growth and RF production were compared between wild-type D. hansenii and a RF production-impaired metal-tolerant ura3 mutant in the presence of sublethal cobalt(II) concentrations. Debaryomyces hansenii (wild type) exhibits an extended lag phase with an increase in RF synthesis. Supplementation of exogenous uracil shortened the lag phase at the highest concentration of cobalt(II) used, suggesting that uracil has a possible role in metal acclimation. The D. hansenii ura3 mutant isolated by chemical mutagenesis exhibited a higher level of metal tolerance, no extended lag phase, and no marked increase in RF synthesis. Transformation of the mutant with the URA3 gene isolated from Saccharyomyces cerevisiae or D. hansenii did not restore wild-type characteristics, suggesting a second mutation that impairs RF oversynthesis. Our results demonstrate that growth, metal sensitivity, and RF biosynthesis are linked. PMID:18026221

  12. UV fragmentation and ultrafast dynamics of trinuclear silver/1-methylthymine and silver/1-methyluracil metal-base pairs in an ion trap

    NASA Astrophysics Data System (ADS)

    Nosenko, Yevgeniy; Riehn, Christoph; Klopper, Wim

    2016-08-01

    We report on gas phase UV action spectroscopy and photodynamics of [Ag3(1MT-H/1MU-H)2]+ comprised of a linear silver string and two deprotonated 1-methyl-thymine/uracil (1MT/1MU) ligands. We applied pump-probe femtosecond laser photofragmentation in an electrospray ion trap mass spectrometer and high-level ab initio calculations at the level of approximate coupled-cluster singles-doubles theory. The experimental UV band at 283/275 nm is assigned to a red shifted 1ππ∗ nucleobase located transition. Relaxation of the 1ππ∗ state occurs with time constants of 0.2/1.1 ps and 0.2/4.2 ps for the 1MT and 1MU complexes, respectively, on a similar ultrafast time scale as non-metalated uracil derivatives.

  13. TGF-β triggers HBV cccDNA degradation through AID-dependent deamination.

    PubMed

    Qiao, Ying; Han, Xiaoxu; Guan, Gefei; Wu, Na; Sun, Jianbo; Pak, Vladimir; Liang, Guoxin

    2016-02-01

    The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a viral center molecule for HBV infection and persistence. However, the cellular restriction factors of HBV cccDNA are not well understood. Here, we show that TGF-β can induce nuclear viral cccDNA degradation and hypermutation via activation-induced cytidine deaminase (AID) deamination activity in hepatocytes. This suppression by TGF-β is abrogated when AID or the activity of uracil-DNA glycosylase (UNG) is absent, which indicates that AID deamination and the UNG-mediated excision of uracil act in concert to degrade viral cccDNA. Moreover, the HBV core protein promotes the interaction between AID and viral cccDNA. Overall, our results indicate a novel molecular mechanism that allows cytokine TGF-β to restrict viral nuclear cccDNA in innate immunity, thereby suggesting a novel method for potentially eliminating cccDNA.

  14. Blocking cyclobutane pyrimidine dimer formation by steric hindrance.

    PubMed

    Vendrell-Criado, Victoria; Lhiaubet-Vallet, Virginie; Yamaji, Minoru; Cuquerella, M Consuelo; Miranda, Miguel A

    2016-04-26

    The efficiency of thymine (Thy) and uracil (Ura) to form cyclobutane pyrimidine dimers (CPDs) in solution, upon UV irradiation differs by one order of magnitude. This could to be partially related to the steric hindrance induced by the methyl at C5 in thymine. The aim of the present work is to establish the influence of a bulky moiety at this position on the photoreactivity of pyrimidines. With this purpose, photosensitization with benzophenone and acetone of a 5-tert-butyl uracil derivative () and the equivalent Thy () has been compared. Introduction of the tert-butyl group completely blocks CPD formation. Moreover, the mechanistic insight obtained by laser flash photolysis is in accordance with the observed photoreactivity. PMID:27112630

  15. Effect of 2-phenoxyethanol upon RNA, DNA and protein biosynthesis in Escherichia coli NCTC 5933.

    PubMed

    Gilbert, P; Beveridge, E G; Crone, P B

    1980-01-01

    The effects of concentrations of 2-phenoxyethanol of negligible bactericidal activity upon the rates of biosynthetic assimilation by Escherichia coli, of 14C-thymidine, 14C-uracil, 14C-phenylalanine and 14C-glucose, were assessed. Increasing the drug concentration from 0.05-0.4% w/v progressively increased inhibition of growth rate, measured as changes in optical density. Thymidine, uracil and glucose assimilation were inhibited to an extent similar to growth rate, whilst phenylalanine incorporation was markedly less sensitive at the lower concentrations (leads to 0.2% w/v). In addition to its previously observed roles in inhibiting oxidative phosphorylation and TCA cycle enzymes, it is suggested that 2-phenoxyethanol can exert a more direct inhibitory action upon DNA and RNA biosynthesis and possibly on protein biosynthesis.

  16. Urea: obligate intermediate of pyrimidine-ring catabolism in Rhodosporidium toruloides.

    PubMed Central

    Thwaites, W M; Davis, C H; Wallis-Biggart, N; Wondrack, L M; Abbott, M T

    1979-01-01

    Urea has been shown to be an obligate intermediate in and the penultimate product of the catabolism of pyrimidine-ring nitrogen in Rhodosporidium toruloides (Rhodotorula). One of a series of mutants selected for its inability to utilize uracil as a sole source of nitrogen was unable to utilize urea also. The mutant accumulated urea and failed to form 14CO2 during supplementation with [2-14C]uracil. Radioautograms from the resulting cell extracts and media failed to reveal expected intermediates. Cell-free extracts of the mutant were shown to lack urease activity. Revertants of the mutant were essentially wild type in all tested attributes. Elements of the reductive pathway for pyrimidine catabolism are present in Rhodosporidium (O. A. Milstein and M. L. Bekker, J. Bacteriol. 127: 1-6, 1976), but is has not been determined whether this pathway is involved with production of urea. Images PMID:571431

  17. Rapid microwave-enhanced synthesis of C5-alkynyl pyranonucleosides as novel cytotoxic antitumor agents.

    PubMed

    Dimopoulou, Athina; Manta, Stella; Kiritsis, Christos; Gkaragkouni, Dimitra-Niki; Papasotiriou, Ioannis; Balzarini, Jan; Komiotis, Dimitri

    2013-03-01

    A microwave-assisted, one-pot, coupling reaction for the synthesis of C5-alkynyl-uracil and cytosine glucopyranonucleosides has been developed. The reaction is carried out under standard Sonogashira coupling conditions from glucopyranonucleosides of 5-iodouracil or 5-iodocytosine and various terminal alkynes. All compounds were evaluated for their cytostatic and antiviral activity. The 5-phenylethynyluracil pyranonucleoside derivative 6a showed the most promising cytostatic activity (50% inhibitory concentration in the lower micromolar range). No meaningful antiviral activity was recorded.

  18. Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues.

    PubMed

    Killelea, Tom; Ghosh, Samantak; Tan, Samuel S; Heslop, Pauline; Firbank, Susan J; Kool, Eric T; Connolly, Bernard A

    2010-07-13

    Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3'-5' proofreading exonuclease, observed with deaminated bases at this location. A beta-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3' single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.

  19. 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

  20. The nucleoside uridine isolated in the gas phase.

    PubMed

    Peña, Isabel; Cabezas, Carlos; Alonso, José L

    2015-03-01

    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 (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.

  1. 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.

  2. 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. PMID:24500331

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Hydrolysis of α-Chloro-Substituted 2- and 4-Pyridones: Rate Enhancement by Zwitterionic Structure

    PubMed Central

    Tan, Ronald C.; Vien, Janie Q. T.; Wu, Weiming

    2011-01-01

    The hydrolysis of α-chloro-N-methyl-4-pyridone was found to be more than five times faster than that of α-chloro-N-methyl-2-pyridone. Structural studies of 2- and 4-pyridones have revealed the higher polarity and greater extent of zwitterionic content in 4-pyridone. The results are thus consistent with the hypothesis that polarization and higher zwitterionic content in the heterocyclic structures enhances the rate of hydrolysis in α-substituted pyridone and uracil derivatives. PMID:22178555

  8. Hydrolysis of α-chloro-substituted 2- and 4-pyridones: rate enhancement by zwitterionic structure.

    PubMed

    Tan, Ronald C; Vien, Janie Q T; Wu, Weiming

    2012-01-15

    The hydrolysis of α-chloro-N-methyl-4-pyridone was found to be more than five times faster than that of α-chloro-N-methyl-2-pyridone. Structural studies of 2- and 4-pyridones have revealed the higher polarity and greater extent of zwitterionic content in 4-pyridone. The results are thus consistent with the hypothesis that polarization and higher zwitterionic content in the heterocyclic structures enhances the rate of hydrolysis in α-substituted pyridone and uracil derivatives. PMID:22178555

  9. A general approach to the synthesis of 5-S-functionalized pyrimidine nucleosides and their analogues.

    PubMed

    Kananovich, Dzmitry G; Reino, Alli; Ilmarinen, Kaja; Rõõmusoks, Marko; Karelson, Mati; Lopp, Margus

    2014-08-14

    A general and efficient approach was developed for the introduction of S-functionality at the C-5 position of cytosine and uracil nucleosides and their analogues. The key step is a palladium-catalyzed C-S coupling of the corresponding 5-bromo nucleoside derivative and alkyl thiol. The butyl 3-mercaptopropionate coupling products were further converted to the corresponding disulphides, the stable precursors of 5-mercaptopyrimidine nucleosides.

  10. 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

  11. A straightforward entry to chiral carbocyclic nucleoside analogues via the enantioselective [3+2] cycloaddition of α-nucleobase substituted acrylates.

    PubMed

    Xie, Ming-Sheng; Wang, Yong; Li, Jian-Ping; Du, Cong; Zhang, Yan-Yan; Hao, Er-Jun; Zhang, Yi-Ming; Qu, Gui-Rong; Guo, Hai-Ming

    2015-08-11

    A straightforward entry to chiral carbocyclic nucleoside analogues has been realized via the enantioselective [3+2] cycloaddition of α-nucleobase substituted acrylates to vinyl cyclopropanes for the first time. With Pd2(dba)3-L5 as the catalyst, carbocyclic purine, uracil, and thymine nucleoside analogues with quaternary stereocenters were obtained in excellent yields (up to 99% yield) and good enantioselectivities (up to 92% ee). PMID:26145719

  12. Unmarked gene integration into the chromosome of Mycobacterium smegmatis via precise replacement of the pyrF gene.

    PubMed

    Knipfer, N; Seth, A; Shrader, T E

    1997-01-01

    After integration into the bacterial chromosome an exogenous gene may be stably expressed without continued selection for the recombinant locus. However, chromosomal integration events occur infrequently, requiring the concomitant integration of a drug resistance marker in order to identify colonies of recombinant cells. The generation of a drug-resistant recombinant strain can both reduce the in vivo applicability of the strain and preclude the use of recombinant vectors which use the same drug resistance marker. We have constructed a plasmid, pINT-delta, which allows recombination of exogenous genes onto the Mycobacterium smegmatis chromosome. The exogenous gene completely replaces the pyrF gene and the resultant strain lacks any exogenous drug resistance marker. The methodologies described herein are general and applicable even to those bacteria for which extrachromosomal plasmids are not available. Using pINT-delta we integrated the lacZ gene into the M. smegmatis chromosome via a precise exchange of lacZ and pyrF. The resultant strain was used to demonstrate that the expression of genes integrated at the pyrF locus is repressed twofold by inclusion of uracil in the growth medium. In addition, we used pINT-delta to construct an M. smegmatis strain with a precise deletion of its pyrF locus. This strain, TSm-627, grows normally in rich medium but does not grow in medium lacking uracil. TSm-627 cells allow the pyrF gene to be used as a selectable marker for growth on medium lacking uracil. In TSm-627 cells, the pyrF gene is also useful as a counterselectable marker on complete medium containing 5'-fluoroorotic acid and uracil. Two pyrF-containing plasmids, designed to exploit the new delta pyrF strain, have been constructed and their possible applications to problems in mycobacteriology are discussed. PMID:9169204

  13. Identification and synthesis of new bicyclic acetals from the mountain pine beetle, Dendroctonus ponderosae Hopkins (Col.: Scol.).

    PubMed

    Francke, W; Schröder, F; Philipp, P; Meyer, H; Sinnwell, V; Gries, G

    1996-03-01

    Head-space volatiles obtained from male mountain pine beetles, Dendroctonus ponderosae, were analyzed by coupled GC-MS and chiral gas chromatography. 5-Ethyl-7-methyl-6,8-dioxabicyclo[3.2.1]octane (6) was found as a new naturally occurring isomer of brevicomin (1). In addition, several stereoisomers of 7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octan-2-ol (11) and 1-(5-methyl-6,8-dioxabicyclo[3.2.1]octyl)ethanol (12) could be identified. Relative and absolute configurations of the compounds were determined by unambiguous syntheses, which are described.

  14. Studies on gene control regions X. The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction.

    PubMed Central

    Sista, H S; Loder, R T; Caruthers, M H

    1979-01-01

    Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group. These operators also contained a poly(dA) . poly(dT) tail 8 to 12 base pairs in length at one end. Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical. For example a seven fold reduction in stability was observed for the transversion. Results also suggest that the operator spans at least 21 base pairs. Images PMID:379824

  15. Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase.

    PubMed

    Hortua Triana, Miryam Andrea; Cajiao Herrera, Daniela; Zimmermann, Barbara H; Fox, Barbara A; Bzik, David J

    2016-10-01

    Dihydroorotate dehydrogenase (DHODH) mediates the fourth step of de novo pyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. In Toxoplasma gondii, disruption of the first, fifth, or sixth step of de novo pyrimidine biosynthesis induced uracil auxotrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH of T. gondii (TgDHODH) failed. To further address the essentiality of TgDHODH, mutant gene alleles deficient in TgDHODH activity were designed to ablate the enzyme activity. Replacement of the endogenous DHODH gene with catalytically deficient DHODH gene alleles induced uracil auxotrophy. Catalytically deficient TgDHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show that TgDHODH is essential for the synthesis of pyrimidines and suggest that TgDHODH is required for a second essential function independent of its role in pyrimidine biosynthesis. PMID:27481247

  16. 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.

  17. 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. PMID:23857323

  18. Influence of genotype and nutrition on survival and metabolism of starving yeast.

    PubMed

    Boer, Viktor M; Amini, Sasan; Botstein, David

    2008-05-13

    Starvation of yeast cultures limited by auxotrophic requirements results in glucose wasting and failure to achieve prompt cell-cycle arrest when compared with starvation for basic natural nutrients like phosphate or sulfate. We measured the survival of yeast auxotrophs upon starvation for different nutrients and found substantial differences: When deprived of leucine or uracil, viability declined exponentially with a half-life of <2 days, whereas when the same strains were deprived of phosphate or sulfate, the half-life was approximately 10 days. The survival rates of nongrowing auxotrophs deprived of uracil or leucine depended on the carbon source available during starvation, but were independent of the carbon source during prior growth. We performed an enrichment procedure for mutants that suppress lethality during auxotrophy starvation. We repeatedly found loss-of-function mutations in a number of functionally related genes. Mutations in PPM1, which methylates protein phosphatase 2A, and target of rapamycin (TOR1) were characterized further. Deletion of PPM1 almost completely suppressed the rapid lethality and substantially suppressed glucose wasting during starvation for leucine or uracil. Suppression by a deletion of TOR1 was less complete. We suggest that, similar to the Warburg effect observed in tumor cells, starving yeast auxotrophs wastes glucose as a consequence of the failure of conserved growth control pathways. Furthermore, we suggest that our results on condition-dependent chronological lifespan have important implications for the interpretation and design of studies on chronological aging.

  19. Pyrimidine degradation influences germination seedling growth and production of Arabidopsis seeds

    PubMed Central

    Cornelius, Stefanie; Witz, Sandra; Rolletschek, Hardy; Möhlmann, Torsten

    2011-01-01

    PYD1 (dihydropyrimidine dehydogenase) initiates the degradation of pyrimidine nucleobases and is located in plastids. In this study, a physiological analysis of PYD1 employing T-DNA knockout mutants and overexpressors was carried out. PYD1 knockout mutants were restricted in degradation of exogenously provided uracil and accumulated high uracil levels in plant organs throughout development, especially in dry seeds. Moreover, PYD1 knockout mutants showed delayed germination which was accompanied by low invertase activity and decreased monosaccharide levels. Abscisic acid (ABA) is an important regulator of seed germination, and ABA-responsive genes were deregulated in PYD1 knockout mutants. Together with an observed increased PYD1 expression in wild-type seedlings upon ABA treatment, an interference of PYD1 with ABA signalling can be assumed. Constitutive PYD1 overexpression mutants showed increased growth and higher seed number compared with wild-type and knockout mutant plants. During senescence PYD1 expression increased to allow uracil catabolism. From this it is concluded that early in development and during seed production PYD1 is needed to balance pyrimidine catabolism versus salvage. PMID:21865177

  20. Formation of Nucleobases and Other Prebiotic Species from the UV Irradiation of Pyrimidine in Astrophysical Ices

    NASA Astrophysics Data System (ADS)

    Nuevo, M.; Sandford, S. A.; Milam, S. N.; Materese, C. K.; Elsila, J. E.; Dworkin, J. P.

    2011-05-01

    Nucleobases are N-heterocycles which are the informational subunits of DNA and RNA. Biological nucleobases are divided in two types: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin has been confirmed by isotope measurements, but no N-heterocycle has ever been observed in the ISM. Experiments showed that the UV irradiation of pyrimidine mixed in astrophysical ices such as H_2O, NH_3, CH_3OH, or any combination of these at low temperature (20-30 K) leads to the formation of multiple photo-products derived from pyrimidine including the nucleobases uracil and cytosine. Theoretical studies on the formation of uracil confirmed its experimental formation pathway and demonstrated that the H_2O matrix plays a key role in the chemistry [9]. Thymine, however, was not found in any of the samples, though other pyrimidine derivatives, as well as other species of prebiotic interest such as urea and the amino acid glycine, could be identified [8]. We will extend this study to the formation of nucleobases and other prebiotic species from the UV irradiation of pyrimidine in astrophysically relevant ice mixtures containing H_2O, NH_3, CH_3OH, CO, and CO_2.

  1. Loading and pre-loading processes generate a distinct siRNA population in Tetrahymena.

    PubMed

    Mochizuki, Kazufumi; Kurth, Henriette M

    2013-07-01

    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.

  2. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    PubMed

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

  3. Alternative Induction of Meiotic Recombination From Single-Base Lesions of DNA Deaminases

    PubMed Central

    Pauklin, Siim; Burkert, Julia S.; Martin, Julie; Osman, Fekret; Weller, Sandra; Boulton, Simon J.; Whitby, Matthew C.; Petersen-Mahrt, Svend K.

    2009-01-01

    Meiotic recombination enhances genetic diversity as well as ensures proper segregation of homologous chromosomes, requiring Spo11-initiated double-strand breaks (DSBs). DNA deaminases act on regions of single-stranded DNA and deaminate cytosine to uracil (dU). In the immunoglobulin locus, this lesion will initiate point mutations, gene conversion, and DNA recombination. To begin to delineate the effect of induced base lesions on meiosis, we analyzed the effect of expressing DNA deaminases (activation-induced deaminase, AID, and APOBEC3C) in germ cells. We show that meiotic dU:dG lesions can partially rescue a spo11Δ phenotype in yeast and worm. In rec12 Schizosaccharomyces pombe, AID expression increased proper chromosome segregation, thereby enhancing spore viability, and induced low-frequency meiotic crossovers. Expression of AID in the germ cells of Caenorhabditis elegans spo-11 induced meiotic RAD-51 foci formation and chromosomal bivalency and segregation, as well as an increase in viability. RNAi experiments showed that this rescue was dependent on uracil DNA-glycosylase (Ung). Furthermore, unlike ionizing radiation-induced spo-11 rescue, AID expression did not induce large numbers of DSBs during the rescue. This suggests that the products of DNA deamination and base excision repair, such as uracil, an abasic site, or a single-stranded nick, are sufficient to initiate and alter meiotic recombination in uni- and multicellular organisms. PMID:19237686

  4. Uridine homeostatic disorder leads to DNA damage and tumorigenesis.

    PubMed

    Cao, Zhe; Ma, Jun; Chen, Xinchun; Zhou, Boping; Cai, Chuan; Huang, Dan; Zhang, Xuewen; Cao, Deliang

    2016-03-28

    Uridine is a natural nucleoside precursor of uridine monophosphate in organisms and thus is considered to be safe and is used in a wide range of clinical settings. The far-reaching effects of pharmacological uridine have long been neglected. Here, we report that the homeostatic disorder of uridine is carcinogenic. Targeted disruption (-/-) of murine uridine phosphorylase (UPase) disrupted the homeostasis of uridine and increased spontaneous tumorigenesis by more than 3-fold. Multiple tumors (e.g., lymphoma, hepatoma and lung adenoma) occurred simultaneously in some UPase deficient mice, but not in wild-type mice raised under the same conditions. In the tissue from UPase -/- mice, the 2'-deoxyuridine,5'-triphosphate (dUTP) levels and uracil DNA were increased and p53 was activated with an increased phospho-Ser18 p53 level. Exposing cell lines (e.g., MCF-7, RKO, HCT-8 and NCI-H460) to uridine (10 or 30 µM) led to uracil DNA damage and p53 activation, which in turn triggered the DNA damage response. In these cells, phospho-ATM, phospho-CHK2, and phospho-γH2AX were increased by uridine. These data suggest that uridine homeostatic disorder leads to uracil DNA damage and that pharmacological uridine may be carcinogenic.

  5. 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.

  6. Formation of the nucleobases around the star forming region

    NASA Astrophysics Data System (ADS)

    Saha, Rajdeep; Majumdar, Liton; Das, Ankan; Chakrabarti, Sandip K.; Chakrabarti, Sonali

    2013-06-01

    Chemistry of the dense and cold interstellar clouds are mainly dominated by the ion-molecular and radical-radical interactions though some neutral-neutral reactions are also barrierless and feasible at this condition. The base pairs of RNA are guanine (G) & uracil (U) (G-U pair) and adenine (A) & cytosine (C) (A-C pair). We perform quantum chemical calculations to predict the energetically most economical as well as favorable root for the formation of major bases of the nucleic acids. The outcome of this quantum chemical calculations could be used into our hydro-chemical model to obtain the abundances of some of the important bases of RNA during the formation of a proto star. It is well known that the thymine (T) and uracil (U) are the two nucleobases which are not common in DNA and RNA. Our quantum chemical calculation suggests that uracil could be produced prior to thymine in our chemical network. These findings could be used to support the RNA world hypothesis.

  7. 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.

  8. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    PubMed

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

  9. In situ biodosimetric experiment for space applications.

    PubMed

    Goldschmidt, Gergely; Kovaliczky, Eva; 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.

  10. Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy.

    PubMed

    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

  11. Structural Basis of Substrate Specificity in Geobacter metallireducens SMUG1.

    PubMed

    Zhang, Zhemin; Shen, Jiemin; Yang, Ye; Li, Jing; Cao, Weiguo; Xie, Wei

    2016-06-17

    Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are critical to maintain genome integrity, in which the base excision repair pathway plays an essential role. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for removing the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Geobacter metallireducens SMUG1 (GmeSMUG1) is an interesting family 3 enzyme in the UDG superfamily, with dual substrate specificities for DNA with uracil or xanthine. In contrast, the mutant G63P of GmeSMUG1 has exclusive activity for uracil, while N58D is inactive for both substrates, as we have reported previously. However, the structural bases for these substrate specificities are not well understood. In this study, we solved a series of crystal structures of WT and mutants of GmeSMUG1 at relatively high resolutions. These structures provide insight on the molecular mechanism of xanthine recognition for GmeSMUG1 and indicate that H210 plays a key role in xanthine recognition, which is in good agreement with the results of our EMSA and activity assays. More importantly, our mutant structures allow us to build models to rationalize our previous experimental observations of altered substrate activities of these mutants. PMID:27071000

  12. Nonreplicating, Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

    PubMed Central

    2015-01-01

    Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection by Toxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80 genetic background by targeting the deletion of the orotidine 5′-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion of OMPDC induced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80 Δompdc mutants stimulated a fully protective CD8+ T cell-dependent immunity that prevented acute infection by type I and type II strains of T. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdc mutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine. PMID:25776745

  13. Pyrimidine Biosynthesis in Lactobacillus leichmannii

    PubMed Central

    Hutson, Judith Y.; Downing, Mancourt

    1968-01-01

    Tracer studies of pyrimidine biosynthesis in Lactobacillus leichmannii (ATCC 7830) indicated that, while aspartate is utilized in the usual manner, the guanido carbon of arginine, rather than carbon dioxide, is utilized as a pyrimidine precursor. The guanido carbon of arginine also contributes, to some extent, to the carbon dioxide pool utilized for purine biosynthesis. The enzyme of the first reaction leading from arginine to pyrimidines, arginine deiminase, was investigated in crude bacterial extracts. It was inhibited by thymidylic acid and purine ribonucleotides, and to a lesser extent by purine deoxynucleotides and deoxycytidylic acid. Under the assay conditions employed, a number of nucleotides had no effect on the enzyme activity of the aspartate transcarbamylase of L. leichmannii. Growth of the cells in media containing uracil, compared to growth in media without uracil, resulted in a four- to fivefold decrease in the concentrations of aspartate transcar-bamylase and dihydroorotase and a twofold increase in the concentration of arginine deiminase, as estimated from specific enzyme activity in crude extracts of the cells. A small increase in specific enzyme activity of ornithine transcarbamylase and carbamate kinase was also observed in extracts obtained from cells grown on uracil. No appreciable change in concentration of any of the five enzymes studied was detected when the cells were grown in media containing thymidine or guanylic acid. A hypothetical scheme which suggests a relationship between the control of purine and pyrimidine biosynthesis in this bacterium and which is consistent with the experimental results obtained is presented. PMID:5686000

  14. 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.

  15. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    PubMed Central

    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

    2013-01-01

    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 multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs 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. PMID:23972113

  16. 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

  17. 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

  18. 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.

  19. Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. II. Reactivity with hsa-coupled, uridine-containing, monophosphoric ribodinucleotides.

    PubMed

    Alarcón-Segovia, D; Fishbein, E; Estrada-Parra, S; García-Ortigoza, E

    1976-03-01

    Sera from patients with scleroderma have been found to have anti-RNA antibodies which react with human serum albumin (HSA)-coupled uridine and uridine monophosphate (UMP) and are inhibited by uracil, uridine and UMP. Scleroderma sera react uniformly with 5'-polyuridylic acid (poly(U)) and fail to react with polyadenylic, polyuridylic acid poly(A) - poly(U)) which is also indicative of their uracil specificity. Anti-RNA antibodies found in systemic lupus erythematosus (SLE) are immunochemically different from those found in scleroderma in that, instead of being uniformly specific to uracil, they are markedly heterogeneous and may react with uracil, uridine and/or UMP. SLE sera frequently react with poly(A) - poly(U), indicating also their ability to recognize the double helical structure of double-stranded RNA. Thirty-seven scleroderma and thirty-four SLE sera from as many patients with either of these conditions were tested against HSA-coupled, uridine-containing monophosphoric dinucleotides in an attempt to characterize further their anti-RNA antibodies. Scleroderma sera were found to react primarily with dinucleotides in which uridine was the base proximal to the carrier protein and, except for sera that also contained antibodies to adenosine which reacted with UpA, they failed to react with dinucleotides in which uridine was in a terminal position only. Reaction with dinucleotides in which uridine was proximal to the carrier protein could be inhibited by uracil but not by the corresponding terminal base. Some lupus sera were found to react with both dinucleotides that contain the same bases in opposite sequence, e.g. ApU and UpA, while others were found to react with only one of the sequences. They were also found to react more frequently with dinucleotides in which HSA was coupled to a base other than uridine, suggesting that the reaction is primarily due to anti-DNA antibodies. Because immunization with dinucleotides coupled to protein prepared by the same

  20. 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...

  1. 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…

  2. The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, tha...

  3. Alteration of the DNA methylation status of donor cells impairs the developmental competence of porcine cloned embryos

    PubMed Central

    HUAN, Yan Jun; WU, Zhan Feng; ZHANG, Ji Guang; ZHU, Jiang; XIE, Bing Teng; WANG, Jian Yu; LI, Jing Yu; XUE, Bing Hua; KONG, Qing Ran; LIU, Zhong Hua

    2015-01-01

    Nuclear reprogramming induced by somatic cell nuclear transfer is an inefficient process, and donor cell DNA methylation status is thought to be a major factor affecting cloning efficiency. Here, the role of donor cell DNA methylation status regulated by 5-aza-2'-deoxycytidine (5-aza-dC) or 5-methyl-2'-deoxycytidine-5'-triphosphate (5-methyl-dCTP) in the early development of porcine cloned embryos was investigated. Our results showed that 5-aza-dC or 5-methyl-dCTP significantly reduced or increased the global methylation levels and altered the methylation and expression levels of key genes in donor cells. However, the development of cloned embryos derived from these cells was reduced. Furthermore, disrupted pseudo-pronucleus formation and transcripts of early embryo development-related genes were observed in cloned embryos derived from these cells. In conclusion, our results demonstrated that alteration of the DNA methylation status of donor cells by 5-aza-dC or 5-methyl-dCTP disrupted nuclear reprogramming and impaired the developmental competence of porcine cloned embryos. PMID:26537205

  4. 40 CFR 180.579 - Fenamidone; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... of the fungicide, fenamidone, including its metabolites and degradates, in or on the following... residues of the fungicide fenamidone, including its metabolites and degradates, in or on the following...-one, 3,5-dihydro-5-methyl-2-(methylthio)-5-phenyl-3 (phenylamino)-,(S)-), and its metabolite...

  5. 40 CFR 180.579 - Fenamidone; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... of the fungicide, fenamidone, including its metabolites and degradates, in or on the following... residues of the fungicide fenamidone, including its metabolites and degradates, in or on the following...-one, 3,5-dihydro-5-methyl-2-(methylthio)-5-phenyl-3 (phenylamino)-,(S)-), and its metabolite...

  6. 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...

  7. 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…

  8. 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...

  9. 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…

  10. 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...

  11. 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...

  12. 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)

  13. 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…

  14. HYDROLOGIC CONDITIONS AFFECTING THE TROPOSPHERIC FLUX OF VINCLOZOLIN AND ITS DEGRADATION PRODUCTS

    EPA Science Inventory

    A laboratory chamber was used to determine hydrologic conditions that lead to the tropospheric flux of a suspected anti-androgenic dicarboximide fungicide, vinclozolin (3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-oxzoli-dine-2,4-dione) and three degradation products from sterilized...

  15. Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation

    PubMed Central

    Stavnezer, Janet; Linehan, Erin K.; Thompson, Mikayla R.; Habboub, Ghaith; Ucher, Anna J.; Kadungure, Tatenda; Tsuchimoto, Daisuke; Nakabeppu, Yusaku; Schrader, Carol E.

    2014-01-01

    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. PMID:24927551

  16. Mineral catalysis of the formation of the phosphodiester bond in aqueous solution: The possible role of montmorillonite clays

    NASA Astrophysics Data System (ADS)

    Ferris, James P.; Ertem, Gözen; Kamaluddin; Agarwal, Vipin; Hua, Lu Lin

    The binding of adenosine to Na+-montmorillonite 22A is greater than 5'-AMP, at neutral pH. Adenine derivatives bind more strongly to the clay than the corresponding uracil derivatives. These data are consistent with the protonation of the adenine by the acidic clay surface and a cationic binding of the protonated ring to the anionic clay surface. Other forces must be operative in the binding of uracil derivatives to the clay since the uracil ring system is not basic. The reaction of the 5'-AMP with water soluble carbodiimide in the presence of Na+-montmorillonite results in the formation of 2',5'-pApA (18.9%), 3',5'-pApA (11%), and AppA (4.8%). When poly(U) is used in place of the clay the product yields are 2',5',-pApA (15.5%), 3',5'-pApA (3.7%) and AppA (14.9%). The cyclic nucleotide, c(pA)2 is also formed when poly(U) is used. AppA is the principal reaction product when neither clay nor poly(U) is present in the reaction mixture. When 2'-deoxy-5'-AMP reacts with carbodiimide in the presence of Na+-montmorillonite 22A the products are dpApA (4.8%), dAppApA (4.5%) and dAppA (17.4%). Cyclic 3',5'-dAMP is the main product (14%) of the reaction of 2'-deoxy-3'-AMP.

  17. [Determination of the interaction kinetics between meloxicam and β-cyclodextrin using the quantitative high-performance affinity chromatography coupled with mass spectrometry].

    PubMed

    Wang, Cai-fen; Li, Zhuo; Wang, Xiao-bo; Li, Hai-yan; Zhang, Ji-wen; Sun, Li-xin

    2015-09-01

    The association rate constant and dissociation rate constant are important parameters of the drug-cyclodextrin supermolecule systems, which determine the dissociation of drugs from the complex and the further in vivo absorption of drugs. However, the current studies of drug-cyclodextrin interactions mostly focus on the thermodynamic parameter of equilibrium constants (K). In this paper, a method based on quantitative high performance affinity chromatography coupled with mass spectrometry was developed to determine the apparent dissociation rate constant (k(off,app)) of drug-cyclodextrin supermolecule systems. This method was employed to measure the k(off,app) of meloxicam and acetaminophen. Firstly, chromatographic peaks of drugs and non-retained solute (uracil) on β-cyclodextrin column at different flow rates were acquired, and the retention time and variance values were obtained via the fitting the peaks. Then, the plate heights of drugs (H(R)) and uracil (H(M,C)) were calculated. The plate height of theoretical non-retained solute (H(M,T)) was calculated based on the differences of diffusion coefficient and the stagnant mobile phase mass transfer between drugs and uracil. Finally, the k(off,app) was calculated from the slope of the regression equation between (H(R)-H(M,T)) and uk/(1+k)2, (0.13 ± 0.00) s(-1) and (4.83 ± 0.10) s(-1) for meloxicam and acetaminophen (control drug), respectively. In addition, the apparent association rate constant (k(on,app)) was also calculated through the product of K (12.53 L x mol(-1)) and k(off,app). In summary, it has been proved that the method established in our study was simple, efficiently fast and reproducible for investigation on the kinetics of drug-cyclodextrin interactions. PMID:26757555

  18. Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism.

    PubMed

    Turner, R J; Lu, Y; Switzer, R L

    1994-06-01

    A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5' to 3' order: a 151-nucleotide (nt) untranslated leader; pyrR, encoding a 20-kDa protein; a 173-nt intercistronic region; pyrP, encoding a 46-kDa protein; a 145-nt intercistronic region; and eight overlapping cistrons encoding all of the six enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via an attenuation mechanism. There are three transcription terminators within the operon, each of which is preceded by another stem-loop structure, the antiterminator, whose formation would prevent formation of the terminator stem-loop. These are located in the leader, the pyrR-pyrP intercistronic region, and the pyrP-pyrB intercistronic region. Northern (RNA) blot analysis has identified transcripts of lengths which coincide with termination at these proposed attenuation sites and whose relative abundances vary in the expected pyrimidine-dependent manner. Each antiterminator contains a 50-base conserved sequence in its promoter-proximal half. Various transcriptional fusions of the pyr promoter and surrounding sequences to promoterless reporter genes support an attenuation mechanism whereby when pyrimidines are abundant, the PyrR protein binds to the conserved sequence in the pyr mRNA and disrupts the antiterminator, permitting terminator hairpin formation and promoting transcription termination. Deletion of pyrR from the chromosome resulted in the constitutive, elevated expression of aspartate transcarbamylase, which is encoded by pyrB, the third gene in the operon. Complementation of an E. coli upp mutant, as well as direct enzymatic assay, has demonstrated that pyrR also confers uracil phosphoribosyltransferase activity. Analysis of pyrR and upp deletion mutants demonstrated that upp, not pyrR, encodes the quantitatively important uracil phosphoribosyltransferase activity. The pyrP gene probably encodes an integral membrane

  19. 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

  20. 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.

  1. 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. PMID:17560278

  2. A multi-faceted analysis of RutD reveals a novel family of α/β hydrolases

    PubMed Central

    Knapik, Aleksandra A.; Petkowski, Janusz J.; Otwinowski, Zbyszek; Cymborowski, Marcin T.; Cooper, David R.; Majorek, Karolina A.; Chruszcz, Maksymilian; Krajewska, Wanda M.; Minor, Wladek

    2012-01-01

    The rut pathway of pyrimidine catabolism is a novel pathway that allows pyrimidine bases to serve as the sole nitrogen source in suboptimal temperatures. The rut operon in E. coli evaded detection until 2006, yet consists of seven proteins named RutA, RutB, etc. through RutG. The operon is comprised of a pyrimidine transporter and six enzymes that cleave and further process the uracil ring. Herein, we report the structure of RutD, a member of the α/β hydrolase superfamily, which is proposed to enhance the rate of hydrolysis of aminoacrylate, a toxic side product of uracil degradation, to malonic semialdehyde. Although this reaction will occur spontaneously in water, the toxicity of aminoacrylate necessitates catalysis by RutD for efficient growth with uracil as a nitrogen source. RutD has a novel and conserved arrangement of residues corresponding to the α/β hydrolase active site, where the nucleophile’s spatial position occupied by Ser, Cys or Asp of the canonical catalytic triad is replaced by histidine. We have used a combination of crystallographic structure determination, modeling and bioinformatics, to propose a novel mechanism for this enzyme. This approach also revealed that RutD represents a previously undescribed family within the α/β hydrolases. We compare and contrast RutD with PcaD, which is the closest structural homolog to RutD. PcaD is a 3-oxoadipate-enol-lactonase-with a classic arrangement of residues in the active site. We have modeled a substrate in the PcaD active site and proposed a reaction mechanism. PMID:22641504

  3. Mineral catalysis of the formation of the phosphodiester bond in aqueous solution: the possible role of montmorillonite clays.

    PubMed

    Ferris, J P; Ertem, G; Kamaluddin; Agarwal, V; Hua, L L

    1989-01-01

    The binding of adenosine to Na(+)-montmorillonite 22A is greater than 5'-AMP, at neutral pH. Adenine derivatives bind more strongly to the clay than the corresponding uracil derivatives. These data are consistent with the protonation of the adenine by the acidic clay surface and a cationic binding of the protonated ring to the anionic clay surface. Other forces must be operative in the binding of uracil derivatives to the clay since the uracil ring system is not basic. The reaction of the 5'-AMP with water soluble carbodiimide in the presence of Na(+)-montmorillonite results in the formation of 2',5'-pApA (18.9%), 3',5'-pApA (11%), and AppA (4.8%). When poly(U) is used in place of the clay the product yields are 2',5'-pApA (15.5%), 3',5'-pApA (3.7%) and AppA (14.9%). The cyclic nucleotide, c(pA)2 is also formed when poly(U) is used. AppA is the principal reaction product when neither clay nor poly(U) is present in the reaction mixture. When 2'-deoxy-5'-AMP reacts with carbodiimide in the presence of Na(+)-montmorillonite 22A the products are dpApA (4.8%), dAppApA (4.5%) and dAppA (17.4%). Cyclic 3',5'-dAMP is the main product (14%) of the reaction of 2'-deoxy-3'-AMP.

  4. Radical Pathways for the Prebiotic Formation of Pyrimidine Bases from Formamide.

    PubMed

    Nguyen, Huyen Thi; Jeilani, Yassin A; Hung, Huynh Minh; Nguyen, Minh Tho

    2015-08-20

    The prebiotic formation of nucleobases, the building blocks of RNA/DNA, is of current interest. Highly reactive radical species present in the atmosphere under irradiation have been suggested to be involved in the prebiotic synthesis of nucleobases from formamide (FM). We studied several free radical reaction pathways for the synthesis of pyrimidine bases (cytosine, uracil, and thymine) from FM under cold conditions. These pathways are theoretically determined using density functional theory (DFT) computations to examine their kinetic and thermodynamic feasibilities. These free radical reaction pathways share some common reaction types such as H-rearrangement, (•)H/(•)OH/(•)NH2 radical loss, and intramolecular radical cyclization. The rate-determining steps in these pathways are characterized with low energy barriers. The energy barriers of the ring formation steps are in the range of 3-7 kcal/mol. Although DFT methods are known to significantly underestimate the barriers for addition of (•)H radical to neutral species, many of these reactions are highly exergonic with energy release of -15 to -52 kcal/mol and are thus favorable. Among the suggested pathways for formation of cytosine (main route, routes 7a and 1a), uracil (main route, routes 7b and 1b), and thymine (main route and route 26a), the main routes are in general thermodynamically more exergonic and more kinetically favored than other alternative routes with lower overall energy barriers. The reaction energies released following formation of cytosine, uracil, and thymine from FM via the main radical routes amount to -59, -81, and -104 kcal/mol, respectively. Increasing temperature induces unfavorable changes in both kinetic and thermodynamic aspects of the suggested routes. However, the main routes are still more favored than the alternative pathways at the temperature up to the boiling point of FM. PMID:26196536

  5. Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid.

    PubMed

    Schouten, K A; Weiss, B

    1999-12-01

    Endonuclease V (deoxyinosine 3'-endonuclease) of Escherichia coli K-12 is a putative DNA repair enzyme that cleaves DNA's containing hypoxanthine, uracil, or mismatched bases. An endonuclease V (nfi) mutation was tested for specific mutator effects on a battery of trp and lac mutant alleles. No marked differences were seen in frequencies of spontaneous reversion. However, when nfi mutants were treated with nitrous acid at a level that was not noticeably mutagenic for nfi(+) strains, they displayed a high frequency of A:T-->G:C, and G:C-->A:T transition mutations. Nitrous acid can deaminate guanine in DNA to xanthine, cytosine to uracil, and adenine to hypoxanthine. The nitrous acid-induced A:T-->G:C transitions were consistent with a role for endonuclease V in the repair of deaminated adenine residues. A confirmatory finding was that the mutagenesis was depressed at a locus containing N(6)-methyladenine, which is known to be relatively resistant to nitrosative deamination. An alkA mutation did not significantly enhance the frequency of A:T-->G:C mutations in an nfi mutant, even though AlkA (3-methyladenine-DNA glycosylase II) has hypoxanthine-DNA glycosylase activity. The nfi mutants also displayed high frequencies of nitrous acid-induced G:C-->A:T transitions. These mutations could not be explained by cytosine deamination because an ung (uracil-DNA N-glycosylase) mutant was not similarly affected. However, these findings are consistent with a role for endonuclease V in the removal of deaminated guanine, i.e., xanthine, from DNA. The results suggest that endonuclease V helps to protect the cell against the mutagenic effects of nitrosative deamination.

  6. The ability to accumulate deoxyuridine triphosphate and cellular response to thymidylate synthase (TS) inhibition

    PubMed Central

    Webley, S D; Welsh, S J; Jackman, A L; Aherne, G W

    2001-01-01

    Thymidylate synthase (TS) is an important enzyme catalysing the reductive methylation of dUMP to dTMP that is further metabolized to dTTP for DNA synthesis. Loss of viability following TS inhibition occurs as a consequence of depleted dTTP pools and at least in some cell lines, accumulation of dUTP and subsequent misincorporation of uracil into DNA. The expansion in dUTP pools is largely determined by the expression of the pyrophosphatase, dUTPase. Our previous work has shown that following TS inhibition the ability to accumulate dUTP was associated with an earlier growth inhibitory effect. 3 human lung tumour cell lines and HT29 human colon tumour cells transfected with dUTPase have been used to investigate the relationship between loss of viability following TS inhibition and dUTP accumulation. Cell cycle arrest typical of TS inhibition was an early event in all cell lines and occurred irrespective of the ability to accumulate dUTP or p53 function. However, a large expansion of dUTP pools was associated with mature DNA damage (4 h) and an earlier loss of viability following TS inhibition compared to cells in which dUTP pools were not expanded. In A549 cells damage to mature DNA may have been exacerbated by significantly higher activity of the excision repair enzyme, uracil-DNA glycosylase. Consistent with results using different inhibitors of TS, transfection of dUTPase into HT29 cells significantly reduced the cytotoxicity of a 24 h but not 48 h exposure to ZD9331. Although loss of viability can be mediated through dTTP deprivation alone, the uracil misincorporation pathway resulted in an earlier commitment to cell death. The relevance of this latter pathway in the clinical response to TS inhibitors deserves further investigation. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11487279

  7. Novel Opportunities for Thymidylate Metabolism as a Therapeutic Target

    PubMed Central

    Wilson, Peter M.; Fazzone, William; LaBonte, Melissa J.; Deng, Jinxia; Neamati, Nouri; Ladner, Robert D.

    2008-01-01

    For over 40 years, the fluoropyrimidine 5-fluorouracil (5-FU) has remained the central agent in therapeutic regimens employed in the treatment of colorectal cancer (CRC) and is frequently combined with the DNA-damaging agent's oxaliplatin and irinotecan increasing response rates and improving overall survival. However, many patients will derive little or no benefit from treatment, highlighting the need to identify novel therapeutic targets to improve the efficacy of current 5-FU-based chemotherapeutic strategies. Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi providing substrate for thymidylate synthase (TS) and DNA synthesis and repair. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA as uracil is lethal. Importantly, uracil misincorporation represents an important mechanism of cytotoxicity induced by the TS-targeted class of chemotherapeutic agents including 5-FU. A growing body of evidence suggests that dUTPase is an important mediator of response to TS-targeted agents. In this manuscript we present further evidence demonstrating that elevated expression of dUTPase can protect breast cancer cells from the expansion of the intracellular uracil pool, translating to reduced growth inhibition following treatment with 5-FU. We therefore report the implementation of in silico drug development techniques to identify and develop small molecule inhibitors of dUTPase. As 5-FU and the oral 5-FU pro-drug capecitabine remain central agents in the treatment of a variety of malignancies, the clinical utility of a small molecule inhibitor to dUTPase represents a viable strategy to improve the clinical efficacy of these mainstay chemotherapeutic agents. PMID:18790783

  8. Genetic Dissection of Pyrimidine Biosynthesis and Salvage in Leishmania donovani*

    PubMed Central

    Wilson, Zachary N.; Gilroy, Caslin A.; Boitz, Jan M.; Ullman, Buddy; Yates, Phillip A.

    2012-01-01

    Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis. PMID:22367196

  9. Simultaneous quantification and splenocyte-proliferating activities of nucleosides and bases in Cervi cornu Pantotrichum

    PubMed Central

    Zong, Ying; Wang, Yu; Li, Hang; Li, Na; Zhang, Hui; Sun, Jiaming; Niu, Xiaohui; Gao, Xiaochen

    2014-01-01

    Background: Cervi Cornu Pantotrichum has been a well known traditional Chinese medicine, which is young horn of Cervus Nippon Temminck (Hualurong: HLR). At present, the methods used for the quality control of Cervi Cornu Pantotrichum show low specificity. Objective: To describe a holistic method based on chemical characteristics and splenocyte-proliferating activities to evaluate the quality of HLR. Materials and Methods: The nucleosides and bases from HLR were identified by high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), and six of them were chosen to be used for simultaneous HPLC quantification according to the results of proliferation of mouse splenocytes in vitro. Results: In this study, eight nucleosides and bases have been identified. In addition, uracil, hypoxanthine, uridine, inosine, guanosine, and adenosine were chosen to be used for simultaneous HPLC quantification. Simultaneous quantification of these six substances was performed on ten groups of HLR under the condition of a TIANHE Kromasil C18 column (5 μm, 4.6 mm × 250 mm i.d.) and a gradient elution of water and acetonitrile. Of the ten groups, HLR displayed the highest total nucleoside contents (TNC, sum of adenosine and uracil, 0.412 mg/g) with the strongest splenocyte-proliferating activities. Conclusion: These results suggest that TNC (such as particularly highly contained adenosine and uracil) in HLR has a certain correlation with the activity of splenocyte-proliferating, and it may be used as a quality control for HLR. This comprehensive method could be applied to other traditional Chinese medicines to ameliorate their quality control. PMID:25422536

  10. Ubiquitous Water-Soluble Molecules in Aquatic Plant Exudates Determine Specific Insect Attraction

    PubMed Central

    Sérandour, Julien; Reynaud, Stéphane; Willison, John; Patouraux, Joëlle; Gaude, Thierry; Ravanel, Patrick; Lempérière, Guy; Raveton, Muriel

    2008-01-01

    Plants produce semio-chemicals that directly influence insect attraction and/or repulsion. Generally, this attraction is closely associated with herbivory and has been studied mainly under atmospheric conditions. On the other hand, the relationship between aquatic plants and insects has been little studied. To determine whether the roots of aquatic macrophytes release attractive chemical mixtures into the water, we studied the behaviour of mosquito larvae using olfactory experiments with root exudates. After testing the attraction on Culex and Aedes mosquito larvae, we chose to work with Coquillettidia species, which have a complex behaviour in nature and need to be attached to plant roots in order to obtain oxygen. This relationship is non-destructive and can be described as commensal behaviour. Commonly found compounds seemed to be involved in insect attraction since root exudates from different plants were all attractive. Moreover, chemical analysis allowed us to identify a certain number of commonly found, highly water-soluble, low-molecular-weight compounds, several of which (glycerol, uracil, thymine, uridine, thymidine) were able to induce attraction when tested individually but at concentrations substantially higher than those found in nature. However, our principal findings demonstrated that these compounds appeared to act synergistically, since a mixture of these five compounds attracted larvae at natural concentrations (0.7 nM glycerol, <0.5 nM uracil, 0.6 nM thymine, 2.8 nM uridine, 86 nM thymidine), much lower than those found for each compound tested individually. These results provide strong evidence that a mixture of polyols (glycerol), pyrimidines (uracil, thymine), and nucleosides (uridine, thymidine) functions as an efficient attractive signal in nature for Coquillettidia larvae. We therefore show for the first time, that such commonly found compounds may play an important role in plant-insect relationships in aquatic eco-systems. PMID:18841203

  11. 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.

  12. 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

  13. 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.

  14. Pyrimidine Biosynthesis Is Not an Essential Function for Trypanosoma brucei Bloodstream Forms

    PubMed Central

    Munday, Jane C.; Donachie, Anne; Morrison, Liam J.; de Koning, Harry P.

    2013-01-01

    Background African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite. Methodology/Principal Findings Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line. Conclusions/Significance Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal. PMID:23505454

  15. Placental FKBP5 genetic and epigenetic variation is associated with infant neurobehavioral outcomes in the RICHS cohort.

    PubMed

    Paquette, Alison G; Lester, Barry M; Koestler, Devin C; Lesseur, Corina; Armstrong, David A; Marsit, Carmen J

    2014-01-01

    Adverse maternal environments can lead to increased fetal exposure to maternal cortisol, which can cause infant neurobehavioral deficits. The placenta regulates fetal cortisol exposure and response, and placental DNA methylation can influence this function. FK506 binding protein (FKBP5) is a negative regulator of cortisol response, FKBP5 methylation has been linked to brain morphology and mental disorder risk, and genetic variation of FKBP5 was associated with post-traumatic stress disorder in adults. We hypothesized that placental FKBP5 methylation and genetic variation contribute to gene expression control, and are associated with infant neurodevelopmental outcomes assessed using the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scales (NNNS). In 509 infants enrolled in the Rhode Island Child Health Study, placental FKBP5 methylation was measured at intron 7 using quantitative bisulfite pyrosequencing. Placental FKBP5 mRNA was measured in a subset of 61 infants by quantitative PCR, and the SNP rs1360780 was genotyped using a quantitative allelic discrimination assay. Relationships between methylation, expression and NNNS scores were examined using linear models adjusted for confounding variables, then logistic models were created to determine the influence of methylation on membership in high risk groups of infants. FKBP5 methylation was negatively associated with expression (P = 0.08, r = -0.22); infants with the TT genotype had higher expression than individuals with CC and CT genotypes (P = 0.06), and those with CC genotype displayed a negative relationship between methylation and expression (P = 0.06, r = -0.43). Infants in the highest quartile of FKBP5 methylation had increased risk of NNNS high arousal compared to infants in the lowest quartile (OR 2.22, CI 1.07-4.61). TT genotype infants had increased odds of high NNNS stress abstinence (OR 1.98, CI 0.92-4.26). Placental FKBP5 methylation reduces expression in a genotype

  16. Indole Alkaloids from the Sea Anemone Heteractis aurora and Homarine from Octopus cyanea.

    PubMed

    Shaker, Kamel H; Göhl, Matthias; Müller, Tobias; Seifert, Karlheinz

    2015-11-01

    The two new indole alkaloids 2-amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1), 2-amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one (2), and auramine (3) have been isolated from the sea anemone Heteractis aurora. Both indole alkaloids were synthesized for the confirmation of the structures. Homarine (4), along with uracil (5), hypoxanthine (6), and inosine (7) have been obtained from Octopus cyanea. PMID:26567952

  17. [Effect of N-methyl-N-beta-chloroethylhydrazine and its benzaldehydhydrazone on RNA-and protein synthesis as well as metabolism of synchronously growing yeast cells (Saccharomyces cerevisiae) (author's transl)].

    PubMed

    Braun, R; Lange, H; Mangold, U; Mangold, R

    1976-01-01

    N-methyl-N-beta-chloroethylhydrazine and its benzaldehydhydrazone inhibit the influx of [6-3H]-uracil and L-[U-14C]leucin in yeast cells as well as the incorporation in RNA and protein. Experiments with synchronously and asynchronously growing cells showed the N-methyl-N-beta-chloroethylhydrazine essentially influences the dividing phase while the benzaldehydhydrazone is more effective in the growth phase. The effect of these two substances on respiration, glycolysis and the concentration of fructose-1,6-diphosphate, triose phosphate, and adenosine triphosphate is small.

  18. Chemical Constituents of Cordyceps cicadae.

    PubMed

    Chu, Zhi-Bo; Chang, Jun; Zhu, Ying; Sun, Xun

    2015-12-01

    One new bifuran derivative (1), together with fourteen known compounds, were isolated from Cordyceps cicadae X. Q. Shing. The known compounds included nine nucleosides, uracil (2), uridine (3), 2'-deoxyuridine (4), 2'-deoxyinosine (5), guanosine (6), 2'-deoxyguanosine (7), thymidine (8), adenosine (9), and 2'-deoxyadenosine (10); three amino acids tryptophan (11), phenylalanine (12), and tyrosine (13); and two dopamine analogues N-acetylnoradrenaline (14) and its dimer, trans-2-(3',4'-dihydroxyphenyl)-3-acetylamino-7-(N-acetyl-2"-amino-ethylene)-1,4-benzodioxane (15). Their structures were decisively elucidated by spectroscopic analysis, including 1D and 2D NMR techniques. PMID:26882686

  19. Indole Alkaloids from the Sea Anemone Heteractis aurora and Homarine from Octopus cyanea.

    PubMed

    Shaker, Kamel H; Göhl, Matthias; Müller, Tobias; Seifert, Karlheinz

    2015-11-01

    The two new indole alkaloids 2-amino-1,5-dihydro-5-(1H-indol-3-ylmethyl)-4H-imidazol-4-one (1), 2-amino-5-[(6-bromo-1H-indol-3-yl)methyl]-3,5-dihydro-3-methyl-4H-imidazol-4-one (2), and auramine (3) have been isolated from the sea anemone Heteractis aurora. Both indole alkaloids were synthesized for the confirmation of the structures. Homarine (4), along with uracil (5), hypoxanthine (6), and inosine (7) have been obtained from Octopus cyanea.

  20. PHYSIOLOGICAL ROLE OF 70S RIBOSOMES IN BACTERIA*

    PubMed Central

    Algranati, Israel D.; Gonzalez, Nelida S.; Bade, Ernesto G.

    1969-01-01

    Evidence is presented indicating that free 70S ribosomes are real components of Bacillus stearothermophilus and Escherichia coli in exponential and stationary phases of growth. After pulses of radioactive leucine or uracil were given, protein synthesis was instantaneously stopped by chloramphenicol, or polypeptide growth was allowed to proceed to completion in slowly cooled cells. Analysis of label distribution in sucrose gradients showed that 70S monomers, and not subunits, are final products of translation. A ribosome cycle for protein synthesis in bacteria is proposed. PMID:4894332