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Sample records for 5-methyl uracil

  1. Pharmacokinetics of 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)uracil in woodchucks.

    PubMed

    Witcher, J W; Boudinot, F D; Baldwin, B H; Ascenzi, M A; Tennant, B C; Du, J F; Chu, C K

    1997-10-01

    1-(2-Fluoro-5-methyl-beta-L-arabinofuranosyl)uracil (L-FMAU) is a nucleoside analog with potent in vitro activity against hepatitis B virus (HBV) and Epstein-Barr virus. The purpose of this study was to characterize the disposition of L-FMAU following oral and intravenous administration in the woodchuck animal model. The numerous similarities between woodchuck hepatitis virus and HBV infection justify the use of the woodchuck as an animal model for preclinical studies of anti-HBV agents in vivo. Woodchucks were given 25 mg of L-FMAU per kg of body weight intravenously and orally. Concentrations of L-FMAU in urine and plasma were determined by high-performance liquid chromatography. Following intravenous administration of 25 mg of L-FMAU per kg to woodchucks, total clearance was moderate, averaging 0.23 +/- 0.07 liter/h/kg. Renal clearance and nonrenal clearance averaged 0.13 +/- 0.08 and 0.10 +/- 0.06 liter/h/kg, respectively. The steady-state volume of distribution averaged 0.99 +/- 0.17 liter/kg, indicative of intracellular distribution of the nucleoside. The terminal-phase half-life of L-FMAU following intravenous administration averaged 6.2 +/- 2.0 h, and mean residence time averaged 4.5 +/- 0.8 h. Absorption of L-FMAU after oral administration was incomplete, and bioavailability was approximately 20%. Concentrations of L-FMAU in plasma remained above the in vitro 50% effective concentration of 0.026 microg/ml for HBV (C. K. Chu, T. Ma, K. Shanmuganathan, C. Wang, Y. Xiang, S. B. Pai, G.-Q. Yao, J.-P. Sommadossi, and Y.-C. Cheng, Antimicrob. Agents Chemother. 39:979-981, 1995) for 24 h after both intravenous and oral administration of 25 mg of L-FMAU per kg. PMID:9333045

  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. In search of uracil derivatives as bioactive agents. Uracils and fused uracils: Synthesis, biological activity and applications.

    PubMed

    Pałasz, Aleksandra; Cież, Dariusz

    2015-06-01

    This review article is an effort to summarize recent developments in researches providing uracil derivatives with promising biological potential. This article also aims to discuss potential future directions on the development of more potent and specific uracil analogues for various biological targets. Uracils are considered as privileged structures in drug discovery with a wide array of biological activities and synthetic accessibility. Antiviral and anti-tumour are the two most widely reported activities of uracil analogues however they also possess herbicidal, insecticidal and bactericidal activities. Their antiviral potential is based on the inhibition of key step in viral replication pathway resulting in potent activities against HIV, hepatitis B and C, the herpes viruses etc. Uracil derivatives such as 5-fluorouracil or 5-chlorouracil were the first pharmacological active derivatives to be generated. Poor selectivity limits its therapeutic application, resulting in high incidences of gastrointestinal tract or central nervous toxicity. Numerous modifications of uracil structure have been performed to tackle these problems resulting in the development of derivatives exhibiting better pharmacological and pharmacokinetic properties including increased bioactivity, selectivity, metabolic stability, absorption and lower toxicity. Researches of new uracils and fused uracil derivatives as bioactive agents are related with modifications of substituents at N(1), N(3), C(5) and C(6) positions of pyrimidine ring. This review is an endeavour to highlight the progress in the chemistry and biological activity of the uracils, predominately after the year 2000. In particular are presented synthetic methods and biological study for such analogues as: 5-fluorouracil or 5-chlorouracil derivatives, tegafur analogues, arabinopyranonucleosides of uracil, glucopyranonucleosides of uracil, liposidomycins, caprazamycins or tunicamycins, tritylated uridine analogues, nitro or cyano

  4. Anionic derivatives of uracil: fragmentation and reactivity.

    PubMed

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

    2014-09-01

    Uracil is an essential biomolecule for terrestrial life, yet its prebiotic formation mechanisms have proven elusive for decades. Meteorites have been shown to contain uracil and the interstellar abundance of aromatic species and nitrogen-containing molecules is well established, providing support for uracil's presence in the interstellar medium (ISM). The ion chemistry of uracil may provide clues to its prebiotic synthesis and role in the origin of life. The fragmentation of biomolecules provides valuable insights into their formation. Previous research focused primarily on the fragmentation and reactivity of cations derived from uracil. In this study, we explore deprotonated uracil-5-carboxylic acid and its anionic fragments to elucidate novel reagents of uracil formation and to characterize the reactivity of uracil's anionic derivatives. The structures of these fragments are identified through theoretical calculations, further fragmentation, experimental acidity bracketing, and reactivity with several detected and potential interstellar species (SO2, OCS, CS2, NO, N2O, CO, NH3, O2, and C2H4). Fragmentation is achieved through collision induced dissociation (CID) in a commercial ion trap mass spectrometer, and all reaction rate constants are measured using a modification of this instrument. Experimental data are supported by theoretical calculations at the B3LYP/6-311++G(d,p) level of theory. Lastly, the astrochemical implications of the observed fragmentation and reaction processes are discussed. PMID:25036757

  5. Decameric uracil complexes around Li+.

    PubMed

    Zins, Emilie-Laure; Pepe, Claude; Schröder, Detlef

    2010-07-01

    Electrospray ionization (ESI) in combination with mass spectrometry (MS) experiments were carried out to study decameric uracil complexes cationized with Li(+) ion. A previous study has shown that, under specific experimental conditions, a particularly intense peak of the decamer U(10)Li(+) is formed, which was referred to as an indication for so-called 'magic number' cluster. In order to gain more insight on the structure of this decameric complex, here, we report experimental studies concerning the kinetics of the fragmentation. In accordance with the new experimental data, structural models were constructed and fully optimized using ab initio and density functional theory quantum chemistry calculations. The theoretical study allowed us to propose a stable gas-phase structure which is compatible with all experimental findings. PMID:20564575

  6. Novel Energetic Compounds Based on 5-Methyl-1-Aminotetrazole

    NASA Astrophysics Data System (ADS)

    Tang, Yongxing; Yang, Hongwei; Ju, Xuehai; Lu, Chunxu; Cheng, Guangbin

    2015-04-01

    Two energetic compounds based on 5-methyl-1-aminotetrazole were prepared. The trinitroethyl moiety and furazan ring were introduced to tetrazole, respectively. The trinitroethyl moiety can increase the oxygen balance, and the furazan ring can improve the detonation properties. The structures of both compounds were confirmed by X-ray crystallography. Their thermal stability and explosive performance were also investigated.

  7. Genetic variants in uracil processing enzymes are associated with abnormal DNA uracil content

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction. Maintenance of DNA integrity through DNA repair is critical in cancer prevention. Among the enzymes involved in DNA repair are those that prevent or correct uracil misincorporation in DNA. Repair of misincorporated uracil is important since it can cause double-stranded DNA breaks and o...

  8. 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

  9. Heteroorganic furan derivatives. 61. Trimethyl (5-methyl-2-furyl) silane and trimethyl (5-methyl-2-furyl) german

    SciTech Connect

    Lukevits, E.; Ignatovich, L.M.; Iovel', I.G.; Gol'dberg, Yu.Sh.; Shimanskaya, M.V.

    1987-07-01

    The transformations of trimethyl (5-methyl-2-furyl)silane and trimethyl (5-methyl-2-furyl) germane were studied upon vapor-phase oxidation by atmospheric oxygen on a V-Mo-Ag-O catalyst. Under these conditions, trimethyl(5-formyl-2-furyl)-silane and trimethyl (5-formyl-2-furyl) germane are formed albeit in only 5-7% yield. This low yield is a consequence of the thermal instability of the starting compounds and the aldehydes formed. The oxidation of 2-methyl-5-tert-butylfuran was studied under comparable conditions. The corresponding aldehyde was obtained in 30% yield. A scheme was proposed for the catalytic oxidation of 5-substituted 2-methylfurans.

  10. Tautomerism of uracil probed via infrared spectroscopy of singly hydrated protonated uracil.

    PubMed

    Bakker, Joost M; Sinha, Rajeev K; Besson, Thierry; Brugnara, Maurizio; Tosi, Paolo; Salpin, Jean-Yves; Maître, Philippe

    2008-12-01

    Tautomerism of the nucleobase uracil is characterized in the gas phase through IR photodissociation spectroscopy of singly hydrated protonated uracil created with tandem mass spectrometric methods in a commercially available Fourier transform ion cyclotron resonance mass spectrometer. Protonated uracil ions generated by electrospray ionization are re-solvated in a low-pressure collision cell filled with a mixture of water vapor seeded in argon. Their structure is investigated by IR photodissociation spectroscopy in the NH and OH stretching region (2500-3800 cm(-1)) with a tabletop IR laser source and in the 1000-2000 cm(-1) range with a free-electron laser. In both regions the IR photodissociation spectrum exhibits well-resolved spectral signatures that point to the presence of two different types of structure for monohydrated protonated uracil, which result from the two lowest-energy tautomers of uracil. Ab initio calculations confirm that no water-catalyzed tautomerization occurs during the re-solvation process, indicating that the two protonated forms of uracil directly originate from the electrospray process. PMID:18998657

  11. 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

  12. The 6-thioguanine/5-methyl-2-pyrimidinone base pair.

    PubMed Central

    Rappaport, H P

    1988-01-01

    As part of a program to determine the physical possibility of expanding the number of types of base pairs in DNA, the pairing stabilities of the analog bases 6-thioguanine (GS) and 5-methyl-2-pyrimidinone (TH) in oligodeoxynucleotides were measured. Procedures were developed to synthesize oligodeoxynucleotides with the analog bases. The sequences of the synthesized oligomers were T-C-G-A-C-G-G-X-Y-C-C-G. An enzymatic procedure was developed to measure relative association constants of oligomer pairs with the self complementary reference oligomer, X = A and Y = T, K(T/A) = K. The results were K(C/G) = (5 +/- .5)K, K(TH/GS) = K/(1 +/- .5), K(T/G) = K/(9 +/- 3), K(TH/G) = K/(25 +/- 5), K(C/GS) less than K/30, K(TH/A) less than K/40, K(T/GS) less than K/40, K(C/A) less than K/40. The results with the standard bases are consistent with other methods of measurement. The stability of the base pair GS/TH is approximately the same as the standard base pair A/T. PMID:3412886

  13. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    PubMed

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. PMID:21371942

  14. 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.

  15. Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs.

    PubMed

    Krokan, H; Wittwer, C U

    1981-06-11

    Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translation of calf thymus DNA. The standard substrate was double-stranded DNA with [3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3'end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP; in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP, and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides. PMID:7279657

  16. On the Electronically Excited States of Uracil

    SciTech Connect

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

    2008-10-09

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

  17. Identification and characterization of human uracil phosphoribosyltransferase (UPRTase).

    PubMed

    Li, Jixi; Huang, Shengdong; Chen, Jinzhong; Yang, Zhenxing; Fei, Xiangwei; Zheng, Mei; Ji, Chaoneng; Xie, Yi; Mao, Yumin

    2007-01-01

    Uracil phosphoribosyltransferase, which catalyzes the conversion of uracil and 5-phosphoribosyl-1-R-diphosphate to uridine monophosphate, is important in the pyrimidine salvage pathway and is an attractive target for rational drug design by incorporation of prodrugs that are lethal to many parasitic organisms specifically. So far, uracil phosphoribosyltransferase has been reported in Arabidopsis thaliana only, not in mammals. In this study, a novel uracil phosphoribosyltransferase family cDNA encoding a 309 amino acid protein with a putative uracil phosphoribosyltransferase domain was isolated from the human fetal brain library. It was named human UPRTase (uracil phosphoribosyltransferase). The ORF of human UPRTase gene was cloned into pQE30 and expressed in Escherichia coli M15. The protein was purified by Ni-NTA affinity chromatography, but UPRTase activity could not be detected by spectrophotometry. RT-PCR analysis showed that human UPRTase was strongly expressed in blood leukocytes, liver, spleen, and thymus, with lower levels of expression in the prostate, heart, brain, lung, and skeletal muscle. Subcellular location of UPRTase-EGFP fusion protein revealed that human UPRTase was distributed in the nucleus and cytoplasm of AD293 cells. Evolutional tree analyses of UPRTases or UPRTase-domain-containing proteins showed that UPRTase was conserved in organisms. UPRTases of archaebacteria or eubacterium had UPRTase activity whereas those higher than Caenorhabditis elegans, which lacked two amino acids in the uracil-binding region, had no UPRTase activity. This means that human UPRTase may have enzymatic activity with another, unknown, factor or have other activity in pyrimidine metabolism. PMID:17384901

  18. 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.

  19. 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

  20. 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

  1. 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 ...

  2. Defective repair of uracil causes telomere defects in mouse hematopoietic cells.

    PubMed

    Vallabhaneni, Haritha; Zhou, Fang; Maul, Robert W; Sarkar, Jaya; Yin, Jinhu; Lei, Ming; Harrington, Lea; Gearhart, Patricia J; Liu, Yie

    2015-02-27

    Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity. PMID:25572391

  3. 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.

  4. Polymorphisms in uracil-processing genes, but not one-carbon nutrients, are associated with altered DNA uracil concentrations in an urban Puerto Rican population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background. Five genes - UNG, SMUG1, MBD4, TDG and DUT - are involved in repair or prevention of uracil misincorporation into DNA, an anomaly that can cause mutagenic events leading to cancer. Little is known about the determinants of uracil misincorporation, including the effects of single-nucleoti...

  5. Targeting tumors with nonreplicating Toxoplasma gondii uracil auxotroph vaccines.

    PubMed

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

    2013-09-01

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

  6. Targeting tumors with nonreplicating Toxoplasma gondii uracil auxotroph vaccines

    PubMed Central

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

    2013-01-01

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

  7. Enhancement of Borrelia burgdorferi PCR by uracil N-glycosylase.

    PubMed Central

    Loewy, Z G; Mecca, J; Diaco, R

    1994-01-01

    Uracil DNA glycosylases are DNA repair enzymes present in virtually every organism. These enzymes function by excising from DNA uracil residues resulting from either misincorporation of dUMP residues by a DNA polymerase or deamination of cytosine. Recently, the enzyme has been exploited in PCRs as a means for controlling carryover contamination from previously amplified DNA. When the enzyme is used in amplifications of Borrelia burgdorferi target sequences, we have observed an enhancement in signal detected by a microwell plate DNA hybridization assay. This increase in signal is dependent upon the length of the target, is titratable with enzyme concentration, and has been observed with amplifications performed with both symmetric and asymmetric PCR profiles. The enhancement is shown to occur at the level of the target genomic DNA. PMID:8126168

  8. Effect of uracil on pullulan production by Aureobasidium pullulans CGMCC1234.

    PubMed

    Sheng, Long; Zhu, Guilan; Tong, Qunyi

    2014-01-30

    Effect of uracil on the pullulan production, biomass and uridine phosphorylase (UPase) activity was studied in this research. Uracil was found to enhance pullulan accumulation and the addition time of uracil was crucial to pullulan production. Pullulan yield of 49.07 g/L was achieved by adding 5mM uracil at 48 h, by comparison to 37.72 g/L obtained with the control. UPase activity could not be detected at early growth stage of Aureobasidium pullulans, but stimulated by added uracil at logarithmic phase and stationary phase. The time course study on the fermentation of pullulan demonstrates that pullulan production was not closely associated with biomass accumulation. Results indicate that the increased pullulan yield brought by uracil was correlated with UPase activity. PMID:24299794

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

    SciTech Connect

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

    2014-01-14

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

  10. Synthesis and triplex formation of oligonucleotides containing 8-thioxodeoxyadenosine and 5-methyl-2-thiodeoxycytosine.

    PubMed

    Ohkubo, Akihiro; Miyata, Kenichi; Tsunoda, Hirosuke; Seio, Kohji; Sekine, Mitsuo

    2009-01-01

    For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides (TFO) containing 8-thioxodeoxyadenine (s(8)A) residues in place of the protonated cytosines (Cs) required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)A exhibited much stronger hybridization ability than C under neutral conditions when four s(8)A bases were arranged in a consecutive sequence. Moreover, we also synthesized TFOs containing 5-methyl-2-thiocytosines and examined their ability of triplex formation. PMID:19749240

  11. 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

  12. FTIR and Raman spectra and fundamental frequencies of 5-halosubstituted uracils: 5-X-uracil (X = F, Cl, Br and I)

    NASA Astrophysics Data System (ADS)

    Singh, J. S.

    2012-02-01

    FTIR and Raman spectra of 5-halosubstituted uracils (5-X-uracil; X = F, Cl, Br and I) were recorded in the region 200-4000 cm -1. Assuming under the Cs point group, the distribution of normal mode of vibrations between the two species as planar (a') and non-planar (a″) are given by 21a' + 9a″, of which also correspond to the 30 modes of uracil moiety and the electro negativity of halogen group substitution causes some where mixing/shifting in their modes with other modes. The ring breathing and kekule stretching modes are observed in lower magnitudes compared to those of uracil which could be due to mass effect of halogen atom in place of the hydrogen atom. The C-X (X = F, Cl, Br and 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.

  13. Incorporation of exogenous uracil by Cryptosporidium parvum in vitro.

    PubMed Central

    Upton, S J; Tilley, M; Mitschler, R R; Oppert, B S

    1991-01-01

    Oocysts of Cryptosporidium parvum were used to infect Madin-Darby bovine kidney cells. Cultures were incubated in a reduced-oxygen atmosphere in candle jars or in a 5% CO2-95% air atmosphere. At 72 h, parasites were quantitated microscopically and found to be enhanced 5.5-fold in the reduced-oxygen atmosphere. Using candle jars, we then determined that C. parvum was amenable to [3H]uracil incorporation assays and easily quantitated with this method. Images PMID:2056042

  14. Resonance formation in low energy electron scattering from uracil

    NASA Astrophysics Data System (ADS)

    Mašín, Zdeněk; Gorfinkiel, Jimena D.

    2014-05-01

    We present detailed ab initio results for resonance formation in low energy electron scattering from uracil obtained with the R-matrix method. We identify a larger number of resonances than any previous theoretical study. Most of these resonances have core-excited shape character and appear to be associated to the ring structure of the molecule. Their link to DEA spectra and to the resonances present in electron scattering from pyrimidine are discussed. Contribution to the Topical Issue "Electron and Positron Induced Processes", edited by Michael Brunger, Radu Campeanu, Masamitsu Hoshino, Oddur Ingólfsson, Paulo Limão-Vieira, Nigel Mason, Yasuyuki Nagashima and Hajime Tanuma.

  15. Pyrrhotite catalyzes the synthesis of uracil under hydrothermal conditions

    NASA Astrophysics Data System (ADS)

    López Ibáñez de Aldecoa, A.; Menor-Salván, C.

    2013-09-01

    The hypothesis of a prebiotic origin for metabolic cycles in hydrothermal systems gained interest during last years. The experimental approach to support this hypothesis was oriented mainly to the formation of organic molecules in iron sulfide mineral surfaces from inorganic precursors. In this work, we explore the behavior of previously formed, prebiotically plausible organic molecules in iron sulfide rich, low temperature hydrothermal environments. Using urea as a starting point, we found that uracil and other nitrogen heterocycles could be synthesized using water-urea solution as precursor in a packed pyrrhotite bed reactor, simulating hydrothermal conditions.

  16. Dynamics of uracil and 5-fluorouracil in DNA.

    PubMed

    Parker, Jared B; Stivers, James T

    2011-02-01

    The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death. Each of these enzymes uses an extrahelical base recognition mechanism, and previous studies with UNG have shown that extrahelical recognition is facilitated by destabilized base pairs possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of base pairs containing 5-FU and U are an important unknown in understanding the role of these enzymes in damage recognition and prodrug activation. The pH dependence of the (19)F NMR chemical shift of 5-FU imbedded in a model trinucleotide was used to obtain a pK(a) = 8.1 for its imino proton (10 °C). This is about 1.5 units lower than the imino protons of uracil or thymine and indicates that at neutral pH 5-FU exists significantly as an ionized tautomer that can mispair with guanine during DNA replication. NMR imino proton exchange measurements show that U/A and 5-FU/A base pairs open with rate constants (k(op)) that are 6- and 13-fold faster than a T/A base pair in the same sequence context. In contrast, these same base pairs have apparent opening equilibrium constants (αK(op)) that differ by less than a factor of 2, indicating that the closing rates (k(cl)) are enhanced by nearly equal amounts as k(op). These dynamic measurements are consistent with the previously proposed kinetic trapping model for extrahelical recognition by UNG. In this model, the enhanced intrinsic opening rates of destabilized base pairs allow the bound

  17. Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions

    PubMed Central

    Maul, Robert W; Saribasak, Huseyin; Martomo, Stella A; McClure, Rhonda L; Yang, William; Vaisman, Alexandra; Gramlich, Hillary S; Schatz, David G; Woodgate, Roger; Wilson, David M; Gearhart, Patricia J

    2013-01-01

    Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA. PMID:21151102

  18. 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. PMID:24036044

  19. 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)

    NASA Astrophysics Data System (ADS)

    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.

  20. Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

    PubMed Central

    Shalhout, Sophia; Haddad, Dania; Sosin, Angela; Holland, Thomas C.; Al-Katib, Ayad; Martin, Alberto

    2014-01-01

    Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG−/− cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG−/−splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels. PMID:25154417

  1. [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

  2. 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

  3. 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

  4. Kinetic parameters of uracil dosimeter in simulated extraterrestrial UV radiation

    NASA Astrophysics Data System (ADS)

    Kovács, G.; Gróf, P.; Bérces, A.; Patel, M. R.; Lammer, H.; Rontó, Gy.

    Studies of the solar UV environment on Earth 2.0 Gyr to 3.8 Gyr ago suggest that the terrestrial atmosphere was essentially anoxic, resulting in an ozone column abundance insufficient for protecting the planetary surface in the UV-B (280 nm - 315 nm) and the UV-C (200 nm - 280 nm) ranges. Since, short wavelength solar UV radiation in the UV-B and UV-C range penetrated through the atmosphere to the unprotected early Earth's surface, associated biological consequences may be expected. We discuss experimental data obtained as follows: Radiation sources applied were solar simulator and high power deuterium lamp, the wavelength were adjusted by interference filters (210, 230, 250 nm) and the irradiances were measured by OL754 spectroradiometer. The photo-reverse effect depends highly on the wavelength of the exposed radiation. Shorter wavelength UV radiation of about 200 nm is strongly effective in monomerization, while the longer wavelengths prefer the production of dimerization. In case of polychromatic light, like in space or on a planetary surface which is unprotected by an ozone layer the two processes run parallel. We could demonstrate experimentally, for the case of a uracil thin-layer that the photo-reaction process of the nucleotides can be both dimerization and the reverse process: monomerization. These results are important for the study of solar UV effects on organisms in the early terrestrial environment as well as for the search for life on Mars since we can show that biological harmful effects can also be reduced by shorter wavelength UV radiation, which is of importance in reducing DNA damages provoked by wavelengths longer than about 240 nm. Our earlier results showed that dimerization of the pyrimidin base uracil can be described by a first order kinetics, and this reaction gives the possibility to determine the dose of the UV source applied. This work is a theoretical and experimental approach to the relevant parameters of the first order kinetics.

  5. 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

  6. The role of the environment in the ion induced fragmentation of uracil.

    PubMed

    Markush, Pal; Bolognesi, Paola; Cartoni, Antonella; Rousseau, Patrick; Maclot, Sylvain; Delaunay, Rudy; Domaracka, Alicja; Kocisek, Jaroslav; Castrovilli, Mattea C; Huber, Bernd A; Avaldi, Lorenzo

    2016-06-22

    The fragmentation of uracil molecules and pure and nano-hydrated uracil clusters by (12)C(4+) ion impact is investigated. This work focuses on the fragmentation behavior of complex systems and the effect of the environment. On the one hand, it is found that the environment in the form of surrounding uracil or water molecules has a significant influence on the fragmentation dynamics, providing an overall 'protective' effect, while on the other hand we observe the opening of specific fragmentation channels. In particular, we report on the first observation of a series of hydrated fragments. This indicates a strong interaction between uracil and water molecules, holding the water clusters bound to the observed molecular fragments. PMID:27271080

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

    PubMed

    Jafari, M T; Khayamian, T

    2009-06-01

    A novel technique, corona discharge ion mobility spectrometry (CD-IMS), was developed for the qualitative and quantitative determination of 2-furfural (F) and 5-methyl-2-furfural (MF) in aqueous solutions. The limits of detection (LODs) were 5.3 x 10(-3) microg/mL for F and 6.7 x 10(-3) microg/mL for MF. The linear dynamic ranges of 1.16 x 10(-2) to 1.04 microg/mL and 2.20 x 10(-2) to 1.10 microg/mL were obtained for F and MF, respectively. The relative standard deviation was below 12% for both compounds. In addition to analysis of the individual compound, simultaneous determination of F and MF was also investigated. It was realized that F imposes a matrix effect on the MF signal and vice versa. The standard addition method was used to deal with the matrix effect. The recovery of the compounds in the synthetic samples validates the capability of the method. PMID:19531891

  8. Designing and developing S100P inhibitor 5-methyl cromolyn for pancreatic cancer therapy.

    PubMed

    Arumugam, Thiruvengadam; Ramachandran, Vijaya; Sun, Duoli; Peng, Zhenghong; Pal, Ashutosh; Maxwell, David S; Bornmann, William G; Logsdon, Craig D

    2013-05-01

    We have previously shown that the antiallergic drug cromolyn blocks S100P interaction with its receptor receptor for advanced glycation end product (RAGE) and improves gemcitabine effectiveness in pancreatic ductal adenocarcinoma (PDAC). However, the concentration required to achieve its effectiveness was high (100 μmol/L). In this study, we designed and synthesized analogs of cromolyn and analyzed their effectiveness compared with the parent molecule. An ELISA was used to confirm the binding of S100P with RAGE and to test the effectiveness of the different analogs. Analog 5-methyl cromolyn (C5OH) blocked S100P binding as well as the increases in NF-κB activity, cell growth, and apoptosis normally caused by S100P. In vivo C5OH systemic delivery reduced NF-κB activity to a greater extent than cromolyn and at 10 times lesser dose (50 mg vs. 5 mg). Treatment of mice-bearing syngeneic PDAC tumors showed that C5OH treatment reduced both tumor growth and metastasis. C5OH treatment of nude mice bearing orthotopic highly aggressive pancreatic Mpanc96 cells increased the overall animal survival. Therefore, the cromolyn analog, C5OH, was found to be more efficient and potent than cromolyn as a therapeutic for PDAC. PMID:23303403

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

    PubMed

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

    2016-09-01

    Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS. PMID:27342374

  10. 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

  11. 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.

  12. Synthesis, structure and biological activity of nickel(II) complexes of 5-methyl 2-furfural thiosemicarbazone.

    PubMed

    Jouad, E M; Larcher, G; Allain, M; Riou, A; Bouet, G M; Khan, M A; Thanh, X D

    2001-09-01

    5-Methyl 2-furfuraldehyde thiosemicarbazone (M5HFTSC) with nickel(II) leads to three types of complexes: [Ni(M5HFTSC)(2)X(2)], [Ni(M5FTSC)(2)] and [Ni(M5FTSC)(2)] x 2DMF. In the first type the ligand remains in thione form, while in the two other, the anionic thiolato form is involved. The species [Ni(M5HFTSC)(2)X(2)] has been characterized spectroscopically. The structures of [Ni(M5FTSC)(2)] x 2DMF and [Ni(M5FTSC)(2)] have been solved using X-ray diffraction. Biological studies of [Ni(M5HFTSC)(2)Cl(2)] have been carried out in vitro for antifungal activity on human pathogenic fungi, Aspergillus fumigatus and Candida albicans, and in vivo for toxicity on mice. The results are compared to those of the ligand, the metal salt and a similar copper complex [Cu(M5HFTSC)Cl(2)]. PMID:11566328

  13. 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

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

    PubMed

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

    2016-02-18

    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

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

    PubMed Central

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

    2016-01-01

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

  16. Uracil-induced signaling pathways for DUOX-dependent gut immunity

    PubMed Central

    Lee, Kyung-Ah; Kim, Boram; You, Hyejin; Lee, Won-Jae

    2015-01-01

    ABSTRACT Intestinal dual oxidase (DUOX) activation is the first line of host defense against enteric infection in Drosophila. DUOX enzymatic activity is mainly controlled by phospholipase C-β (PLCβ)-dependent calcium mobilization, whereas DUOX gene expression is mainly controlled by the MEKK1-p38 mitogen-activated protein kinase pathway. Furthermore, bacterial-derived uracil molecules act as ligands for DUOX activation. However, our current understanding of uracil-induced signal transduction pathways remain incomplete. We have recently found that uracil stimulates Hedgehog signaling, which in turn upregulates cadherin99C (Cad99C) expression in enterocytes. Cad99C molecules, along with PLCβ and protein kinase C, induce the formation of signaling endosomes that facilitate intracellular calcium mobilization for DUOX activity. These observations illustrate the complexity of signaling cascades in uracil-induced signaling pathways. Here, we further demonstrated the role of lipid raft formation and calmodulin-dependent protein kinase-II on endosome formation and calcium mobilization, respectively. Moreover, we will provide a brief discussion on two different models for uracil recognition and uracil-induced DUOX activation in Drosophila enterocytes. PMID:26655037

  17. 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

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

    PubMed

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

    2016-05-12

    C5-methylation of cytosines is strongly correlated with UV-induced mutations detected in skin cancers. Mutational hot-spots appearing at TCG sites are due to the formation of pyrimidine cyclobutane dimers (CPDs). The present study, performed for the model DNA duplex (TCGTA)3·(TACGA)3 and the constitutive single strands, examines the factors underlying the effect of C5-methylation on pyrimidine dimerization at TCG sites. This effect is quantified for the first time by quantum yields ϕ. They were determined following irradiation at 255, 267, and 282 nm and subsequent photoproduct analysis using HPLC coupled to mass spectrometry. C5-methylation leads to an increase of the CPD quantum yield up to 80% with concomitant decrease of that of pyrimidine(6-4) pyrimidone adducts (64PPs) by at least a factor of 3. The obtained ϕ values cannot be explained only by the change of the cytosine absorption spectrum upon C5-methylation. The conformational and electronic factors that may affect the dimerization reaction are discussed in light of results obtained by fluorescence spectroscopy, molecular dynamics simulations, and quantum mechanical calculations. Thus, it appears that the presence of an extra methyl on cytosine affects the sugar puckering, thereby enhancing conformations of the TC step that are prone to CPD formation but less favorable to 64PPs. In addition, C5-methylation diminishes the amplitude of conformational motions in duplexes; in the resulting stiffer structure, ππ* excitations may be transferred from initially populated exciton states to reactive pyrimidines giving rise to CPDs. PMID:27075054

  19. A computational study of adenine, uracil, and cytosine adsorption upon AlN and BN nano-cages

    NASA Astrophysics Data System (ADS)

    Baei, Mohammad T.; Taghartapeh, Mohammad Ramezani; Lemeski, E. Tazikeh; Soltani, Alireza

    Density-functional theory calculations are used to investigate the interaction of Al12N12 and B12N12 clusters with the adenine (A), uracil (U), and cytosine (C) molecules. The current calculations demonstrate that these hybrid adsorbent materials are able to adsorb the adenine, uracil, and cytosine molecules through exothermic processes. Our theoretical results reveal improvement in the adsorption of adenine, uracil, and cytosine on Al12N12 and B12N12. It is observed that B12N12 is highly sensitive to adenine, uracil, and cytosine compared with Al12N12 to serve as a biochemical sensor.

  20. 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.

  1. Prebiotic Synthesis of 5-Substituted Uracils: A Bridge Between the RNA World and the DNA-Protein World

    NASA Astrophysics Data System (ADS)

    Robertson, Michael P.; Miller, Stanley L.

    1995-05-01

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

  2. Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid.

    PubMed

    Piper, J R; McCaleb, G S; Montgomery, J A; Kisliuk, R L; Gaumont, Y; Sirotnak, F M

    1986-06-01

    Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on

  3. FTIR and Raman spectra and fundamental frequencies of 5-halosubstituted uracils: 5-X-uracil (X=F, Cl, Br and I).

    PubMed

    Singh, J S

    2012-02-15

    FTIR and Raman spectra of 5-halosubstituted uracils (5-X-uracil; X=F, Cl, Br and I) were recorded in the region 200-4000cm(-1). Assuming under the C(s) point group, the distribution of normal mode of vibrations between the two species as planar (a') and non-planar (a″) are given by 21a'+9a″, of which also correspond to the 30 modes of uracil moiety and the electro negativity of halogen group substitution causes some where mixing/shifting in their modes with other modes. The ring breathing and kekule stretching modes are observed in lower magnitudes compared to those of uracil which could be due to mass effect of halogen atom in place of the hydrogen atom. The C-X (X=F, Cl, Br and 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. PMID:22169026

  4. An efficient prebiotic synthesis of cytosine and uracil

    NASA Astrophysics Data System (ADS)

    Robertson, Michael P.; Miller, Stanley L.

    1995-06-01

    IN contrast to the purines1 3, the routes that have been proposed for the prebiotic synthesis of pyrimidines from simple precursors give only low yields. Cytosine can be synthesized from cyano-acetylene and cyanate4,5; the former precursor is produced from a spark discharge in a CH4/N2 mixture4,5 and is an abundant interstellar molecule6. 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 explored7 is the reaction of cyanoacetaldehyde (formed by hydrolysis of cyanoacetylene8) with urea. But at low concentrations of urea, this reaction produces no detectable quantities of cytosine7. 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 world9.

  5. 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.

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

    SciTech Connect

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

    2015-06-02

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

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

    DOE 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

  8. 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. PMID:22356544

  9. Isolation and identification of 5-hydroxyl-5-methyl-2-hexenoic acid from Actinoplanes sp. HBDN08 with antifungal activity.

    PubMed

    Zhang, Ji; Wang, Xiang-Jing; Yan, Yi-Jun; Jiang, Ling; Wang, Ji-Dong; Li, Bao-Ju; Xiang, Wen-Sheng

    2010-11-01

    A bioactivity-guided approach was employed to isolate and determine the chemical identity of bioactive constituents with antifungal activity from Actinoplanes sp. HBDN08. The structure of the antifungal metabolite was elucidated as 5-hydroxyl-5-methyl-2-hexenoic acid on the basis of spectral analysis. This compound showed strong in vitro antifungal activity against Botrytis cinerea, Cladosporium cucumerinum and Corynespora cassiicola, with an IC(50) of 32.45, 27.17, and 30.66 mg/L, respectively; however, it only moderately inhibited hyphal growth of Rhizoctonia solani with an IC(50) of 61.64 mg/L. The in vivo antifungal activity under greenhouse conditions demonstrated that 5-hydroxyl-5-methyl-2-hexenoic acid could effectively control the diseases caused by B. cinerea, C. cucumerinum and C. cassiicola with 71.42%, 78.63% and 65.13% control values at 350 mg/L, respectively. This strong antifungal activity suggests that 5-hydroxyl-5-methyl-2-hexenoic acid might be a promising candidate for new antifungal agents. PMID:20584599

  10. A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil

    PubMed Central

    Greagg, Martin A.; Fogg, Mark J.; Panayotou, George; Evans, Steven J.; Connolly, Bernard A.; Pearl, Laurence H.

    1999-01-01

    Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G⋅C → A⋅U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea. PMID:10430892

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. 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

  13. Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

    PubMed Central

    Endres, Matthias; Biniszkiewicz, Detlev; Sobol, Robert W.; Harms, Christoph; Ahmadi, Michael; Lipski, Andreas; Katchanov, Juri; Mergenthaler, Philipp; Dirnagl, Ulrich; Wilson, Samuel H.; Meisel, Andreas; Jaenisch, Rudolf

    2004-01-01

    Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia. PMID:15199406

  14. 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

  15. 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.

  16. Nanohydration of uracil: emergence of three-dimensional structures and proton-induced charge transfer.

    PubMed

    Bacchus-Montabonel, Marie-Christine; Calvo, Florent

    2015-04-21

    Stepwise hydration of uracil has been theoretically revisited using different methods ranging from classical force fields to quantum chemical approaches. Hydration initially begins within the uracil plane but proceeds at four molecules into three-dimensional configurations or even water clusters next to the nucleobase. The relative stability between the various structures is significantly affected by zero-point energy and finite temperature (entropy) effects and also gives rise to markedly different responses to an excitation by an impinging high-energy proton. In particular, charge transfer to the molecular complex is dramatically altered in collisions toward the coating cluster but barely modified for peripheral hydration patterns. PMID:25793649

  17. Enhancement of infrared absorption of low-temperature uracil thin films by a nanostructured silver surface

    NASA Astrophysics Data System (ADS)

    Ivanov, A. Yu.; Stepanian, S. G.; Adamowicz, L.; Karachevtsev, V. A.

    2016-02-01

    Enhancement of infrared absorption (SEIRA) of adsorbed biological molecules by a nanostructured metal surface is one of the main routes to increasing the sensitivity of modern optical biosensors. The FTIR absorption spectra of thin films of the RNA base uracil deposited on low-temperature substrate (T = 6 K) with nanoscale silver structures were investigated in the spectral range 2700-600 cm-1. It was shown that the intensity of the absorption bands corresponding to νCO stretching vibrations (range 1800-1600 cm-1) of uracil (Ur) thin films increases 3-4 fold. For multilayer films, the influence of the nanostructures on the vibrational spectra weakens, and for the film layers more than 15 nm away from the surface, the enhancement is essentially absent. The energies and the vibrational spectra of the complexes of uracil monomers and dimers with 20-atom tetrahedral silver nanoclusters were calculated by the quantum-mechanical method DFT/B3LYP. The most stable complexes have the coordination bond between the top of the silver tetrahedron and the oxygen of the carbonyl group C4O. It was found that the formation of such complexes significantly (3-5 fold) enhances the intensity of the νC4O stretching vibration of uracil, while the intensities of the βNH, βCH and ring bending vibrations do not increase significantly.

  18. Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome.

    PubMed Central

    Seal, G; Brech, K; Karp, S J; Cool, B L; Sirover, M A

    1988-01-01

    Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome. Images PMID:3353381

  19. 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...

  20. 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.

  1. Aldol Reactions of Axially Chiral 5-Methyl-2-(o-aryl)imino-3-(o-aryl)-thiazolidine-4-ones.

    PubMed

    Erol Gunal, Sule; Dogan, Ilknur

    2016-01-01

    Axially chiral 5-methyl-2-(o-aryl)imino-3-(o-aryl)-thiazolidine-4-ones have been subjected to aldol reactions with benzaldehyde to produce secondary carbinols which have been found to be separable by HPLC on a chiral stationary phase. Based on the reaction done on a single enantiomer resolved via a chromatographic separation from a racemic mixture of 5-methyl-2-(α-naphthyl)imino-3-(α-naphthyl)-thiazolidine-4-one by HPLC on a chiral stationary phase, the aldol reaction was shown to proceed via an enolate intermediate. The axially chiral enolate of the thiazolidine-4-one was found to shield one face of the heterocyclic ring rendering face selectivity with respect to the enolate. The selectivities observed at C-5 of the ring varied from none to 11.5:1 depending on the size of the ortho substituent. Although the aldol reaction proceeded with a lack of face selectivity with respect to benzaldehyde, recrystallization returned highly diastereomerically enriched products. PMID:27322237

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

    PubMed

    Robertson, M P; Miller, S L

    1995-05-01

    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. PMID:7732378

  3. 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

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

    PubMed

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

    2015-07-01

    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

  5. Ion-forming processes on 248 NM laser excitation of uracil and methyl-monosubstituted uracils: A time-resolved transient conductivity study in aqueous solution

    NASA Astrophysics Data System (ADS)

    Görner, Helmut; Schulte-Frohlinde, Dietrich

    1995-04-01

    Uracil, thymine and 1-, 3-, and 6-methyluracil were studied by time-resolved optical and conductometric methods after 248 nm excitation with 20 ns laser pulses. The transient conductivity in argon-saturated aqueous solution, showing a maximum increase ( Δκmax) during the pulse, is ascribed to the generation of hydrated electrons (e aq-) and protons. Biphotonic photoionization as the primary process is inferred from the almost linear dependence of Δκmax on the square of the laser pulse intensity ( IL2). The quantum yield, obtained from either Δκmax or optical detection of e aq-, e.g. gfe- = 0.02 for uracil at pH 7 and IL = 12 MW cm -2, varies by a factor of about two for the five pyrimidines. The neutralization kinetics depend strongly on pH and the concentrations of laser-induced e aq- and H +, i.e. on IL. At pH 6-7 the Δκ signal decays by second-order kinetics. Under argon the electron adds to the (methyl)uracil and neutralization occurs by reaction of the radical anion with a proton, which originates from a fast decay of the radical cation. Virtually the same conductivity pattern was found for the neutralization reaction of OH - and H + under N 2O. In the acidic pH range the decay changes to first-order kinetics due to reaction of H + with e aq- under argon or with OH - under N 2O. In the alkaline pH range OH - release is involved in the relaxation process resulting from the radical cation after excitation of the conjugate base. No indication of a specific spatial correlation of the charged species, as proposed earlier by Grossweiner for other systems, was found.

  6. 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.

  7. 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

  8. 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.

  9. Isolation and mapping of a uracil-sensitive mutant of Salmonella typhimurium.

    PubMed

    Bussey, L B; Ingraham, J L

    1982-01-01

    A uracil-sensitive mutant of Salmonella typhimurium was isolated by diethyl sulfate mutagenesis and penicillin counterselection. This mutation identifies a new Salmonella gene that is well separated from the structural genes for arginine and pyrimidine biosynthesis. The use-1 mutation was located between the ilv gene cluster (isoleucine-valine operon) and hisR (structural gene for histidine tRNA) at 83 map units. PMID:7048028

  10. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  12. 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

  13. NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

    PubMed Central

    Hussain, Shobbir; Sajini, Abdulrahim A.; Blanco, Sandra; Dietmann, Sabine; Lombard, Patrick; Sugimoto, Yoichiro; Paramor, Maike; Gleeson, Joseph G.; Odom, Duncan T.; Ule, Jernej; Frye, Michaela

    2013-01-01

    Summary Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs), yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs). Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders. PMID:23871666

  14. 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-...

  15. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 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-...

  16. Base excision repair of both uracil and oxidatively damaged bases contribute to thymidine deprivation-induced radiosensitization

    SciTech Connect

    Allen, Bryan G.; Johnson, Monika; Marsh, Anne E.; Dornfeld, Kenneth J. . E-mail: kenneth-dornfeld@uiowa.edu

    2006-08-01

    Purpose: Increased cellular sensitivity to ionizing radiation due to thymidine depletion is the basis of radiosensitization with fluoropyrimidine and methotrexate. The mechanism responsible for cytotoxicity has not been fully elucidated but appears to involve both the introduction of uracil into, and its removal from, DNA. The role of base excision repair of uracil and oxidatively damaged bases in creating the increased radiosensitization during thymidine depletion is examined. Methods and Materials: Isogenic strains of S. cerevisiae differing only at loci involved in DNA repair functions were exposed to aminopterin and sulfanilamide to induce thymidine deprivation. Cultures were irradiated and survival determined by clonogenic survival assay. Results: Strains lacking uracil base excision repair (BER) activities demonstrated less radiosensitization than the parental strain. Mutant strains continued to show partial radiosensitization with aminopterin treatment. Mutants deficient in BER of both uracil and oxidatively damaged bases did not demonstrate radiosensitization. A recombination deficient rad52 mutant strain was markedly sensitive to radiation; addition of aminopterin increased radiosensitivity only slightly. Radiosensitization observed in rad52 mutants was also abolished by deletion of the APN1, NTG1, and NTG2 genes. Conclusion: These data suggest radiosensitization during thymidine depletion is the result of BER activities directed at both uracil and oxidatively damaged bases.

  17. 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

  18. 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

  19. Determination of optimal conditions for 5-methyl-benzotriazole biodegradation with activated sludge communities by dilution of the inoculum.

    PubMed

    Yuan, Heyang; Herzog, Bastian; Helmreich, Brigitte; Lemmer, Hilde; Müller, Elisabeth

    2014-07-15

    The aerobic biodegradation of 5-methyl-benzotriazole (5-TTri) was optimized using lab-scale setups and activated sludge communities (ASC) collected from three wastewater treatment plants (WWTP) MBR-MH, CAS-E and CAS-M being different in their treatment technologies. ASC inocula were diluted to rule out non-biodegrading species and incubated under two nutrient conditions: A) mineral salt media (MSM) and B) carbon and nitrogen supplied MSM giving MSM-CN. 5-TTri removal with the ASC ranged from 60% to 100% in only 10 days. 100 μL suspended biomass from the biodegrading setups was subsequently plated on solid media to eliminate possible activated sludge remnants. After growth occurred, mixed colonies were harvested and inoculated in fresh liquid MSM containing 20 mg L(-1) 5-TTri. These bacterial consortia showed good 5-TTri removal in MSM-CN rather than in MSM, indicating nutrient supply being required for efficient biodegradation. In addition, experiments with high 5-TTri concentrations ranging from 20 to 1,000 mg L(-1) were conducted in both, MSM and MSM-CN and the maximal 5-TTri removal capacity of the ASC evaluated. 50 mg L(-1) 5-TTri was still removed in both media whereas 100 mg L(-1) was solely removed in MSM-CN. 5-TTri biodegradation patterns also indicated that 5-TTri might be co-metabolized by microbial consortia. Furthermore, experiments with gradient-solid-media-plates showed 5-TTri to be inhibitory for the ASC in concentrations above 50 mg L(-1) and revealed the optimal conditions regarding carbon and nitrogen concentration and pH value for effective 5-TTri biodegradation by ASC. Nitrogen proved a crucial factor for enhancing organisms' biodegradation capacity with an optimal pH around 7 while carbon showed no such effect. PMID:24287305

  20. 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.

  1. 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. PMID:26045555

  2. Zooming into pi-stacked manifolds of nucleobases: ionized states of dimethylated uracil dimers.

    PubMed

    Zadorozhnaya, Anna A; Krylov, Anna I

    2010-02-01

    The electronic structure of 1,3-dimethyluracil and its dimer is characterized by ab initio calculations. The methylation eliminates the H-bonded isomers and allows one to focus on the pi-stacked manifold. In the neutral species, methylation increases the binding energy by 3-4 kcal/mol and reduces the lowest ionization energy (IE) by 0.6 eV. Other valence IEs are also red-shifted and the relative state ordering is the same as in uracil; however, the magnitude of the effect varies from 0.37 to 0.86 eV. The largest shifts are observed for the states with large contributions from lone pairs of nitrogens, which are primary substitution sites. The effect of stacking interactions on IEs is similar in methylated and non-methylated dimers: the lowest IE is red-shifted by 0.37 and 0.35 eV relative to the respective monomers. The splittings between other pairs of dimer states derived from the in-phase and out-of-phase combinations of the monomers' states are also similar to non-methylated uracil dimers, except for the states that include a large weight of nitrogen lone pairs. Because of the nonuniform effect on both monomers' levels and the shifts, the relative order of the ionized states in the dimer changes, relative to that of the non-methylated uracil dimer. The ionized stacked isomers show two different relaxation patterns--several isomers form structures with the delocalized hole stabilized by the orbital overlap, whereas others relax to the structures with the localized hole stabilized by electrostatic interactions. Electronic spectra of the ionized species at the neutral and cation geometries are presented and discussed. PMID:20055394

  3. Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.

    PubMed

    Abellón-Ruiz, Javier; Waldron, Kevin J; Connolly, Bernard A

    2016-07-17

    Archaeal family-D DNA polymerases (Pol-D) comprise a small (DP1) proofreading subunit and a large (DP2) polymerase subunit. Pol-D is one of the least studied polymerase families, and this publication investigates the enzyme from Archaeoglobus fulgidus (Afu Pol-D). The C-terminal region of DP2 contains two conserved cysteine clusters, and their roles are investigated using site-directed mutagenesis. The cluster nearest the C terminus is essential for polymerase activity, and the cysteines are shown to serve as ligands for a single, critical Zn(2+) ion. The cysteines farthest from the C terminal were not required for activity, and a role for these amino acids has yet to be defined. Additionally, it is shown that Afu Pol-D activity is slowed by the template strand hypoxanthine, extending previous results that demonstrated inhibition by uracil. Hypoxanthine was a weaker inhibitor than uracil. Investigations with isolated DP2, which has a measurable polymerase activity, localised the deaminated base binding site to this subunit. Uracil and hypoxanthine slowed Afu Pol-D "in trans", that is, a copied DNA strand could be inhibited by a deaminated base in the alternate strand of a replication fork. The error rate of Afu Pol-D, measured in vitro, was 0.24×10(-5), typical for a polymerase that has been proposed to carry out genome replication in the Archaea. Deleting the 3'-5' proofreading exonuclease activity reduced fidelity twofold. The results presented in this publication considerably increase our knowledge of Pol-D. PMID:27320386

  4. Synthesis, characterization and application of new azo dyes derived from uracil for polyester fibre dyeing

    NASA Astrophysics Data System (ADS)

    Yazdanbakhsh, Mohamad-reza; Abbasnia, Masoumeh; Sheykhan, Mehdi; Ma'mani, Leila

    2010-08-01

    Some novel uracil derived azo compounds were synthesized by diazotization of substituted aromatic amines, amidine- and guanidine-like amines such as 2-aminopyridine and 2-aminopyrimidine, ortho-hydroxy aniline and ortho-hydroxy naphthyl amines and coupling reaction with 6-amino-1,3-dimethyluracil. Structures of the dyes were fully characterized by spectroscopic techniques (UV, 1H NMR, 13C NMR, CHN and IR). The dyes were applied to polyester, affording orange-yellow shades and the wash fastness of the dyeings was excellent.

  5. 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.

  6. 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

  7. 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

  8. Functionalization of single-walled carbon nanotubes with uracil, guanine, thymine and L-alanine

    NASA Astrophysics Data System (ADS)

    Silambarasan, D.; Iyakutti, K.; Vasu, V.

    2014-06-01

    Experimental investigation of functionalization of oxidized single-walled carbon nanotubes (OSWCNTs) with three nucleic acid bases such as uracil, guanine, thymine and one amino acid, L-alanine is carried out. Initially, the SWCNTs are oxidized by acid treatment. Further, the oxidized SWCNTs are effectively functionalized with aforementioned biological compounds by ultrasonication. The diameter of OSWCNTs has increased after the adsorption of biological compounds. The cumulative Π-Π stacking, hydrogen bond and polar interaction are the key factors to realize the adsorption. The amount of adsorption of each biological compound is estimated. The adsorption of guanine is more among all the four biological compounds.

  9. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

    PubMed Central

    Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

    2013-01-01

    HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

  10. Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed

    PubMed Central

    Weeks, L D; Zentner, G E; Scacheri, P C; Gerson, S L

    2014-01-01

    Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG+/+ and UNG−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG−/− cells accumulated significantly greater uracil and DSBs compared with UNG+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG+/+ cells. Furthermore, DSBs in UNG+/+ and UNG−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG+/+ and UNG−/− cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG−/− but not UNG+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death. PMID:24503537

  11. Syntheses, spectral, X-ray and DFT studies of 5-benzyl-N-phenyl-1,3,4-thiadiazol-2-amine, 2-(5-phenyl-1,3,4-thiadiazol-2-yl) pyridine and 2-(5-methyl-1,3,4-thiadiazole-2-ylthio)-5-methyl-1,3,4-thiadiazole obtained by Mn(II) catalyzed reactions

    NASA Astrophysics Data System (ADS)

    Dani, R. K.; Bharty, M. K.; Kushawaha, S. K.; Paswan, S.; Prakash, Om; Singh, Ranjan K.; Singh, N. K.

    2013-12-01

    New compounds 5-benzyl-N-phenyl-1,3,4-thiadiazol-2-amine (Bptha, 1), 2-(5-phenyl-1,3,4-thiadiazol-2-yl) pyridine (Pthp, 2) and 2-(5-methyl-1,3,4-thiadiazole-2-ylthio)-5-methyl-1,3,4-thiadiazole (Mtmth, 3) have been synthesized and characterized with the aid of elemental analyses, IR, NMR and single crystal X-ray data. The structure of compounds 1, 2 and 3 are stabilized via intramolecular as well as intermolecular hydrogen bonding and crystallize in monoclinic system with space group P 1, P21/n and P 1, respectively. During the course of reaction, the substituted thiosemicarbazide/thiohydrazide get cyclized into the corresponding thiadiazole in the presence of manganese(II) nitrate via loss of H2O to yield compounds 1 and 2. However condensation occurred in the case of 5-methyl-1,3,4-thiadiazole-2-thiol which yielded 2-(5-methyl-1,3,4-thiadiazole-2-ylthio)-5-methyl-1,3,4-thiadiazole (3) by loss of one mole of H2S from two moles of 5-methyl-1,3,4-thiadiazole-2-thiol in the presence of manganese(II) acetate. The geometry optimization has been performed using DFT method and geometrical parameters thus obtained for the compounds have been compared with their single crystal X-ray data. The negative values of HOMO and LUMO energies for the molecules indicate that they are stable. The electronic transition from the ground state to the excited state due to a transfer of electrons from the HOMO to LUMO levels is mainly associated with the π⋯π transition.

  12. 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

  13. 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.

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

    PubMed

    Sun, Shuai; Brown, Alex

    2015-04-30

    The resonance Raman spectra of the 5-halogenated (F, Cl, and Br) uracils are simulated via the Herzberg-Teller (HT) short-time dynamics formalism. The gradient of the S1 excited state is computed at the CAMB3LYP/aug-cc-pVTZ level of theory in the conductor-like polarizable continuum model for water (C-PCM, H2O), based on the equilibrium geometry determined using PBE0/aug-cc-pVTZ in H2O (C-PCM). The simulated resonance Raman spectra show good agreement with the experimental spectra in terms of both peak positions and intensities. The differences between the resonance Raman spectra of the three 5-halogenated uracils, caused by the effect of halogen substitution, are examined in terms of ground-state normal-mode eigenvectors and excited-state Cartesian gradients, according to the HT formalism. The differences in the normal-mode eigenvectors and excited-state Cartesian gradients between 5-fluorouracil and 5-chlorouracil are used to interpret the dissimilarity between their resonance Raman spectra. Meanwhile, the similarity between the spectra of 5-chlorouracil and 5-bromouracil is explained by the correspondence between their normal modes and excited-state gradients. PMID:25856119

  15. 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.

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

    PubMed

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

    2010-10-01

    A detailed experimental and computational study of the absorption and fluorescence spectra of 5-aminouracil (5 AU) and 6-aminouracil (6 AU) in aqueous solution is reported. The lowest energy band of the steady-state absorption spectra of 5 AU is considerably red-shifted, noticeably less intense, and broader than its counterpart in uracil (U). On the contrary, the 6 AU lowest energy absorption peak is close in energy to that of U, but it is much narrower and the transition is much more intense. The emission properties of 5 AU, 6 AU, and U are also very different. Both amino-substituted compounds exhibit indeed a much larger Stokes shift as compared to U, and the emission band of 5 AU is much narrower than that of 6 AU. Those features are fully rationalized with the help of PCM/TD-PBE0 calculations in aqueous solution and MS-CASPT2/CASSCF calculations in the gas phase. A stable minimum on the potential energy surface of the lowest energy bright state is found for 5 AU, both in the gas phase and in aqueous solution. For 6 AU a barrierless path leads to the conical intersection with the ground electronic state, but a nonplanar plateau region is predicted in aqueous solution, which is responsible for the very large Stokes shift. Some general considerations on the excited-state dynamics of uracil derivatives are also reported. PMID:20831146

  17. Hydrogen-Bonding Complexes of 5-Azauracil and Uracil Derivatives in Organic Medium.

    PubMed

    Diez-Martinez, Alba; Kim, Eun-Kyong; Krishnamurthy, Ramanarayanan

    2015-07-17

    Uracil derivatives form strong complexes with complementary 2,4-diaminotriazine and adenine compounds, whereas derivatives of 5-azauracil (2,4-dioxotriazine) are known to form weak complexes in aqueous medium. However, herein we report that in organic medium (CDCl3), the 5-azauracil moiety forms hydrogen-bond-mediated complexes with complementary 2,4-diaminotriazine and adenine compounds, with strengths comparable to those formed by uracil compounds. Such dichotomous base-pairing behavior of the 5-azauracil moiety, in organic versus aqueous media, is found to be consistent with the ionization of the 5-azauracil moiety in aqueous medium leading to competitive interference from water molecules (via solvation), which is absent (lack of such ionization and solvent interference) in organic medium. This discriminating role of solvent (e.g., water) could have been an important factor in the selection of molecules, based on their physicochemical properties, and subsequently in the emergence of potential primordial informational oligomers that would have played a role in the origins of life. PMID:26098835

  18. Improved line frequencies for the nucleic acid base uracil for a radioastronomical search

    NASA Astrophysics Data System (ADS)

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

    2006-11-01

    Aims.We report new laboratory spectroscopic data on the rotational spectrum of the pyrimidine nucleic acid base uracil (C4H4N2O2). The dataset has been extended both into the microwave region and towards mm-wavelengths with the aim of providing accurate transition rest frequencies for astrophysical searches. Methods: .The microwave measurements have been performed with a molecular beam Fourier transform microwave spectrometer in the frequency range from 9-19 GHz and the mm-wave band transitions have been recorded with a Stark-modulated free jet spectrometer up to 100 GHz. The global dataset has been analysed with a standard S-reduced Hamiltonian and precise spectroscopic parameters up to quartic order have been obtained. The hyperfine structure due to the two 14N nuclei has been resolved in the microwave measurements and the nuclear quadrupole coupling constants could be derived to high accuracy. Results: .Based on the new laboratory data and analysis, highly precise rotational transition rest frequencies are available for astrophysical important lines of uracil up to 300 GHz.

  19. Uracil on Cu(110): A quantitative structure determination by energy-scanned photoelectron diffraction

    NASA Astrophysics Data System (ADS)

    Duncan, D. A.; Unterberger, W.; Kreikemeyer-Lorenzo, D.; Woodruff, D. P.

    2011-07-01

    The local adsorption site of the nucleobase uracil on Cu(110) has been determined quantitatively by energy-scanned photoelectron diffraction (PhD). Qualitative inspection of the O 1s and N 1s soft x-ray photoelectron spectra, PhD modulation spectra, and O K-edge near-edge x-ray adsorption fine structure indicate that uracil bonds to the surface through its nitrogen and oxygen constituent atoms, each in near atop sites, with the molecular plane essentially perpendicular to surface and aligned along the close packed [1overline 1 0] azimuth. Multiple scattering simulations of the PhD spectra confirm and refine this geometry. The Cu-N bondlength is 1.96 ± 0.04 Å, while the Cu-O bondlengths of the two inequivalent O atoms are 1.93 ± 0.04 Å and 1.96 ± 0.04 Å, respectively. The molecule is twisted out of the [1overline 1 0]direction by 11 ± 5°.

  20. 5-(2,6-difluorobenzyl)oxymethyl-5-methyl-3-(3-methylthiophen-2-yl)- 1,2-isoxazoline as a useful rice herbicide.

    PubMed

    Hwang, In Taek; Kim, Hyoung Rae; Jeon, Dong Ju; Hong, Kyung Sik; Song, Jong Hwan; Cho, Kwang Yun

    2005-11-01

    5-(2,6-difluorobenzyl)oxymethyl-5-methyl-3-(3-methylthiophen-2-yl)-1,2-isoxazoline derivative was synthesized, and its herbicidal activity was assessed under glasshouse and flooded paddy conditions. 5-(2,6-Difluorobenzyl)oxymethyl-5-methyl-3-(3-methylthiophen-2-yl)-1,2-isoxazoline demonstrated good rice selectivity and potent herbicidal activity against annual weeds at 125 g of a.i. ha(-1) under greenhouse conditions. Soil application of this compound showed complete control of barnyard-grass to the fourth leaf stage at 250 g of a.i. ha(-1). Field trials indicated that this compound controlled annual weeds rapidly with a good tolerance on transplanted rice seedlings by post-emergence and soil application. This compound showed a low mammalian and environmental toxicity in various toxicological tests. PMID:16248565

  1. 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.

  2. 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

  3. Synthesis of 5-methyl-5-deaza nonclassical antifolates as inhibitors of dihydrofolate reductases and as potential antipneumocystis, antitoxoplasma, and antitumor agents.

    PubMed

    Gangjee, A; Shi, J; Queener, S F; Barrows, L R; Kisliuk, R L

    1993-10-29

    A series of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines 4-9 were synthesized as 5-deaza nonclassical antifolates containing trimethoxy, dichloro-, or trichlorophenyl substitutions and a N-H, N-CH3, or N-CHO at the 10-position. The compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii), rat liver (RL), and Lactobacillus casei (L. casei); as inhibitors of T. gondii and P. carinii cell growth in culture; and as antitumor agents. The compounds were prepared by modifications of procedures for classical 5-deaza folates. 2,4-Diamino-5-methyl-6-[(3',4',5'-trimethoxy-N- methylanilino)methyl]pyrido[2,3-d]pyrimidine (5a) exhibited high potency as well as selectivity (compared to RL DHFR) for P. carinii and T. gondii DHFR. Compound 5a is one of the most potent and selective nonclassical folate inhibitors of T. gondii DHFR known. The N-10 formyl analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]pyrido-[2,3-d]pyrimidine (6a) had decreased potency, but it maintained high selectivity for T. gondii DHFR. The corresponding chloro-substituted analogues maintained potency or had decreased potency; N-10 substitution did not increase potency or selectivity to the extent observed in the 3',4',5'-trimethoxy series. Partial reduction of the B ring to afford the dihydro analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]-5,8-dihydropyrido[2,3-d]pyrimidine (7), its 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine analogue 8, and 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino)methyl]-5,6,7, 8- tetrahydropyrido[2,3-d]pyrimidine (9) resulted in a significant decrease in potency. In T. gondii cell culture inhibitory studies, 2,4-diamino-5-methyl-6-[(3',4',5'- trimethoxyanilino)methyl]pyrido[2,3-d]pyrimidine (4a), 5a, and 6a were less potent compared to their DHFR inhibitory potencies. Against P. carinii cells in culture, 4a and 5a at 10

  4. Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

    SciTech Connect

    Singh, Prem; Talawar, Ramappa K.; Krishna, P. D. V.; Varshney, Umesh; Vijayan, M.

    2006-12-01

    Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected. Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis.

  5. Electrophilic 5-Substituted Uracils as Potential Radiosensitizers: A Density Functional Theory Study.

    PubMed

    Makurat, Samanta; Chomicz-Mańka, Lidia; Rak, Janusz

    2016-08-18

    Although 5-bromo-2'-deoxyuridine (5BrdU) possesses significant radiosensitizing power in vitro, clinical studies do not confirm any advantages of radiotherapy employing 5BrdU. This situation calls for a continuous search for efficient radiosensitizers. Using the proposed mechanism of radiosensitization by 5BrdU, we propose a series of 5-substituted uracils, XYU, that should undergo efficient dissociative electron attachment. The DFT-calculated thermodynamic and kinetic data concerning the XYU degradations induced by electron addition suggests that some of the scrutinized derivatives have much better characteristics than 5BrdU itself. Synthesis of these promising candidates for radiosensitizers, followed by studies of their radiosensitizing properties in DNA context, and ultimately in cancer cells, are further steps to confirm their potential applicability in anticancer treatment. PMID:27156191

  6. Ultrafast dynamics of uracil and thymine studied using a sub-10 fs deep ultraviolet laser.

    PubMed

    Xue, Bing; Yabushita, Atsushi; Kobayashi, Takayoshi

    2016-06-22

    Single 9.6 fs deep ultraviolet pulses with a spectral range of 255-290 nm are generated by a chirped-pulse four-wave mixing technique for use as pump and probe pulses. The electronic excited state and vibrational dynamics are simultaneously observed for an aqueous solution of uracil and thymine over the full spectral range using a 128-channel lock-in amplifier detector. Two probe photon energy-dependent lifetimes gradually increasing with the probe photon energy are obtained from the decay dynamics data. Ultrafast decay dynamics through the conical intersection is assigned from the first excited ππ* to the final ground state involving the nπ* states. Vibrational modes of the electronic ground state and excited states can be observed, which are strongly coupled to the decay dynamics of the electronic excited state. PMID:27299165

  7. 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.

  8. 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

  9. Synthesis, DNA binding and antiviral activity of new uracil, xanthine, and pteridine derivatives.

    PubMed

    El-Sabbagh, Osama I; El-Sadek, Mohamed E; El-Kalyoubi, Samar; Ismail, Ibrahim

    2007-01-01

    Some new 6-amino-1,3-dimethyl-5-(substituted methylidene)aminouracils were synthesized. Most of them were cyclized with triethyl orthoformate as a one-carbon source to afford 1,3-dime-thyl-6-substituted pteridine derivatives. Certain uracils gave xanthine instead of the expected pteridine derivatives upon using another one-carbon source such as triethyl orthoacetate or triethyl orthobenzoate. The nucleic acid binding assay revealed that some new compounds showed high affinity, chelation, and fragmentation of nucleic acids whether DNA or RNA contrary to acyclovir that has affinity to DNA only. The antiviral activity of these novel compounds showed that compounds 2e and 2f reduced the cytopathogencity of Peste des petits ruminant virus (PPRV) on Vero cell culture by 60 and 50%, respectively. PMID:17206606

  10. Sites of adsorption of adenine, uracil, and their corresponding derivatives on sodium montmorillonite.

    PubMed

    Perezgasga, L; Serrato-Díaz, A; Negrón-Mendoza, A; De Pablo Galán, L; 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, pK(a) of the adsorbate, and Van der Waals interactions. PMID:16010992

  11. Enantioselective route to 5-methyl- and 5,7-dimethyl-6,7-dihydro-5H-dibenz[c,e]azepine: secondary amines with switchable axial chirality.

    PubMed

    Pira, Silvain L; Wallace, Timothy W; Graham, Jonathan P

    2009-04-01

    (-)-5-Methyl-6,7-dihydro-5H-dibenz[c,e]azepine 4, a new secondary amine featuring an axis-center stereochemical relay, was prepared enantioselectively from 2'-acetylbiphenyl-2-carboxylic acid, using (R)-2-phenylglycinol as an auxiliary for the control of both elements of chirality. The biaryl axis in 4 preferentially adopts the aS-configuration, with the methyl substituent pseudoequatorial, but conversion into the corresponding N-Boc derivative locks the axis into the aR-configuration, as predicted on the basis of molecular mechanics calculations. PMID:19275220

  12. Synthesis and crystal structure of copper (II) uracil ternary polymeric complex with 1,10-phenanthroline along with the Hirshfeld surface analysis of the metal binding sites for the uracil ligand

    NASA Astrophysics Data System (ADS)

    Patil, Yogesh Prakash; Nethaji, Munirathinam

    2015-02-01

    The study of models for "metal-enzyme-substrate" interaction has been a proactive area of research owing to its biological and pharmacological importance. In this regard the ternary copper uracil complex with 1,10-phenanthroline represents metal-enzyme-substrate system for DNA binding enzymes. The synthesis of the complex, followed by slow evaporation of the reaction mixture forms two concomitant solvatomorph crystals viz., {[Cu(phen)(μ-ura)(H2O)]n·H2O (1a)} and {[Cu(phen)(μ-ura)(H2O)]n·CH3OH (1b)}. Both complexes are structurally characterized, while elemental analysis, IR and EPR spectra were recorded for 1b (major product). In both complexes, uracil coordinates uniquely via N1 and N3 nitrogen atom acting as a bidentate bridging ligand forming a 1-D polymer. The two solvatomorphs were quantitatively analyzed for the differences with the aid of Hirshfeld surface analysis.

  13. Synthesis of Substituted N-[4(5-Methyl/phenyl-1,3,4-oxadiazol-2-yl)-3,6-dihydropyridin-1(2H)-yl]benzamide/benzene Sulfonamides as Anti-Inflammatory and Anti-Cancer Agents

    PubMed Central

    Gangapuram, Madhavi; Redda, Kinfe K.

    2010-01-01

    Fourteen novel substituted N-[4(5-methyl/phenyl-1,3,4-oxadiazol-2-yl)-3,6-dihydropyridin-1(2H)-y1] benzamide/benzene sulfonamides (11a–n) were synthesized in fair to good yields via sodium borohydride reduction of the corresponding substituted N-(benzoylimino)-4-(5-methyl/5-phenyl-1,3,4-oxadiazol-2yl) pyridinium ylide (10a–n) in absolute ethanol. PMID:20526413

  14. 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

  15. Synthesis and antiviral activity of novel acyclic nucleoside analogues of 5-(1-azido-2-haloethyl)uracils.

    PubMed

    Kumar, R; Sharma, N; Nath, M; Saffran, H A; Tyrrell, D L

    2001-11-22

    We present the discovery of a novel category of 5-substituted acyclic pyrimidine nucleosides as potent antiviral agents. A series of 1-[(2-hydroxyethoxy)methyl] (5-7), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl] (8-10), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl] (11-13) derivatives of 5-(1-azido-2-haloethyl)uracil were synthesized and evaluated for their biological activity in cell culture. 1-[4-Hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azido-2-chloroethyl)uracil (12) was the most effective antiviral agent in the in vitro assays against DHBV (EC(50) = 0.31-1.55 microM) and HCMV (EC(50) = 3.1 microM). None of the compounds investigated showed any detectable toxicity to several stationary and proliferating host cells. PMID:11708924

  16. 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

  17. Formation of Uracil from the Ultraviolet Photo-Irradiation of Pyrimidine in Pure H2O Ices

    NASA Astrophysics Data System (ADS)

    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.

  18. Excess Electron Attachment Induces Barrier-Free Proton Transfer in Anionic Complexes of Thymine and Uracil with Formic Acid

    SciTech Connect

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

    2004-06-03

    The anionic complexes of formic acid with uracil and thymine reveal broad features in photoelectron spectroscopy (PES) experiments with maxima at 1.7 and 1.1 eV, respectively. The results of quantum chemical calculations suggest that electron vertical detachment energies (VDE) of 1.6-1.9 eV correspond to anionic structures in which a proton has been transferred from the carboxylic group of the formic acid to the O8 atom of uracil or thymine. Smaller values of VDE (0.8 to 1.3 eV) correspond to chemically untransformed complexes, in which anionic uracil or thymine interacts through two hydrogen bonds with the carboxylic group of the intact formic acid. The recorded spectra and the results of quantum chemical calculations suggest that both nucleic acid bases undergo barrier-free proton transfer in anionic complexes with formic acid. The difference in experimental spectra of UF- and TF- provides an indication that the methyl group of thymine could make a difference in the intermolecular proton transfer.

  19. 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

  20. 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

  1. Solvation free energies of molecules. The most stable anionic tautomers of uracil

    PubMed Central

    Haranczyk, Maciej; Gutowski, Maciej; Warshel, Arieh

    2008-01-01

    Anionic states of nucleic acid bases are suspected to play a role in the radiation damage processes of DNA. Our recent studies suggested that the excess electron attachment to the nucleic acid bases can stabilize some rare tautomers, i.e. imine-enamine tautomers and other tautomers with a proton being transferred form nitrogen sites to carbon sites (with respect to the canonical tautomer). So far, these new anionic tautomers have been characterized by the gas phase electronic structure calculations and photoelectron spectroscopy experiments. In the current contribution we explore the effect of water solvation on the stability of the new anionic tautomers of uracil. The accurate free energies of solvation are calculated in a two step approach. The major contribution was calculated using the classical free energy perturbation adiabatic charging approach, where it is assumed that the solvated molecule has the charge distribution given by the polarizable continuum model. In the second step the free energy of solvation is refined by taking into account the real, average solvent charge distribution. This is done using our accelerated QM/MM simulations, where the QM energy of the solute is calculated in the mean potential averaged over many MD steps. We found that in water solution three of the recently identified anionic tautomers are 6.5 – 3.6 kcal/mol more stable than the anion of the canonical tautomer. PMID:18654684

  2. Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation.

    PubMed

    Xue, Jian-Huang; Xu, Gui-Fang; Gu, Tian-Peng; Chen, Guo-Dong; Han, Bin-Bin; Xu, Zhi-Mei; Bjørås, Magnar; Krokan, Hans E; Xu, Guo-Liang; Du, Ya-Rui

    2016-01-01

    In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. To search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation. PMID:26620559

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-01

    The study of interactions between metal ions and nucleobases, nucleosides, nucleotides, or nucleic acids has become an active research area in chemical, biological, and therapeutic fields. In this respect, the coordination behavior of nucleobase derivatives to transition metals was studied in order to get a better understanding about DNA-metal interactions in in vitro and in vivo systems. Two nucleobase derivatives, 3-benzoyl-1-[3-(thymine-1-yl)propamido]thiourea and 3-benzoyl-1-[3-(uracil-1-yl)propamido]thiourea, were synthesized and their dissociation constants were determined at different temperatures and 0.3 ionic strength. Potentiometric studies were carried out on the interaction of the derivatives towards some divalent metals in 50% v/v ethanol-water containing 0.3 mol.dm(-3) KCl, at five different temperatures. The formation constants of the metal complexes for both ligands follow the order: Cu(2+) > Ni(2+) > Co(2+) > Zn(2+) > Pb(2+) > Cd(2+) > Mn(2+). The thermodynamic parameters were estimated; the complexation process has been found to be spontaneous, exothermic, and entropically favorable. PMID:27049340

  5. Valence and diffuse-bound anions of noble-gas complexes with uracil

    NASA Astrophysics Data System (ADS)

    Streit, Lívia; Dolgounitcheva, O.; Zakrzewski, V. G.; Ortiz, J. V.

    2012-11-01

    Valence-bound (VB) and diffuse-bound (DB) anions of noble-gas (Ar, Kr, and Xe) complexes with uracil have been studied with ab initio methods. MP2 optimizations revealed minima corresponding to anions of both kinds in each case. Coupled-cluster singles and doubles with perturbative triples, CCSD(T), and electron propagator single-point calculations were performed in order to assess vertical and adiabatic electron detachment energies of these complexes. Ab initio electron propagator calculations employed the outer valence Green's function and partial third-order approximations, and the algebraic diagrammatic construction in third order. Basis set effects have been systematically examined. DB anions of all three complexes were adiabatically bound, with calculated adiabatic electron attachment energies below 0.06 eV. Corresponding vertical electron detachment energies were below 0.1 eV. As to VB anions, only the Xe complex had a positive adiabatic electron detachment energy, of 0.01 eV, with a corresponding vertical electron detachment energy of 0.6 eV. These computational findings are consistent with the interpretation of results previously obtained experimentally by Hendricks et al.

  6. On the gas phase fragmentation of protonated uracil: a statistical perspective.

    PubMed

    Rossich Molina, Estefanía; Salpin, Jean-Yves; Spezia, Riccardo; Martínez-Núñez, Emilio

    2016-06-01

    The potential energy surface of protonated uracil has been explored by an automated transition state search procedure, resulting in the finding of 1398 stationary points and 751 reactive channels, which can be categorized into isomerizations between pairs of isomers, unimolecular fragmentations and bimolecular reactions. The use of statistical Rice-Ramsperger-Kassel-Marcus (RRKM) theory and Kinetic Monte Carlo (KMC) simulations allowed us to determine the relative abundances of each fragmentation channel as a function of the ion's internal energy. The KMC/RRKM product abundances are compared with novel mass spectrometry (MS) experiments in the collision energy range 1-6 eV. To facilitate the comparison between theory and experiments, further dynamics simulations are carried out to determine the fraction of collision energy converted into the ion's internal energy. The KMC simulations show that the major fragmentation channels are isocyanic acid and ammonia losses, in good agreement with experiments. The third predominant channel is water loss according to both theory and experiments, although the abundance obtained in the KMC simulations is very low, suggesting that non-statistical dynamics might play an important role in this channel. Isocyanic acid (HNCOH(+)) is also an important product in the KMC simulations, although its abundance is only significant at internal energies not accessible in the MS experiments. PMID:27194127

  7. [Clinical study of Peptide-cocktail vaccination with tegafur-uracil/leucovorin for advanced colorectal cancer].

    PubMed

    Sugiura, Fumiaki; Inoue, Keisuke; Okuno, Kiyotaka; Sukegawa, Yasushi

    2012-11-01

    cDNA microarray technology coupled with laser microdissection has been used to identify human leukocyte antigen(HLA)-A24-restricted epitope peptides as potential targets for cancer vaccination in colorectal cancer patients. These antigenic peptides were derived from 2 different testis cancer antigens, ring finger protein 43(RNF43) and translocase of outer mitochondrial membrane 34(TOMM34). We conducted a clinical trial of vaccines against colorectal cancer specific peptides(RNF43 and TOMM34) with tegafur-uracil/Leucovorin( UFT/LV) for the treatment of advanced or recurrent colorectal cancer. The vaccinations were well tolerated without any adverse events. The highest long-term survival was observed in the group showing cytolytic T-lymphocyte (CTL) responses against both RNF43 and TOMM34, followed by the group showing CTL responses against only RNF43 or only TOMM34. A new study has been planned in order to obtain more immunological responses, and we have started a clinical trial of vaccines against multiple peptides[RNF43, TOMM34, forkhead box protein M1(FOXM1), maternal embryonic leucine zipper kinase(MELK), holliday junction recognition protein(HJURP), vascular endothelial growth factor receptor (VEGFR)1, and VEGFR2]by using UFT/LV for the treatment of advanced or recurrent colorectal cancer. PMID:23267878

  8. [Drug-induced liver injury associated with uracil/tegafur + folinate therapy].

    PubMed

    Kawasaki, Atsushi; Mimatsu, Kenji; Kuboi, Youichi; Kano, Hisao; Oida, Takatsugu; Amano, Sadao

    2009-11-01

    We report a case of drug-induced liver injury associated with uracil/tegafur(UFT) + folinate (Uzel) therapy. A 73-year-old woman had undergone right hemicolectomy with ascending colon cancer in April 2008. Postoperative diagnosis was tub2, pSS, pN0, fStage II. We administered UFT + Uzel therapy(UFT 500 mg/day, Uzel 75 mg/day)as adjuvant chemotherapy after operation for 6 weeks. Two weeks after the first drug administration, she visited our hospital complaining of anorexia and general malaise. Laboratory data showed elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). She was admitted to the hospital with a diagnosis of grade 3 liver dysfunction. We supposed the cause of this liver dysfunction to be a drug-induced liver injury associated with UFT + Uzel, and administered drugs to the patient for liver protection. With time, the symptoms and elevation of AST, ALT and ALP recovered to the normal level. This case was diagnosed as drug-induced allergic liver injury by UFT, because the result of the drug lymphocyte stimulate test (DLST) was positive. PMID:19920397

  9. Actions of human DNA glycosylases on uracil-containing DNA, methylated DNA and their reconstituted chromatins.

    PubMed

    Ishiwata, K; Oikawa, A

    1979-07-26

    Extracts of human lymphoblastoid cells catalyzed complete release of uracil (Ura) from PBS1 DNA, which contains Ura instead of thymine as a normal component (Ura-DNA), and 3-methyladenine (3-MeAde) from DNA methylated with methyl methanesulfonate (Me-DNA). These two activities, Ura-DNA glycosylase and 3-MeAde-DNA glycosylase, differed in heat stability. Cell extracts released Ura more rapidly and 3-MeAde more slowly from alkali-denatured preparations of Ura- and Me-DNA, respectively, than from native DNA's. On incubation with reconstituted chromatins, prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein by salt gradient dialysis, cell extracts released all the Ura but only about half of the 3-MeAde residues, although both these chromatins were degraded by micrococcal nuclease until about half of the nucleotides became acid soluble. The activities of Ura-DNA and 3-MeAde-DNA glycosylase of xeroderma pigmentosum cells were similar to those of normal cells. PMID:465495

  10. 5-Methylation of Cytosine in CG:CG Base-Pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir; Olson, Wilma; Li, Yun

    2014-03-01

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

  11. 5-Methylation of Cytosine in CG:CG Base-pair Steps: A Physicochemical Mechanism for the Epigenetic Control of DNA Nanomechanics

    PubMed Central

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

    2014-01-01

    Van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile ‘opening’ mode and two shear ‘sliding’ and ‘tearing’ modes in the orthogonal plane. The stacking interactions of the methyl groups globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. Additionally, the indirect effects of the methyl groups on possible base-pair steps neighboring CG:CG are observed to be of comparable importance to their direct effects on CG:CG. The results have implications for the epigenetic control of DNA mechanics. PMID:24313757

  12. QSAR study of some 5-methyl/trifluoromethoxy- 1H-indole-2,3-dione-3-thiosemicarbazone derivatives as anti-tubercular agents

    PubMed Central

    Shahlaei, M.; Fassihi, A.; Nezami, A.

    2009-01-01

    In the present study, quantitative relationships between molecular structure and anti-tubercular activity of some 5-methyl/trifluoromethoxy-1H-indole-2,3-dione-3-thiosemicarbazone derivatives were discovered. The detailed application of an efficient linear method and principal component regression (PCR) for the evaluation of quantitative structure activity relationships of the studied compounds is demonstrated. Components produced by principal component analysis were used as the input for a linear model development. Results indicate a linear relationship between the principal components obtained from molecular descriptors and the inhibitory activity of this set of molecules. The maximum variance in the activity of the molecules in PCR method was 73%. The performance of the developed model was tested by several validation methods. PMID:21589807

  13. 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.

  14. Synthesis, characterization and intramolecular proton transfer of 3,3";-dihydroxy-4,4";-[5-methyl-1,3-phenylenebis(nitrilomethylidyne)]-bis-phenol

    NASA Astrophysics Data System (ADS)

    Eshtiagh-Hosseini, Hossein; Beyramabadi, S. Ali; Morsali, Ali; Mirzaei, Masoud; Chegini, Hamed; Elahi, Morteza; Naseri, Mohammad Ali

    2014-08-01

    A newly synthesized Schiff base, 3,3";-dihydroxy-4,4";-[5-methyl-1,3-phenylenebis(nitrilomethylidyne)]-bis-phenol, was characterized experimentally. Its geometries optimization, tautomerization, assignment of the IR bands and NMR chemical shifts were calculated by using density functional theory (DFT) method. In addition, the atoms in molecules (AIM) analysis was employed for investigation of its tautomerization. Four possible tautomers of the investigated Schiff base were optimized in both of the gas and solution phases. The Schiff base has no planar structure, but each of the benzene rings is in a separate plane. In the most stable tautomer, the phenolic protons of the two sbnd OH groups are engaged in the intramolecular-hydrogen bond with the azomethine nitrogens. Good consistency between the theoretical and experimental results confirms validity of the optimized geometry. Also, kinetics and mechanism of the intramolecular-proton transfer of the studied Schiff base was demonstrated theoretically.

  15. Synthesis and spin-trapping properties of a trifluoromethyl analogue of DMPO: 5-methyl-5-trifluoromethyl-1-pyrroline N-oxide (5-TFDMPO).

    PubMed

    Karoui, Hakim; Nsanzumuhire, Céline; Le Moigne, François; Hardy, Micael; Siri, Didier; Derat, Etienne; Rockenbauer, Antal; Ouari, Olivier; Tordo, Paul

    2014-04-01

    The 5-diethoxyphosphonyl-5-methyl-1-pyrroline N-oxide superoxide spin adduct (DEPMPO-OOH) is much more persistent (about 15 times) than the 5,5-dimethyl-1-pyrroline N-oxide superoxide spin adduct (DMPO-OOH). The diethoxyphosphonyl group is bulkier than the methyl group and its electron-withdrawing effect is much stronger. These two factors could play a role in explaining the different half-lifetimes of DMPO-OOH and DEPMPO-OOH. The trifluoromethyl and the diethoxyphosphonyl groups show similar electron-withdrawing effects but have different sizes. We have thus synthesized and studied 5-methyl-5-trifluoromethyl-1-pyrroline N-oxide (5-TFDMPO), a new trifluoromethyl analogue of DMPO, to compare its spin-trapping performance with those of DMPO and DEPMPO. 5-TFDMPO was prepared in a five-step sequence by means of the Zn/AcOH reductive cyclization of 5,5,5-trifluoro-4-methyl-4-nitropentanal, and the geometry of the molecule was estimated by using DFT calculations. The spin-trapping properties were investigated both in toluene and in aqueous buffer solutions for oxygen-, sulfur-, and carbon-centered radicals. All the spin adducts exhibit slightly different fluorine hyperfine coupling constants, thereby suggesting a hindered rotation of the trifluoromethyl group, which was confirmed by variable-temperature EPR studies and DFT calculations. In phosphate buffer at pH 7.4, the half-life of 5-TFDMPOOOH is about three times shorter than for DEPMPO-OOH and five times longer than for DMPO-OOH. Our results suggest that the stabilization of the superoxide adducts comes from a delicate balance between steric, electronic, and hydrogen-bonding effects that involve the β group, the hydroperoxyl moiety, and the nitroxide. PMID:24590621

  16. 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

  17. [A CASE OF ADVANCED BLADDER NEUROENDOCRINE CARCINOMA (SMALL CELL CARCINOMA) SIGNIFICANTLY IMPROVED BY LOW DOSE OF ORAL TEGAFUR-URACIL].

    PubMed

    Nomi, Hayahito; Takahara, Kiyoshi; Minami, Koichiro; Maenosono, Ryoichi; Matsunaga, Tomohisa; Yoshikawa, Yuki; Tsujino, Takuya; Hirano, Hajime; Inamoto, Teruo; Yamamoto, Ikuhisa; Tsuji, Motomu; Kiyama, Satoshi; Azuma, Haruhito

    2015-10-01

    A 81-old-woman underwent a transurethral resection of bladder tumor (TURBT) at a nearby hospital in April 2011. The diagnosis was invasive urothelial carcinoma, G3 with a component of bladder small cell carcinoma, T1 or more. She was recommended to visit our hospital for combined modality therapy of bladder cancer, but she refused the treatment for over one year. In May 2012, she came to our hospital with the chief complaint of pain at urination. Cystoscopy revealed non-papillary sessile tumor in the top of the bladder, and CT scan demonstrated the presence of the right obturator lymph nodes swollen up to 1.2 cm in size. The second TURBT was performed and the diagnosis was bladder small cell carcinoma (pT3N2M0) according to urothelial cancer guidelines of the Japanese Urological Association (JUA). Because she strongly refused hospitalization anymore, we started daily oral intake of low dose Tegafur-Uracil (100 mg) for the treatment. After one month, the serum Neuron-Specific Enolase (NSE; tumor maker of small cell cancer) level was elevated to 27.6 ng/ml and the right obturator lymph node was enlarged up to 1.9 cm. Therefore, the Trgafur-Uracil dose was increased to 200 mg daily. After then, the serum NSE level was decreased to 15.5 ng/ml following reduction in size of the obturator lymph nodes with partial response in December 2013. After two years of follow-up period, her regular urine test showed normal findings, and no apparent recurrence was detected on urinary bladder with MRI and Cystoscopy. This is a case of advanced bladder small cell carcinoma significantly improved by oral administration of Tegafur-Uracil 200 mg/day for over 2 years. PMID:26717786

  18. [A case of sigmoid colon cancer in a patient undergoing hemodialysis treated by tegafur/uracil/folinate].

    PubMed

    Miyazawa, Tomonori; Chida, Tadasu; Hasegawa, Shigeru; Iwafuchi, Yo-Ichi; Kamimura, Akira; Watanabe, Gen

    2008-01-01

    The patient was a 59-year-old man who had undergone hemodialysis because of chronic renal failure. The patient was diagnosed as colonic perforation due to stricture of sigmoid colon cancer, and sigmoidectomy was performed. Since colonic perforation and imperfect lymphnode dissection might increase the risk of recurrence, the patient underwent 6 courses of tegafur/uracil/folinate(UFT/LV)chemotherapy. No adverse events occurred in the course of chemotherapy. UFT/LV chemotherapy may be administered safely with careful management even for a patient on hemodialysis. PMID:18195547

  19. 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. PMID:26172334

  20. 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

  1. 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.

  2. 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

  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. PMID:26763002

  4. 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

  5. A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase

    SciTech Connect

    Meyer-Siegler, K.; Mauro, D.J.; Seal, G.; Wurzer, J.; DeRiel, J.K.; Sirover, M.A. )

    1991-10-01

    The authors have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a {beta}-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated M{sub r} 36,050 and pI 8.7, corresponding to the M{sub r} 37,000 and pI 8.1 of purified human placental UDG. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 378-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated form it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.

  6. 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.

  7. 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

  8. Electrostatic interactions play an essential role in DNA repair and cold-adaptation of uracil DNA glycosylase.

    PubMed

    Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

    2008-03-01

    Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k(cat) and reduced K(m) relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K(m)) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation. PMID:18196298

  9. Protein p56 from the Bacillus subtilis phage ϕ29 inhibits DNA-binding ability of uracil-DNA glycosylase

    PubMed Central

    Serrano-Heras, Gemma; Ruiz-Masó, José A.; del Solar, Gloria; Espinosa, Manuel; Bravo, Alicia; Salas, Margarita

    2007-01-01

    Protein p56 (56 amino acids) from the Bacillus subtilis phage ϕ29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of β-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry. PMID:17698500

  10. 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. PMID:26743004

  11. 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

  12. 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)

    NASA Astrophysics Data System (ADS)

    Singh, J. S.

    2015-02-01

    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

  13. 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

  14. 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

  15. Neuroprotective Effects of the Glutamate Transporter Activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153) following Traumatic Brain Injury in the Adult Rat.

    PubMed

    Karklin Fontana, Andréia Cristina; Fox, Douglas P; Zoubroulis, Argie; Valente Mortensen, Ole; Raghupathi, Ramesh

    2016-06-01

    Traumatic brain injury (TBI) in humans and in animals leads to an acute and sustained increase in tissue glutamate concentrations within the brain, triggering glutamate-mediated excitotoxicity. Excitatory amino acid transporters (EAATs) are responsible for maintaining extracellular central nervous system glutamate concentrations below neurotoxic levels. Our results demonstrate that as early as 5 min and up to 2 h following brain trauma in brain-injured rats, the activity (Vmax) of EAAT2 in the cortex and the hippocampus was significantly decreased, compared with sham-injured animals. The affinity for glutamate (KM) and the expression of glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST) were not altered by the injury. Administration of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a GLT-1 activator, beginning immediately after injury and continuing for 24 h, significantly decreased neurodegeneration, loss of microtubule-associated protein 2 and NeuN (+) immunoreactivities, and attenuated calpain activation in both the cortex and the hippocampus at 24 h after the injury; the reduction in neurodegeneration remained evident up to 14 days post-injury. In synaptosomal uptake assays, MS-153 up-regulated GLT-1 activity in the naïve rat brain but did not reverse the reduced activity of GLT-1 in traumatically-injured brains. This study demonstrates that administration of MS-153 in the acute post-traumatic period provides acute and long-term neuroprotection for TBI and suggests that the neuroprotective effects of MS-153 are related to mechanisms other than GLT-1 activation, such as the inhibition of voltage-gated calcium channels. PMID:26200170

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

    PubMed

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

    2016-02-01

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

  17. 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

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

    PubMed

    Cancilla, Devon A; Baird, J Christopher; Geis, Steven 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. PMID:12503756

  19. Associations of urinary 5-methyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine with phthalate exposure and semen quality in 562 Chinese adult men.

    PubMed

    Pan, Yitao; Jing, Jun; Yeung, Leo W Y; Sheng, Nan; Zhang, Hongxia; Yao, Bing; Dai, Jiayin

    2016-09-01

    5-methyl-2'-deoxycytidine (5mdC) and 5-hydroxymethyl-2'-deoxycytidine (5hmdC), products of DNA methylation and hydroxymethylation processes, have been detected previously in human urine, but their associations with environmental chemicals or healthy outcomes are unclear. The present investigation explored the associations between urinary 5mdC and 5hmdC with phthalate exposure and semen quality. We assessed semen parameters including sperm concentration, motility, and morphology, before measuring urinary 5mdC, 5hmdC and 13 phthalate metabolites among 562 subfertile men from Nanjing, China. Urinary 5mdC and 5hmdC were positively associated with the levels of low molecular weight phthalate metabolites (Low-MWP), high molecular weight phthalate metabolites (High-MWP), and the sum of all phthalate metabolites (ΣPAEs), respectively. Urinary 5mdC was associated with below-reference sperm concentration (odds ratios for increasing quartiles=1.0, 2.2, 3.0, 2.0; p for trend =0.02), sperm motility (1.0, 1.1, 1.9, 1.3; p for trend =0.05), and sperm morphology (1.0, 1.4, 2.3, 1.5; p for trend =0.05). Sperm concentration was associated with the highest quartile of urinary 5hmdC [odds ratio=1.9 (95% CI: 1.1, 3.6)]. Our findings showed significant associations between urinary 5mdC and 5hmdC with phthalate metabolites and semen parameters, which suggested urinary 5mdC and 5hmdC may be promising biomarkers in future epidemiological studies. PMID:27346742

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

    PubMed

    Habtemariam, Solomon; Varghese, George K

    2015-01-01

    Premna serratifolia Linn. (syn: . P. corymbosa (Burm. f.) Merr., P. integrifolia L. and P. obtusifolia R. Br.) is a member of the Verbenaceae family that is extensively used in the Ayurvedic system of medicine in India. As part of our continuous pharmacological and phytochemical studies on medicinal plants, we have screened the methanolic extracts of leaves, root bark (RB) and root wood of P. serratifolia for cytotoxic activity against two cancer cell lines: SHSY-5Y neuroblastoma and B16 melanoma cells. The RB extract that showed promising activity was fractionated using solvents of increasing polarity followed by a combination of Sephadex LH-20 column and Combiflash chromatography as well as HPLC to afford the active principle. Comprehensive spectroscopic analysis including 1D and 2D NMR (COSY, HMQC, HMBC, NOESY) and MS analysis revealed the identity of the isolated compound as 11,12,16-trihydroxy-2-oxo-5-methyl-10-demethyl-abieta-1[10],6,8,11,13-pentene that appears to be a novel compound based on a new diterpene skeleton. The cytotoxic activity of the isolated compound was 21 and 23 times higher than the crude extract against the SHSY-5Y and B16 cells, respectively. The novel compound also possesses in vitro antioxidant effects as evidenced by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging effect where an IC50 value of 20.4 ± 1.3 μM was obtained. In comparison, the positive control, caffeic acid, showed an IC50 value of 14.4 ± 1.6 μM. PMID:25250850

  1. Hydrodynamic and pharmacological characterization of putative alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors solubilized from pig brain.

    PubMed Central

    Wu, T Y; Chang, Y C

    1994-01-01

    L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate >> N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins. PMID:7516151

  2. Hydrodynamic and pharmacological characterization of putative alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors solubilized from pig brain.

    PubMed

    Wu, T Y; Chang, Y C

    1994-06-01

    L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate > N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins. PMID:7516151

  3. 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. PMID:25541404

  4. 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.

  5. Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

    PubMed Central

    Minkah, Nana; Macaluso, Marc; Oldenburg, Darby G.; Paden, Clinton R.; White, Douglas W.; McBride, Kevin M.

    2015-01-01

    ABSTRACT Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNG (ΔUNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68ΔUNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs. IMPORTANCE Herpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the

  6. 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. PMID:24793482

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

    PubMed Central

    Yousif, Ashraf S.; Stanlie, Andre; Begum, Nasim A.

    2014-01-01

    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. PMID:24994819

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

    PubMed

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

    2014-10-01

    Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR. PMID:24994819

  9. The discovery of long-acting saligenin β₂ adrenergic receptor agonists incorporating hydantoin or uracil rings.

    PubMed

    Procopiou, Panayiotis A; Barrett, Victoria J; Bevan, Nicola J; Butchers, Peter R; Conroy, Richard; Emmons, Amanda; Ford, Alison J; Jeulin, Séverine; Looker, Brian E; Lunniss, Gillian E; Morrison, Valerie S; Mutch, Peter J; Perciaccante, Rossana; Ruston, Mark; Smith, Claire E; Somers, Graham

    2011-07-15

    A series of novel, potent and selective human β(2) adrenoceptor agonists incorporating a hydantoin or a uracil ring on the right-hand side phenyl ring of (R)-salmeterol is presented. Hydantoin 12a had long duration of action in vitro on guinea pig trachea, and 12h in guinea pigs in vivo at its EC(90) 25 μM. It had lower oral absorption than salmeterol in rats, and lower bioavailability than salmeterol in vivo in both rats and dogs (2% and 5%, respectively). An improved method for measuring the absorbed fraction of analogues dosed to rats, which considers the glucuronidated fraction is presented. Compound 12a was metabolised in human liver microsomes and hepatocytes to the active hydantoic acid 12m. PMID:21696967

  10. 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

  11. [Clinical trial of a seven-peptide vaccine and tegafur-uracil/leucovorin as combination therapy for advanced colorectal cancer].

    PubMed

    Inoue, Keisuke; Sugiura, Fumiaki; Kogita, Akihiro; Yoshioka, Yasumasa; Sukegawa, Yasushi; Hida, Jinichi; Okuno, Kiyotaka

    2014-10-01

    We conducted a clinical trial of a seven-peptide vaccine in combination with tegafur-uracil/Leucovorin for advanced colorectal cancer. These antigenic peptides were derived from 5 proteins identified as cancer-testis antigens(ring finger protein 43 [RNF43], translocase of outer mitochondrial membrane 34[TOMM34], maternal embryonic leucine zipper kinase[MELK], forkhead box M1[FOXM1], and holliday junction recognition protein[HJURP])and 2 vascular endothelial growth factor receptors(VEGFR1 and VEGFR2). Thirty patients with advanced colorectal cancer were enrolled. We found that 25 patients had Grade 1 injection-site redness/induration and 1 patient had Grade 3 anaphylaxis. Tumor imaging revealed that 3 patients had a partial response (PR), 15 had stable disease(SD)and 12 had progressive disease(PD). This trial showed that treatment with the seven-peptide vaccine and UFT/LV was well tolerated and feasible for advanced colorectal cancer. PMID:25335716

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

    PubMed

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

    2016-07-01

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

  13. 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

  14. 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

  15. 5-Methyl-2-[3-(4-phenylthiazol-2-yl)triazenyl]benzenesulfonic acid as a chromogenic reagent of N-cetylpyridinium chloride Synthesis, mechanism and analytical application.

    PubMed

    He, Xiao-Ling; Wang, Yong-Qiu; Ling, Ke-Qing

    2008-01-15

    Despite that triazene reagents have been widely used for spectrophotometric determinations of cationic surfactants, the mechanism underlying such applications has yet to be studied. We report the synthesis of a new triazene reagent 5-methyl-2-[3-(4-phenylthiazol-2-yl)triazenyl]benzenesulfonic acid (MPTTBSA) and its interaction with N-cetylpyridinium chloride (CPC). The reagent was synthesized by coupling 4-methyl-2-sulfobenzenediazonium salt with 2-amino-4-phenylthiazole. Spectral evidence suggests that the neutral reagent (H(2)R) exists in a triazenium sulfonate zwitterion form. Two ionizations were detected at weak acidic (H(2)R/HR(-), pK(a1) 2.71+/-0.20) and alkaline pH (HR(-)/R(2-), pK(a2) ca. 13.5), respectively. In the presence of 3.5 equivalents of CPC, a 2.71 unit decrease in pK(a1) and a 3.0 unit decrease in pK(a2) were observed. While the optical properties of H(2)R are essentially unaffected, CPC causes a 53nm red shift and a 31nm red shift in maximum absorption wavelengths (lambda(max)), and a 24% increase and a 29% increase in extinction coefficients (epsilon) of HR(-) and R(2-), respectively. These data suggest that CPC forms ion associates with HR(-) and R(2-), but its interaction with H(2)R is weak. Associations of CPC with HR(-) and R(2-) both follow a 3:1 stoichiometry, and the apparent stability constants of the two associates were estimated as 6.02x10(18) and 2.42x10(22)M(-3), respectively. Consistent with their high stability constants, the two ion associates did not show any changes in optical properties under submicellar and micellar conditions. The strict 3:1 association stoichiometry was interpreted in terms of electrostatic-induced, topology-defined pi-stacking and hydrophobic interactions, which not only change the optical properties of the reagent anions, but also provide the driving force to shift the two ionization equilibria to the right and cause the decreases in pK(a). Compared to the first ionization, perturbation of the second

  16. 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

  17. Exploring the role of the 5-substituent for the intrinsic fluorescence of 5-aryl and 5-heteroaryl uracil nucleotides: a systematic study.

    PubMed

    Pesnot, Thomas; Tedaldi, Lauren M; Jambrina, Pablo G; Rosta, Edina; Wagner, Gerd K

    2013-10-01

    Derivatives of UMP (uridine monophosphate) with a fluorogenic substituent in position 5 represent a small but unique class of fluorophores, which has found important applications in chemical biology and biomolecular chemistry. In this study, we have synthesised a series of derivatives of the uracil nucleotides UMP, UDP and UTP with different aromatic and heteroaromatic substituents in position 5, in order to systematically investigate the influence of the 5-substituent on fluorescence emission. We have determined relevant photophysical parameters for all derivatives in this series, including quantum yields for the best fluorophores. The strongest fluorescence emission was observed with a 5-formylthien-2-yl substituent in position 5 of the uracil base, while the corresponding 3-formylthien-2-yl-substituted regioisomer was significantly less fluorescent. The 5-(5-formylthien-2-yl) uracil fluorophore was studied further in solvents of different polarity and proticity. In conjunction with results from a conformational analysis based on NMR data and computational experiments, these findings provide insights into the steric and electronic factors that govern fluorescence emission in this class of fluorophores. In particular, they highlight the interplay between fluorescence emission and conformation in this series. Finally, we carried out ligand-binding experiments with the 5-(5-formylthien-2-yl) uracil fluorophore and a UDP-sugar-dependent glycosyltransferase, demonstrating its utility for biological applications. The results from our photophysical and biological studies suggest, for the first time, a structural explanation for the fluorescence quenching effect that is observed upon binding of these fluorophores to a target protein. PMID:23945704

  18. 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

  19. 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

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

    PubMed

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

    2016-01-01

    5-[3-(2-Aminopyrimidin-4-yl)aminopropyn-1-yl]uracil (Ura(Pyr)) was designed as a new nucleobase to recognize Ade-Thy base pair in double-stranded DNA. We successfully synthesized the dexoynucleoside phosphoramidite having Ura(Pyr) and incorporated it into triplex forming oligonucleotides (TFOs). Melting temperature analysis revealed that introduction of Ura(Pyr) into TFOs could effectively stabilize their triplex structures without loss of base recognition capabilities. PMID:26602276

  1. Mechanism for the abiotic synthesis of uracil via UV-induced oxidation of pyrimidine in pure H2O ices under astrophysical conditions

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

    The UV photoirradiation of pyrimidine in pure H2O ices has been explored using second-order Møller-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 H2O: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.

  2. 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.

  3. 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.

  4. 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

  5. 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. PMID:26980279

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

    PubMed Central

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

    2016-01-01

    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. PMID:26980279

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

    PubMed Central

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

    2014-01-01

    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. PMID:24591630

  8. Effect of 5-alkyl substitution of uracil on the thermal stability of poly [d(A-r5U)] copolymers.

    PubMed Central

    Sági, J; Brahms, S; Brahms, J; Otvös, L

    1979-01-01

    The thermal transition of poly[d(A-r5U)] polydeoxynucleotides (where r was a hydrogen atom, or a methyl, ethyl, n-propyl, n-butyl or n-pentyl group) was studied by measuring the derivative melting profiles of the polymers in the range of 0.01--0.36 M K+, at pH 6.8. According to the Tm values, polydeoxynucleotide analogues show lower thermal stability than poly[d(A-T)] at any counterion concentration applied. At a given salt concentration, Tm of the alkyl analogues decreased as the number of carbon atoms (n) in the r substituent of poly[d(A-r5U)] increased. 1/Tm plotted against against 1/n yielded a linear relationship. Cooperativity of the melting of all poly[d(A--U)] analogues decreased with the increase of salt concentration in the solution. This change depended again on 5-substitution of the uracil moiety of poly[d(A-U)]. Smallest decrease was observed in the case of poly[d(A--U)] whereas largest decrease was shown by poly[d(A-pe5U)] (pe=pentyl group). PMID:461207

  9. 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

  10. 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.

  11. 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.

  12. 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. PMID:24591630

  13. [A case of stage IV colon cancer effectively treated with tegafur uracil/calcium folinate in a patient on hemodialysis].

    PubMed

    Okamoto, Ken; Kobayashi, Michiya; Maeda, Hiromichi; Takeshita, Atsunori

    2012-07-01

    A 61-year-old man on hemodialysis due to chronic renal failure caused by diabetes mellitus was diagnosed as having ascending colon cancer with multiple liver metastases. Colonoscopy revealed a Type 2 tumor located in the ascending colon. Abdominal CT showed hepatic tumors in S8 measuring 8×7 cm and in S6 measuring 5×4 cm, with a number of small tumors in the other sites. In order to prevent a hemorrhage from the colonic tumor, laparoscope-assisted right colectomy was performed. Seventeen days after the operation, oral administration of tegafur uracil(300mg/body/day)and calcium folinate(75mg/body/day)was initiated for the treatment of hepatic metastases. After three courses of treatment, size reduction of the hepatic metastases(the S8 4×3. 5 cm and the S6 2. 5×2. 5 cm)was obtained. Although nine months with seven courses of chemotherapy had passed without significant side effects, the size of hepatic metastases increased. Currently, therefore, CPT-11 and cetuximab are being administered as second-line treatment. PMID:22790059

  14. Thermodynamic Potential for the Abiotic Synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in Hydrothermal Systems

    NASA Astrophysics Data System (ADS)

    Larowe, Douglas E.; Regnier, Pierre

    2008-10-01

    The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine, and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics of biomolecule synthesis were computed using the concentrations of aqueous N2, CO, CO2 and H2 reported in the modern Rainbow hydrothermal system. The concentrations of precursor molecules that can be synthesized are strongly dependent on temperature with larger concentrations prevailing at lower temperatures. Similarly, the thermodynamic drive to synthesize nucleobases, ribose and deoxyribose varies considerably as a function of temperature: all of the biomolecules considered in this study are thermodynamically favored to be synthesized throughout the temperature range from 0°C to between 150°C and 250°C, depending on the biomolecule. Furthermore, activity diagrams have been generated to illustrate that activities in the range of 10-2- 10-6 for nucleobases, ribose and deoxyribose can be in equilibrium with a range of precursor molecule activities at 150°C and 500 bars. The results presented here support the notion that hydrothermal systems could have played a fundamental role in the origin of life, and can be used to plan and constrain experimental investigation of the abiotic synthesis of nucleic-acid related biomolecules.

  15. Circadian rhythm of dihydrouracil/uracil ratios in biological fluids: a potential biomarker for dihydropyrimidine dehydrogenase levels

    PubMed Central

    Jiang, Hao; Lu, Jing; Ji, Jiang

    2004-01-01

    In many cancer patients, 5-fluorouracil (5-FUra) treatment is toxic and even causes death. Nevertheless, all patients are subjected to a standard therapy regimen because there is no reliable way to identify beforehand those patients who are predisposed to 5-FUra-induced toxicity. In this study, we identified the dihydrouracil/uracil (UH2/Ura) ratio in plasma or urine as a potential biomarker reflecting the activity of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in 5-FUra metabolism. UH2/Ura ratios were measured by high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-MS/MS) in both healthy subjects (n=55) and in patients (n=20) diagnosed with grade I/II gestational trophoblastic tumours. In addition, rats (n=18) were used as an animal model to verify a correlation between UH2/Ura ratios and DPD levels in the liver. A significant circadian rhythm was observed in UH2/Ura ratios in healthy subjects, whereas a disrupted rhythm occurred in cancer patients who were continuously infused with a high dose of 5-FUra. In rats, UH2/Ura ratios, liver DPD levels and PBMC DPD levels showed a definite circadian rhythm. Significant linear correlations with liver DPD levels were demonstrated for plasma UH2/Ura ratios (r=0.883, P<0.01), urine UH2/Ura ratios (r=0.832, P<0.01) and PBMC DPD levels (r=0.859, P<0.01). The UH2/Ura ratio in biological fluid was significantly correlated with liver DPD levels; hence, this ratio could be a potential biomarker to identify patients with a deficiency in DPD. PMID:14744810

  16. 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. PMID:24874819

  17. Avirulent uracil auxotrophs based on disruption of orotidine-5'-monophosphate decarboxylase elicit protective immunity to Toxoplasma gondii.

    PubMed

    Fox, Barbara A; Bzik, David J

    2010-09-01

    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 DeltaOMPDC, DeltaUP, and DeltaOMPDC DeltaUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The DeltaUP knockout strain was replication competent and virulent. In contrast, the DeltaOMPDC and DeltaOMPDC DeltaUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the DeltaOMPDC strain but not the DeltaOMPDC DeltaUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the DeltaOMPDC and DeltaOMPDC DeltaUP knockout strains exhibited extreme attenuation in murine virulence (approximately 8 logs). Genetic complementation of the DeltaOMPDC 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 DeltaOMPDC strain or the DeltaOMPDC DeltaUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting DeltaOMPDC and DeltaOMPDC DeltaUP 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

  18. Concomitant Chemoradiotherapy Using Carboplatin, Tegafur-Uracil and Leucovorin for Stage III and IV Head-and-Neck Cancer: Results of GORTEC Phase II Study

    SciTech Connect

    Fesneau, Melanie; Pointreau, Yoann; Chapet, Sophie; Martin, Laurent; Pommier, Pascal; Alfonsi, Marc; Laguerre, Brigitte; Feham, Nasreddine; Berger, Christine; Garaud, Pascal; Calais, Gilles

    2010-01-15

    Purpose: Concomitant chemoradiotherapy is the standard treatment of locally advanced, nonresectable, head-and-neck squamous cell carcinoma. However, the optimal chemotherapy regimen is still controversial. The objective of this Phase II study was to evaluate the feasibility and efficacy of a concomitant treatment using tegafur-uracil, leucovorin, carboplatin, and radiotherapy. Methods and Materials: A total of 77 patients with head-and-neck squamous cell carcinoma Stage III and IVA were enrolled between October 2003 and July 2005. Of the 77 patients, 72 were eligible. They were treated with tegafur-uracil (300 mg/m{sup 2}/d) and leucovorin (75 mg/d) from Days 1 to 19 and from Days 29 to 47 and carboplatin (70 mg/m{sup 2} intravenously for 4 consecutive days), in three cycles every 21 days. Conventional radiotherapy was delivered to a total dose of 70 Gy in 35 fractions. Results: With a mean follow-up of 22.8 months, the 3-year locoregional control, overall survival and disease-free survival actuarial rate was 33.1%, 41.9%, and 27.2%, respectively. The compliance of the treatment was correct. The main acute toxicity was mucositis, with 62% Grade 3-4. Three patients (4.2%) died of acute toxicity. The incidence and severity of late toxicity was acceptable, with 32% Grade 3 and no Grade 4 toxicity. Conclusion: The protocol of concomitant chemoradiotherapy using tegafur-uracil, leucovorin, and carboplatin for locally advanced unresectable head-and-neck squamous cell carcinoma is feasible. The compliance was correct. The incidence and severity of the acute and late toxicities were acceptable, but not improved. The efficacy of this regimen seems equivalent to the main protocols of concurrent chemoradiotherapy. It represents a possible alternative for patients without an intravenous catheter.

  19. 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

  20. 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

  1. Tritritionikkomycin Z, (uracil-5- sup 3 H,pyridyl-2,4- sup 3 H sub 2 ): Radiolabeling of a potent inhibitor of fungal and insect chitin synthetase

    SciTech Connect

    Ando, Tetsu; Tecle, B.; Toia, R.F.; Casida, J.E. )

    1990-08-01

    Nikkomycin Z (NZ) (a potent fungicide, insecticide, miticide, and inhibitor of fungal and insect chitin synthetase) was converted to a mixture of specific mono-, di-, and tribromo derivatives (BrNZ, Br{sub 2}NZ, and Br{sub 3}NZ, respectively) on reaction with N-bromosuccinimide in N,N-dimethylformamide. Substitution by bromine occurred first at the 2-position of the 3-hydroxypyridyl moiety, second at the 5-position of the uracil moiety, and finally at the 4-position of the 3-hydroxypyridyl moiety as observed both for NZ and for mixtures of uridine and 3-hydroxy-6-methylpyridine as model compounds representative of the moieties of NZ. Following fractionation of the various bromonikkomycin derivatives by HPLC, their structures were assigned by NMR, MS, and UV analyses. Catalytic reductive debromination of Br{sub 3}NZ with tritium gas over palladium on carbon gave (uracil-5-{sup 3}H,pyridyl-2,4-{sup 3}H{sub 2})NZ. This material has sufficiently high specific activity ({approximately}60 Ci/mmol) and suitable positions of labeling to study its uptake, distribution, metabolism, and possible target site interactions in fungal and insect systems.

  2. The simian varicella virus uracil DNA glycosylase and dUTPase genes are expressed in vivo, but are non-essential for replication in cell culture

    PubMed Central

    Ward, Toby M.; Williams, Marshall V.; Traina-Dorge, Vicki; Gray, Wayne L.

    2012-01-01

    Neurotropic herpesviruses express viral deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and uracil DNA glycosylase (UDG) enzymes which may reduce uracil misincorporation into viral DNA, particularly in neurons of infected ganglia. The simian varicella virus (SVV) dUTPase (ORF 8) and UDG (ORF 59) share 37.7% and 53.9% amino acid identity, respectively, with varicella-zoster virus (VZV) homologs. Infectious SVV mutants defective in either dUTPase (SVV-dUTPase−) or UDG (SVV-UDG−) activity or both (SVV-dUTPase−/UDG−) were constructed using recA assisted endonuclease cleavage (RARE) and a cosmid recombination system. Loss of viral dUTPase and UDG enzymatic activity was confirmed in CV-1 cells infected with the SVV mutants. The SVV-dUTPase−, SVV-UDG−, and SVV-dUTPase−/UDG− mutants replicated as efficiently as wild-type SVV in cell culture. SVV dUTPase and UDG expression was detected in tissues derived from acutely infected animals, but not in tissues derived from latently infected animals. Further studies will evaluate the pathogenesis of SVV dUTPase and UDG mutants and their potential as varicella vaccines. PMID:19200445

  3. An efficient synthesis of 3-fluoro-5-thio-xylofuranosyl nucleosides of thymine, uracil, and 5-fluorouracil as potential antitumor or/and antiviral agents.

    PubMed

    Tsoukala, Evangelia; Agelis, George; Dolinsek, Jan; Botić, Tanja; Cencic, Avrelija; Komiotis, Dimitri

    2007-05-01

    1,2:5,6-Di-O-isopropylidene-alpha-D-glucofuranose by the sequence of mild oxidation, reduction, fluorination, periodate oxidation, borohydride reduction, and sulfonylation gave 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-toluenesulfonyl-alpha-D-xylofuranose (5). Tosylate 5 was converted to thioacetate derivative 6, which after acetolysis gave 1,2-di-O-acetyl-5-S-acetyl-3-deoxy-3-fluoro-5-thio-D-xylofuranose (7). Condensation of 7 with silylated thymine, uracil, and 5-fluorouracil afforded nucleosides 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) thymine (8), 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) uracil (9), and 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) 5-fluorouracil (10). Compounds 8, 9, and 10 are biologically active against rotavirus infection and the growth of tumor cells. PMID:17337193

  4. 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

  5. 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. PMID:27180296

  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. 5-methyl-tetrahydrofolate and the S-adenosylmethionine cycle in C57BL/6J mouse tissues: gender differences and effects of arylamine N-acetyltransferase-1 deletion.

    PubMed

    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 C(9)-N(10) 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

  8. 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

  9. Excision of 5-halogenated Uracils by Human Thymine DNA Glycosylase: Robust Activity for DNA Contexts other than CpG*

    PubMed Central

    Morgan, Michael T.; Bennett, Matthew T.; Drohat, Alexander C.

    2010-01-01

    Thymine DNA glycosylase (TDG) excises thymine from G·T mispairs, and removes a variety of damaged bases (X), with a preference for lesions in a CpG·X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG·FU, CpG·ClU, and CpG·BrU than for CpG·T. Here, we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (kmax) for G·X substrates depends significantly on the 5′ base pair. For example, kmax decreases by 6-, 11-, and 82-fold for TpG·ClU, GpG·ClU, and ApG·ClU, respectively, as compared to CpG·ClU. For the other G·X substrates, the 5′-neighbor effects have a similar trend but vary in magnitude. The activity for G·FU, G·ClU, and G·BrU, with any 5′-flanking pair, meets and in most cases significantly exceeds the CpG·T activity. Strikingly, hTDG activity is reduced 102.3- to 104.3-fold for A·X relative to G·X pairs, and reduced further for A·X pairs with a 5′ pair other than C·G. The effect of altering the 5′ pair and/or the opposing base (G·X versus A·X) is greater for substrates that are larger (BrdU, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by hTDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions, and in the cytotoxicity of FU, a widely used anticancer agent, are discussed. PMID:17602166

  10. [A case of pathological complete response after treatment with uracil/tegafur (UFT) and folinate (Leucovorin) for liver metastasis of colon cancer].

    PubMed

    Murayama, Ryouta; Nagata, Naoki; Honda, Shinsaku; Fujii, Mio; Sawatsubashi, Takahiro; Shinohara, Ikuyo; Kuroda, Hiroaki; Sako, Tatsuhiko; Sakamoto, Yoshihiko; Sanefuji, Hayato

    2011-12-01

    The patient is a 62-year-old female who underwent a right hemicolectomy for type-2 ascending colon cancer (moderately-differentiated adenocarcinoma, ss, n0, H0, P0, M0, stage II). Six months after the surgery, a solitary metastatic focus was expressed in the liver S3. Because schizophrenia was present concurrently, tegafur and uracil/folinate (UFT/Leucovorin) treatment was selected and performed for 3 months. Because the tumor shrank afterward, a partial hepatectomy was performed to obtain a curative resection. In a pathological examination of the resected focus, cicatricial/necrotic findings were observed, but no cancer cells were observed; hence, it was determined to be a pathological complete response (CR). In regard to chemotherapy for distant metastasis of colorectal cancer, many molecular-targeted agents are being introduced, thus resulting in more treatment options; however, depending on the patient's background, UFT/LV treatment can be an effective treatment option. PMID:22189237

  11. 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

  12. 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.

  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. Tweaking Subtype Selectivity and Agonist Efficacy at (S)-2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) Receptors in a Small Series of BnTetAMPA Analogues.

    PubMed

    Wang, Shuang-Yan; Larsen, Younes; Navarrete, Cristina Vara; Jensen, Anders A; Nielsen, Birgitte; Al-Musaed, Ali; Frydenvang, Karla; Kastrup, Jette Sandholm; Pickering, Darryl S; Clausen, Rasmus Prætorius

    2016-03-10

    A series of analogues of the (S)-2-Amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) receptor agonist BnTetAMPA (5b) were synthesized and characterized pharmacologically in radioligand binding assays at native and cloned AMPA receptors and functionally by two-electrode voltage clamp electrophysiology at the four homomeric AMPA receptors expressed in Xenopus laevis oocytes. The analogues 6 and 7 exhibit very different pharmacological profiles with binding affinity preference for the subtypes GluA1 and GluA3, respectively. X-ray crystal structures of three ligands (6, 7, and 8) in complex with the agonist binding domain (ABD) of GluA2 show that they induce full domain closure despite their low agonist efficacies. Trp767 in GluA2 ABD could be an important determinant for partial agonism of this compound series at AMPA receptors, since agonist efficacy also correlated with the location of the Trp767 side chain. PMID:26862980

  15. 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.

  16. Spectroscopic and structural investigations of 4-bromomethyl-5-methyl-1,3-dioxol-2-one and 4,5-bis(bromomethyl)-1,3-dioxol-2-one by quantum chemical simulations - A comparative study

    NASA Astrophysics Data System (ADS)

    Carthigayan, K.; Arjunan, V.; Anitha, R.; Periandy, S.; Mohan, S.

    2014-01-01

    The FTIR and FT-Raman spectra of 4-bromomethyl-5-methyl-1,3-dioxol-2-one and 4,5-bis(bromomethyl)-1,3-dioxol-2-one have been recorded in the regions 4000-400 and 4000-100 cm-1, respectively. The geometry of the compounds are optimised with B3LYP method using 6-311++G∗∗ and cc-pVTZ basis sets to characterise all the structural parameters. The optimised structural parameters of the most stable geometry are used in the vibrational frequency calculations. The total electron density and molecular electrostatic potential surfaces of the molecules are constructed by NBO analysis using B3LYP/6-311++G∗∗ method to display the electrostatic potential (electron + nuclei) distribution, molecular shape, size, dipole moments and the reactive centres of the molecules. The energy gap between HOMO and LUMO is measured. The influences of methyl and bromomethyl groups on the skeletal vibrations have been investigated. The global and local reactivity descriptors are also determined to provide the informations on the electrophilic, nucleophilic and free radical prone reactive sites of the molecules.

  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. Platinum(IV) coordination compounds containing 5-methyl-1,2,4-triazolo[1,5- a]pyrimidin-7(4 H)-one as nonleaving ligand. Molecular and cytotoxicity in vitro characterization

    NASA Astrophysics Data System (ADS)

    Łakomska, Iwona; Fandzloch, Marzena; Wojtczak, Andrzej; Szłyk, Edward

    2011-08-01

    Novel platinum(IV) coordination compounds with 5-methyl-1,2,4-triazolo[1,5- a]pyrimidin-7(4 H)-one (HmtpO): cis- trans-[PtCl 2(OH) 2(NH 3)(HmtpO)] ( 1), cis- trans-[PtCl 5(HmtpO)][(CH 3) 2NH 2] ( 2) have been prepared and structurally characterized by spectroscopic methods ( 1H, IR and X-ray crystallography ( 2)). The X-ray results indicate that the local geometry around the platinum(IV) centre approximates a typical octahedral arrangement with nitrogen atom N3 of the HmtpO and three chloride atoms in equatorial positions. The remaining two axial positions are occupied by two chlorides. The preliminary assessment of antitumor properties of ( 1) was performed as an in vitro antiproliferative activity against HL-60 human acute promyelocytic leukemia and HCV29T bladder cancer. The cis- trans-[PtCl 2(OH) 2(NH 3)(HmtpO)] ( 1) exhibits higher cytotoxic activity against HL-60 (IC 50 = 6.4 μM) than cisplatin.

  19. 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

  20. 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

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

    PubMed

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

    2014-11-11

    Vibrational spectral analysis of 5-methyl-N-[4-(trifluoromethyl) phenyl]-isoxazole-4-carboxamide is (5MN4TPI4C) molecule was carried out using FT-IR and FT-Raman spectroscopic techniques. The equilibrium geometry, harmonic vibrational wavenumbers, various bonding features have been computed using density functional B3LYP method with 6-311G(d,p) as basis set. The assignments of the vibrational spectra have been carried out with the aid of normal coordinate analysis (NCA) following the scaled quantum mechanical force field methodology (SQMFFM). Stability of the molecule arising from hyper conjugative interactions, charge delocalization has been analyzed using natural bond orbital (NBO) analysis. The non-linear optical (NLO) behavior of 5MN4TPI4C has been studied by determination of the electric dipole moment (μ) and hyperpolarizability (β) by using B3LYP/6-311G(d,p) method. The molecular orbital compositions and their contributions to the chemical bonding are studied by Total density of energy states (TDOS), sum of α and β electron (αβDOS) density of states. Thermodynamic properties (heat capacity, entropy and enthalpy) of the title compound at different temperatures are calculated. PMID:24858355

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

    SciTech Connect

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

    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.

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

    SciTech Connect

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

    1991-11-01

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

  4. 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.

  5. 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

  6. Synthesis, spectral characterisation of 2-(5-methyl-1H-benzimidazol-2-yl)-4-bromo/nitro-phenols and their complexes with zinc(II) ion, and solvent effect on complexation.

    PubMed

    Tavman, Aydin

    2006-02-01

    2-(5-Methyl-1H-benzimidazol-2-yl)-4-bromo/nitro-phenols (HLBr and HLNO2) and their Zn(II) complexes with ZnX2 (X = Cl, I, NO3) were synthesized and characterized by elemental analysis, molar conductivity, IR, 1H and 13C NMR spectra. The OH proton appears near the NH protons in the 1H NMR spectra of the ligands because of the strong intramolecular hydrogen bonding between the OH hydrogen and the C=N nitrogen atoms. The complexation is investigated in ethanol and isopropanol and it is observed that isopropanol is a better solvent than ethanol for the complex forming. HLBr gives harder complexation reaction with Zn(II) according to HLNO2 because of the stronger intramolecular hydrogen bonding in HLBr, and the both ligands react easier with Zn(NO3)2 than ZnCl2 and ZnI2. The Zn(II) complexes of HLBr have 1:1 M:L ratio and ionic character, however, HLNO2 give a non-ionic complex that has 1:2 M:L ratio. In the complexes the phenolic hydrogen is eliminated and a chelate structure is formed. PMID:15978864

  7. Effect of temperature and quencher on the fluorescence of 4-(5-methyl-3-furan-2-yl-benzofuran-2-yl)-7-methyl-chromen-2-one in different solvents

    NASA Astrophysics Data System (ADS)

    Evale, Basavaraj G.; Hanagodimath, S. M.

    2010-05-01

    The effect of temperature on the fluorescence intensity of 4-(5-methyl-3-furan-2-yl-benzofuran-2-yl)-7-methyl-chromen-2-one (MFBMC) in different solvents, has been studied in the temperature range 293-333 K. A mechanism of fluorescence quenching with increase in temperature is discussed in terms of the relative location of lowest 1( ππ*) and 3( nπ*) states, and the energy difference between them. The non-radiative deactivation of excited state in the absence of quencher is temperature-dependent; its activation energy has been found to be 9.453-27.893 kJ mole -1. Further, the fluorescence quenching by aniline was investigated by both steady-state and time-resolved measurement (at 296 K). The quenching is found to be appreciable and shows positive deviation in the Stern-Volmer plots. This could be explained by static-dynamic quenching models. Various rate constants of the bimolecular quenching reaction have been determined by using ground-state complex formation and sphere of action static quenching model. The magnitude of these constants suggests that sphere of action static quenching model agrees very well with experimental results. Further, with the use of finite sink approximation model, it is concluded that the quenching mechanism is diffusion-limited.

  8. 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

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

    1996-11-01

    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

  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. Uracil misincorporation into DNA of leukocytes of young women with positive folate balance depends on plasma vitamin B12 concentrations and methylenetetrahydrofolate reductase polymorphisms. A pilot study.

    PubMed

    Kapiszewska, Maria; Kalemba, Malgorzata; Wojciech, Urszula; Milewicz, Tomasz

    2005-08-01

    Changes in the folate and vitamin B12 status in the body influence the extent of uracil misincorporation (UrMis) into DNA, which is one of the biomarkers of genomic stability and, thus, portends a risk of cancer. In our study, the level of UrMis into DNA was evaluated by the comet assay (using the specific DNA repair enzyme, uracil DNA glycosylase) in leukocytes from blood donated by healthy young women with positive folate balance achieved by 4 weeks of folic acid supplementation (400 microg/day). The nutritional status was evaluated on the basis of nine food diaries recorded by the subjects during two winter months. The data were computerized, and the intake of nutrients and micronutrients was estimated using the DIETA 2 program (Food and Nutrition Institute, Warsaw, Poland) linked to recently updated Polish food tables. The plasma folate and vitamin B12 concentration, as well as methylenetetrahydrofolate reductase (MTHFR) polymorphisms, were evaluated to determine their influence on the level of UrMis into DNA. The mean value of B12 intake for all subjects reached 100% of the Polish recommended dietary allowances (RDA), whereas the mean value of folate intake, before folate supplementation, was 50%, suggesting moderate deficiency. Folic acid supplementation brought the folate intake way above the RDA, and plasma folate concentration in each individual was above the deficient range (mean value 14.67 ng/ml). The UrMis did not correlate with the plasma folate concentration, but the level of UrMis was significantly lower in subjects with plasma vitamin B12 concentration above 400 pg/ml (P=.02) only after folic acid supplementation. The concentration of folate in plasma correlated (P

  11. Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

    PubMed Central

    Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei

    2014-01-01

    ABSTRACT Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme

  12. 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

  13. 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

  14. Steric and electronic effects on arylthiolate coordination in the pseudotetrahedral complexes [(Tp(Ph,Me))Ni-SAr] (Tp(Ph,Me) = hydrotris{3-phenyl-5-methyl-1-pyrazolyl}borate).

    PubMed

    Deb, Tapash; Anderson, Caitlin M; Chattopadhyay, Swarup; Ma, Huaibo; Young, Victor G; Jensen, Michael P

    2014-12-14

    Synthesis and characterization of several new pseudotetrahedral arylthiolate complexes [(Tp(Ph,Me))Ni-SAr] (Tp(Ph,Me) = hydrotris{3-phenyl-5-methyl-1-pyrazolyl}borate; Ar = Ph, 2,4,6-(i)Pr3C6H2, C6H4-4-Cl, C6H4-4-Me, C6H4-4-OMe) are reported, including X-ray crystal structures of the first two complexes. With prior results, two series of complexes are spanned, [(Tp(Ph,Me))Ni-S-2,4,6-RC6H2] (R'' = H, Me, (i)Pr) plus the xylyl analogue [(Tp(Ph,Me))Ni-S-2,6-Me2C6H3], as well as [(Tp(Ph,Me))Ni-S-C6H4-4-Y] (Y = Cl, H, Me, OMe), intended to elucidate steric and/or electronic effects on arylthiolate coordination. In contrast to [(Tp(Me,Me))Ni-SAr] analogues that adopt a sawhorse conformation, the ortho-disubstituted complexes show enhanced trigonal and Ni-S-Ar bending, reflecting the size of the 3-pyrazole substituents. Moreover, weakened scorpionate ligation is implied by spectroscopic data. Little spectroscopic effect is observed in the series of para-substituted complexes, suggesting the observed effects are primarily steric in origin. The relatively electron-rich and encumbered complex [(Tp(Ph,Me))Ni-S-2,4,6-(i)Pr3C6H2] behaves uniquely when dissolved in CH3CN, forming a square planar solvent adduct with a bidentate scorpionate ligand, [(κ(2)-Tp(Ph,Me))Ni(NCMe)(S-2,4,6-(i)Pr3C6H2)]. This adduct was isolated and characterized by X-ray crystallography. Single-point DFT and TD-DFT calculations on a simplified [(κ(2)-Tp)Ni(NCMe)(SPh)] model were used to clarify the electronic spectrum of the adduct, and to elucidate differences between Ni-SAr bonding and spectroscopy between pseudotetrahedral and square planar geometries. PMID:25341014

  15. 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

  16. 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

  17. Therapeutic Usefulness of Postoperative Adjuvant Chemotherapy with Tegafur–Uracil (UFT) in Patients with Breast Cancer: Focus on the Results of Clinical Studies in Japan

    PubMed Central

    Noguchi, Shinzaburo

    2010-01-01

    In Japan, the history of postoperative chemotherapy for breast cancer started with 5-fluorouracil (5-FU), launched in the 1980s. Currently, oral fluoropyrimidine–based regimens indicated for the treatment of breast cancer in Japan include tegafur plus uracil (UFT); tegafur, gimeracil, and oteracil (TS-1); doxifluridine; and capecitabine. In particular, UFT represents an important option for long-term treatment because of minimal adverse events and the potential for long-term maintenance of effective plasma concentrations of 5-FU to inhibit micrometastasis after surgery. Therefore, various clinical studies of postoperative adjuvant chemotherapy with UFT have been conducted in patients with completely resected tumors. Recent studies have shown that UFT prolongs survival after tumor resection in patients with gastric cancer, colorectal cancer, and lung cancer. In patients with breast cancer, large clinical trials of UFT-based postoperative chemotherapy conducted in Japan have shown that UFT is useful for the treatment of intermediate-risk patients with no lymph node metastasis. This paper reviews the results of clinical studies of UFT conducted in Japan to assess the therapeutic usefulness of this oral 5-FU. The types of patients most likely to benefit from UFT are discussed on the basis of currently available evidence and a global consensus of treatment recommendations. The optimal timing of endocrine therapy and strategies for postoperative adjuvant chemotherapy with UFT in patients with breast cancer are also discussed. PMID:20080863

  18. 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

  19. A benchmark study of molecular structure by experimental and theoretical methods: Equilibrium structure of uracil from gas-phase electron diffraction data and coupled-cluster calculations

    NASA Astrophysics Data System (ADS)

    Vogt, Natalja; Khaikin, Leonid S.; Grikina, Olga E.; Rykov, Anatolii N.

    2013-10-01

    The equilibrium structure of uracil, one of the nucleobases, which build nucleic acids, has been determined for the first time by the gas-phase electron diffraction (GED) method. The necessary rovibrational corrections to the experimental internuclear distances have been calculated with quadratic and cubic force constants in the MP2(all)/cc-pVTZ approximation. For the first time, the equilibrium structure has been optimized by the very time-consuming coupled-cluster method with single and double excitations and perturbative treatment of connected triples using the correlation-consistent polarized weighted core-valence triple-zeta basis set with all electrons being correlated (CCSD(T)(all)/cc-pwCVTZ). The optimized structural parameters have been corrected for the diffuse-function effects and extrapolated to the higher basis set (cc-pwCVQZ) using results of MP2 computations (named as best ab initio structure). The GED equilibrium structure remarkably agrees with the best ab initio one as well as with that one derived from microwave (MW) rotational constants by Puzzarini and Barone. Thus, it has been revealed that the precise experiment and coupled-cluster calculations yield the same results when accurate vibrational corrections (including anharmonic ones) are considered in the experimental structural analysis. Moreover, it has been shown that the equilibrium structure derived from the GED data, being in general of one order less accurate than that determined from the MW rotational constants, is still reliable and accurate.

  20. [Two cases of recurrent colorectal cancer treated successfully with folinate/tegafur/uracil (UFT/LV) chemotherapy on an outpatient basis].

    PubMed

    Hosotaki, Kiyoshi; Tabira, Yoichi; Shimamoto, Masato; Tamori, Yasuhiro

    2008-04-01

    We report two cases of recurrent colorectal cancer in two patients who were treated successfully with a combined oral chemotherapeutic agent folinate/tegafur/uracil (UFT/LV) dosage. The first case was a 74-year-old woman who underwent Hartmann operation for colon cancer perforation. One year and 7 months after surgery, a local recurrence was found on the CT scan and endoscopy. It ended in exploratory laparotomy though we were operated on. Chemotherapy using 5-fluorouracil/Leucovorin (5-FU/LV) was performed six times. Sequentially, UFT/LV internal use was managed at our outpatient clinic. This patient has been living without side effects in the first three postoperative years, and without increase in tumors. The second case was a 65-year-old woman who underwent abdominoperineal resection of the rectum for rectal cancer. The histologic stage of disease aggravation of the patient was stageI. Post operatively, 2 years and 6 months later, we recognized a metastasis node on the lung upper right lobe of the patient and her CA19-9 value climbed dramatically. The patient took UFT/LV during outpatient visits to the hospital, the lung node shadow disappeared two months later, and CA19-9 decreased, too. The patient is living without a cancer recurrence now. UFT/LV treatment is one of the effective modalities against recurrence of colorectal cancer from outpatient treatment while maintaining QOL. PMID:18408440

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

    PubMed

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

    2001-01-01

    Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p. PMID:11139494

  2. Development and validation of a rapid and sensitive UPLC-MS/MS method for determination of uracil and dihydrouracil in human plasma.

    PubMed

    Jacobs, Bart A W; Rosing, Hilde; de Vries, Niels; Meulendijks, Didier; Henricks, Linda M; Schellens, Jan H M; Beijnen, Jos H

    2016-07-15

    Quantification of the endogenous dihydropyrimidine dehydrogenase (DPD) substrate uracil (U) and the reaction product dihydrouracil (UH2) in plasma might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity as a result of DPD deficiency. In this paper, we describe the development and validation of a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for quantification of U and UH2 in human plasma. Analytes were extracted by protein precipitation, chromatographically separated on an Acquity UPLC(®) HSS T3 column with gradient elution and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. U was quantified in the negative ion mode and UH2 in the positive ion mode. Stable isotopes for U and UH2 were used as internal standards. Total chromatographic run time was 5min. Validated concentration ranges for U and UH2 were from 1 to 100ng/mL and 10 to 1000ng/mL, respectively. Inter-assay bias and inter-assay precision for U were within ±2.8% and ≤12.4%. For UH2, inter-assay bias and inter-assay precision were within ±2.9% and ≤7.2%. Adequate stability of U and UH2 in dry extract, final extract, stock solution and plasma was demonstrated. Stability of U and UH2 in whole blood was only satisfactory when stored up to 4hours at 2-8°C, but not at ambient temperatures. An accurate, precise and sensitive UPLC-MS/MS assay for quantification of U and UH2 in plasma was developed. This assay is now applied to support clinical studies with fluoropyrimidine drugs. PMID:27179185

  3. Adsorption of adenine, cytosine, thymine, and uracil on sulfide-modified montmorillonite: FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry studies.

    PubMed

    Carneiro, Cristine E A; Berndt, Graciele; de Souza Junior, Ivan G; de Souza, Cláudio M D; Paesano, Andrea; da Costa, Antonio C S; di Mauro, Eduardo; de Santana, Henrique; Zaia, Cássia T B V; Zaia, Dimas A M

    2011-10-01

    In the present work the interactions of nucleic acid bases with and adsorption on clays were studied at two pHs (2.00, 7.00) using different techniques. As shown by Mössbauer and EPR spectroscopies and X-ray diffractometry, the most important finding of this work is that nucleic acid bases penetrate into the interlayer of the clays and oxidize Fe(2+) to Fe(3+), thus, this interaction cannot be regarded as a simple physical adsorption. For the two pHs the order of the adsorption of nucleic acid bases on the clays was: adenine ≈ cytosine > thymine > uracil. The adsorption of adenine and cytosine on clays increased with decreasing of the pH. For unaltered montmorillonite this result could be explained by electrostatic forces between adenine/cytosine positively charged and clay negatively charged. However for montmorillonite modified with Na(2)S, probably van der Waals forces also play an important role since both adenine/cytosine and clay were positively charged. FT-IR spectra showed that the interaction between nucleic acid bases and clays was through NH(+) or NH (2) (+) groups. X-ray diffractograms showed that nucleic acid bases adsorbed on clays were distributed into the interlayer surface, edge sites and external surface functional groups (aluminol, silanol) EPR spectra showed that the intensity of the line g ≈ 2 increased probably because the oxidation of Fe(2+) to Fe(3+) by nucleic acid bases and intensity of the line g = 4.1 increased due to the interaction of Fe(3+) with nucleic acid bases. Mössbauer spectra showed a large decreased on the Fe(2+) doublet area of the clays due to the reaction of nucleic acid bases with Fe(2+). PMID:21717172

  4. 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

  5. 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

  6. 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

  7. 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

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

    SciTech Connect

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

    2008-01-01

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

  9. Effect of leucovorin on the antitumor efficacy of the 5-FU prodrug, tegafur-uracil, in human colorectal cancer xenografts with various expression levels of thymidylate synthase

    PubMed Central

    TSUJIMOTO, HIROAKI; TSUKIOKA, SAYAKA; ONO, SATORU; SAKAMOTO, ETSUKO; SAKAMOTO, KAZUKI; TSUTA, KOHJI; NAKAGAWA, FUMIO; SAITO, HITOSHI; UCHIDA, JUNJI; KINIWA, MAMORU; FUKUSHIMA, MASAKAZU

    2010-01-01

    The combination of oral tegafur-uracil (UFT) with leucovorin (LV) is used to treat patients with stage II to III colon cancer based on the results of postoperative randomized studies in which UFT/LV treatment showed an equivalent efficacy to intravenous 5-FU plus LV therapy. However, whether the addition of LV to UFT can elevate the antitumor activity of UFT in colorectal tumors with high expression levels of thymidylate synthase (TS), which affects 5-FU efficacy, remains to be clarified. This study investigated the effect of LV on the antitumor activity of UFT and/or 5-FU prodrugs in low folate diet-fed nude mice using human colorectal cancer xenografts with various expression levels of TS. The addition of LV to UFT resulted in a 55–79% inhibition of tumor growth among 11 types of colorectal tumor xenograft, whereas UFT alone showed 23–67% antitumor activity. Although there was an inverse relationship between the antitumor effect of UFT alone and UFT plus LV and tumoral TS activity, UFT plus LV appeared to have a more potent antitumor effect than UFT alone on colorectal tumors such as Co-3 and KM12C/5-FU with high expression levels of TS. This finding was confirmed by the significant positive correlation between the relative inhibition ratio of UFT/LV to UFT alone and TS levels in tumors. To investigate the reason for the higher efficacy of UFT/LV on colorectal cancer xenografts with high TS activity, intratumoral levels of reduced folates and a ternary complex of TS after oral UFT with or without LV were measured using Co-3 xenografts. Elevated levels of reduced folates and an increased ternary complex of TS in LV-treated tumors were noted. Our results indicate that a combined therapy of UFT with LV may contribute to the treatment of colorectal cancer patients with low and high expression levels of tumoral TS by increased formation of the ternary complex of TS leading to potentiated antitumor efficacy of UFT. PMID:22870097

  10. 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

  11. 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

  12. 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

  13. 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.

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

    PubMed

    Laurella, Sergio L; Erben, Mauricio F

    2016-07-01

    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 21.9kJ/mol more stable than the conformer reported previously. Our study also includes the closely related species 1-(5-trifluoromethyl- [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol (FMTPN). An intramolecular OH⋯N hydrogen bond determines the conformational behavior of the [1,3,4]thiadiazol-2-yl)-pyrrolidin-2-ol group as demonstrated by Natural Bond Orbital population analysis. PMID:27070529

  15. Ultrafast intersystem crossing dynamics in uracil unravelled by ab initio molecular dynamics† †Electronic supplementary information (ESI) available: Discussion of fitting procedure for decay time constants, assignment of state character for ISC transitions and Cartesian coordinates of molecular geometries. See DOI: 10.1039/c4cp04158e Click here for additional data file.

    PubMed Central

    Richter, Martin; Mai, Sebastian; González, Leticia

    2014-01-01

    Ab initio molecular dynamics simulations have been performed in order to investigate the relaxation dynamics of uracil after UV excitation in gas phase. Intersystem crossing (ISC) has been included for the first time into time-dependent simulations of uracil, allowing the system to relax in the singlet as well as in the triplet states. The results show a qualitatively different picture than similar simulations that include singlet states only. The inclusion of ISC effectively quenches the relaxation to the singlet ground state and instead privileges transitions from the low-lying nπ* state (S1) to a ππ* triplet state (T2) followed by rapid internal conversion to the lowest triplet state. PMID:25301389

  16. 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

  17. 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

  18. 3-Benzyl-5-methyl-1,2-benzoxazole 2-oxide

    PubMed Central

    Anuradha, G.; Gopalsamy, Vasuki; Veera Reddy, A.; Laxminarasimhulu, G.

    2012-01-01

    In the title compound, C15H13NO2, the isoxazole unit and the attached benzene ring are almost coplanar, making a dihedral angle of 1.42 (8)°. The benzyl ring is inclined to the isoxazole ring by 74.19 (8)° and is in a +sc conformation with respect to the benzisoxazole unit. In the crystal, C—H⋯O hydrogen bonds link the mol­ecules, forming zigzag chains propagating along the b axis. There are also π–π inter­actions present involving the isoxazole and benzyl rings [centroid–centroid distance = 3.5209 (10) Å], and C—H⋯π inter­actions involving the benzene ring of the benzoisoxazole unit and the methyl­ene bridging group. PMID:23125740

  19. 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

  20. Solvent Effects on Molecular Aggregation in 4-(5-Heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol and 4-(5-Methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol.

    PubMed

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

    2016-08-18

    The article presents the results of spectroscopic studies of 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C1) and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (C7) in organic solvent solutions. Depending on the concentration of the compound used, three bands were observed in the fluorescence emission spectra of the compounds in DMSO solutions. A single band was observed in methanol, propan-2-ol, or ethanol. The significantly shortened fluorescence lifetimes and the different shapes of circular dichroism (CD) spectra clearly indicate association of the fluorescence effects with the aggregation processes in the analyzed compounds. The differences in the course of the CD spectra also imply an effect of the substituent group structure on the molecule aggregation interactions. Therefore, it has been postulated that the occurrence of the different spectral forms induced by changes in the compound concentration may be related to the aggregation effects of C1 and C7 molecules, which are also induced by differences in the alkyl substituent structure. PMID:27454065

  1. Studies to investigate the in vivo therapeutic window of the gamma-secretase inhibitor N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide (LY411,575) in the CRND8 mouse.

    PubMed

    Hyde, Lynn A; McHugh, Nansie A; Chen, Joseph; Zhang, Qi; Manfra, Denise; Nomeir, Amin A; Josien, Hubert; Bara, Thomas; Clader, John W; Zhang, Lili; Parker, Eric M; Higgins, Guy A

    2006-12-01

    Accumulation of amyloid beta-peptide (Abeta) is considered a key step in the etiology of Alzheimer's disease. Abeta is produced by sequential cleavage of the amyloid precursor protein by beta- and gamma-secretase enzymes. Consequently, inhibition of gamma-secretase provides a promising therapeutic approach to treat Alzheimer's disease. Preclinically, several gamma-secretase inhibitors have been shown to reduce plasma and brain Abeta, although they also produce mechanism-based side effects, including thymus atrophy and intestinal goblet cell hyperplasia. The present studies sought to establish an efficient screen for determining the therapeutic window of gamma-secretase inhibitors and to test various means of maximizing this window. Six-day oral administration of the gamma-secretase inhibitor N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-l-alaninamide (LY411,575) reduced cortical Abeta(40) in young (preplaque) transgenic CRND8 mice (ED(50) approximately 0.6 mg/kg) and produced significant thymus atrophy and intestinal goblet cell hyperplasia at higher doses (>3 mg/kg). The therapeutic window was similar after oral and subcutaneous administration and in young and aged CRND8 mice. Both the thymus and intestinal side effects were reversible after a 2-week washout period. Three-week treatment with 1 mg/kg LY411,575 reduced cortical Abeta(40) by 69% without inducing intestinal effects, although a previously unreported change in coat color was observed. These studies demonstrate that the 3- to 5-fold therapeutic window for LY411,575 can be exploited to obtain reduction in Abeta levels without induction of intestinal side effects, that intermittent treatment could be used to mitigate side effects, and that a 6-day dosing paradigm can be used to rapidly determine the therapeutic window of novel gamma-secretase inhibitors. PMID:16946102

  2. N-(2-hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), a novel, potent, and selective acid pump antagonist for the treatment of gastroesophageal reflux disease.

    PubMed

    Mori, Hiroki; Tonai-Kachi, Hiroko; Ochi, Yasuo; Taniguchi, Yasuhito; Ohshiro, Hiroyuki; Takahashi, Nobuyuki; Aihara, Takeshi; Hirao, Akiko; Kato, Teruhisa; Sakakibara, Minoru; Kurebayashi, Yoichi

    2009-02-01

    Inhibition of H(+),K(+)-ATPase is accepted as the most effective way of controlling gastric acid secretion. However, current acid suppressant therapy for gastroesophageal reflux disease, using histamine H(2) receptor antagonists and proton pump inhibitors, does not fully meet the needs of all patients because of their mechanism of action. This study sought to characterize the in vitro and in vivo pharmacology of a novel acid pump antagonist, N-(2-Hydroxyethyl)-N,2-dimethyl-8-{[(4R)-5-methyl-3,4-dihydro-2H-chromen-4-yl]amino}imidazo[1,2-a]pyridine-6-carboxamide (PF-03716556), and to compare it with other acid suppressants. Porcine, canine, and human recombinant gastric H(+),K(+)-ATPase activities were measured by ion-leaky and ion-tight assay. The affinities for a range of receptors, ion channels, and enzymes were determined to analyze selectivity profile. Acid secretion in Ghosh-Schild rats and Heidenhain pouch dogs were measured by titrating perfusate and gastric juice samples. PF-03716556 demonstrated 3-fold greater inhibitory activity than 5,6-dimethyl-2-(4-fluorophenylamino)-4-(1-methyl-1,2,3,4-tetrahydroisoquinoline-2-yl)pyrimidine (revaprazan), the only acid pump antagonist that has been available on the market, in ion-tight assay. The compound did not display any species differences, exhibiting highly selective profile including the canine kidney Na(+),K(+)-ATPase. Kinetics experiments revealed that PF-03716556 has a competitive and reversible mode of action. More rapid onset of action than 5-methoxy-2-{[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]-sulfinyl}-benzimidazole (omeprazole) and 3-fold greater potency than revaprazan were observed in Ghosh-Schild rats and Heidenhain pouch dogs. PF-03716556, a novel acid pump antagonist, could improve upon or even replace current pharmacological treatment for gastroesophageal reflux disease. PMID:18981288

  3. 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

    2016-01-01

    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 × 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. PMID:27270462

  4. 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

  5. Randomized phase III trial of treatment duration for oral uracil and tegafur plus leucovorin as adjuvant chemotherapy for patients with stage IIB/III colon cancer: final results of JFMC33-0502

    PubMed Central

    Sadahiro, S.; Tsuchiya, T.; Sasaki, K.; Kondo, K.; Katsumata, K.; Nishimura, G.; Kakeji, Y.; Baba, H.; Sato, S.; Koda, K.; Yamaguchi, Y.; Morita, T.; Matsuoka, J.; Usuki, H.; Hamada, C.; Kodaira, S.

    2015-01-01

    Background While adjuvant chemotherapy is preferable for high-risk colon cancer, treatment duration is controversial. Oral uracil and tegafur (UFT)/leucovorin (LV) is widely used as a standard adjuvant chemotherapy for colon cancer in Japan. We conducted a phase III trial to investigate the optimal duration of adjuvant chemotherapy for stage IIB/III colon cancer. Patients and methods Patients with curatively resected stage IIB/III colon cancer were eligible for enrollment in this trial. Patients were registered within 6 weeks after surgery and were randomly assigned to receive UFT/LV for 28 of 35 days for 6 months in the control group or for 5 consecutive days per week for 18 months in the study group. The primary end point was the disease-free survival (DFS), and the secondary end points were overall survival (OS) and safety. Result A total of 1071 patients were registered from 233 centers. A statistically significant difference in DFS was not observed between the study group and the control group; the 5-year DFS was 69% in the study group and 69% in the control group. The 5-year OS was 85% in the study group and 85% in the control group. Conclusion Eighteen-month treatment with UFT/LV did not improve DFS or OS compared with 6-month UFT/LV treatment in patients with stage IIB/III colon cancer. The important finding from this study is that not 18 months but 6 months of treatment is enough for postoperative UFT/LV for stage IIB/III colon cancer. Clinical trial number UMIN-CTR C000000245. PMID:26347106

  6. 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

  7. 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 Central

    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.

    2014-01-01

    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, 200 μM L-BHDU was noncytotoxic over 3 days, 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 0.03 μM, 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 2 h 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. PMID:25051026

  8. 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.

  9. 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. PMID:25051026

  10. Influence of C5-methylation of cytosine on the formation of cyclobutane pyrimidine dimers

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyi; Eriksson, Leif A.

    2005-01-01

    The reaction pathways for thermal and photochemical formation of 5-methylcytosine (m 5C) pyrimidine dimers (CPD) are explored using density functional theory techniques. It is shown that the methylation of cytosine does not contribute to an increased yield of CPDs after UV irradiation due to an even lower excitation energy at the reactant complex of m 5C as compared to cytosine, a larger barrier to reach the decay channel corresponding to the transition state structure along the ground state reaction path, and a higher-lying decay channel.

  11. 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

  12. Proteomic Analysis of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor Complexes*

    PubMed Central

    Kang, Myoung-Goo; Nuriya, Mutsuo; Guo, Yurong; Martindale, Kevin D.; Lee, Daniel Z.; Huganir, Richard L.

    2012-01-01

    The AMPA receptor (AMPA-R) is a major excitatory neurotransmitter receptor in the brain. Identifying and characterizing the neuronal proteins interacting with AMPA-Rs have provided important information about the molecular mechanisms underlying synaptic transmission and plasticity. In this study, to identify more AMPA-R interactors in vivo, we performed proteomic analyses of AMPA-R complexes from the brain. AMPA-R complexes were isolated from the brain through various combinations of biochemical techniques for solubilization, enrichment, and immunoprecipitation. Mass spectrometry analyses of these isolated complexes identified several novel components of the AMPA-R complexes as well as some previously identified components. The identification of these novel components helps to further define the complex mechanisms involved in the regulation of AMPA receptor function and synaptic plasticity. PMID:22753414

  13. 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

  14. Crystal structure of 4,6-di-chloro-5-methyl-pyrimidine.

    PubMed

    Medjani, Meriem; Hamdouni, Noudjoud; Brihi, Ouarda; Boudjada, Ali; Meinnel, Jean

    2015-12-01

    The title compound, C5H4Cl2N2, is essentially planar with an r.m.s. deviation for all non-H atoms of 0.009 Å. The largest deviation from the mean plane is 0.016 (4) Å for an N atom. In the crystal, mol-ecules are linked by pairs of C-H⋯N hydrogen bonds, forming inversion dimers, enclosing an R (2) 2(6) ring motif. PMID:26870504

  15. 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

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

    PubMed

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

    2015-11-01

    The ability of 4-thiouracil to strongly absorb UVA radiation and to populate a reactive triplet state in high yield has enabled its use as a versatile photocrosslinker for nearly 50 years. In this contribution, we present a detailed spectroscopic and photochemical investigation of the 2-thiouracil, 4-thiouracil, and 2,4-dithiouracil series in an effort to further advance this chemistry and to scrutinize the photoreactivity of 2,4-dithiouracil. Our results reveal that excitation of 2,4-dithiouracil leads to intersystem crossing to the triplet manifold in 220 ± 40 fs, which enables the population of the reactive triplet state with near unity yield (ΦT = 0.90 ± 0.15) and ultimately leads to a ca. 50% singlet oxygen generation (ΦΔ = 0.49 ± 0.02)-one of the highest singlet oxygen yields reported to date for a photoexcited thiobase. In addition, the long-lived triplet state of 2,4-dithiouracil reacts efficiently with the nucleic acid base adenine 5'-monophosphate through a direct, oxygen-independent photocycloaddition mechanism and at a rate that is at least 3-fold faster than that of 4-thiouracil under equal conditions. The new physico-chemical insights reported for these RNA-thiobase derivatives are compared to those of the DNA and RNA bases and the DNA-thiobase derivatives. Furthermore, the strong near-visible absorption and increased photoreactivity measured for 2,4-dithiouracil lays a solid foundation for developing RNA-targeted photocrosslinking and phototherapeutic agents that are more effective than those currently available. PMID:26439833

  17. Design, Synthesis, and Fungicidal Activities of Novel 5-Methyl-1H-1,2,3- trizole-4-carboxyl Amide Analogues.

    PubMed

    Wang, Zhen-Jun; Yang, Hui-Hui; Tian, Lei; Zhao, Wei-Guang

    2016-01-01

    Succinate dehydrogenase inhibitors (SDHIs) are fungicides with an amide bond widely used to control plant diseases caused by phytopathogenic fungi. Because of broad spectrum activity of new SDHIs, they have attracted wide attention from the research community. A series of structurally novel SDHIs with a bioactive 1,2,3-triazole moiety were designed and synthesized. Bioactivity screening showed that some of designed N-phenethyl-1,2,3-trizole-4-carboxyl amide analogues exhibited good fungicidal activities toward Sclerotinia sclerotiorum and Botrytis cinerea, while some of Nbenzyl- 1,2,3-trizole-4-carboxyl amide analogues exhibited good fungicidal activities toward Phytophthora capsici and Cercospora arachidicola. EC50 value of compound 5d against Cercospora arachidicola (6.6 µg/mL) was lower than that of chlorothalonil (12.3 µg/mL). PMID:26558376

  18. Synthesis and crystal structure studies of ethyl 5-methyl-1, 3-diphenyl-1H-pyrazole-4-carboxylate

    SciTech Connect

    Chandra,; Babu, E. A. Jithesh; Mahendra, M.; Srikantamurthy, N.; Umesha, K. B.

    2014-04-24

    The title compound, C{sub 19}H{sub 18}N{sub 2}O{sub 2}, was investigated by single crystal X-ray diffraction method. It crystallizes in monoclinic class under the space group P2{sub 1}/c with cell parameters a= 8.4593(4) Å, b=15.6284(6) Å, c=12.4579(5) Å, α=90°, β=98.241(3)°, γ=90° and Z=2. The ethoxycarbonyl group is slightly twisted from the pyrazole ring, and adopts syn-periplanar conformation. The crystal structure is stabilized by intermolecular C-H….O hydrogen bonds, which help in stabilizing the crystal structure.

  19. Synthesis and spectroscopy studies of the inclusion complex of 3-amino-5-methyl pyrazole with beta-cyclodextrin

    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.

  20. 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. PMID:21200723

  1. 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

  2. Crystal structure of 2,2-dimethyl-N-(5-methyl­pyridin-2-yl)propanamide

    PubMed Central

    El-Hiti, Gamal A.; Smith, Keith; Hegazy, Amany S.; Alanazi, Saud A.; Kariuki, Benson M.

    2015-01-01

    There are two mol­ecules in the asymmetric unit of the title compound, C11H16N2O. The pyridine rings and amide groups overlap almost perfectly (r.m.s. overlay fit = 0.053 Å), but the tertiary butyl groups have different orientations: in one mol­ecule, one of the methyl C atoms is syn to the amide O atom [O—C—C—C = −0.8 (3)°] and in the other the equivalent torsion angle is 31.0 (2)°. In the crystal, the two independent mol­ecules are linked by a pair of N—H⋯N hydrogen bonds in the form of an R 2 2(8) loop to form a dimer. A C—H⋯O inter­action connects the dimers into [100] chains. PMID:26090202

  3. 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

  4. 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

  5. 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

  6. 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. PMID:25487342

  7. Replication of the 2,6-diamino-4-hydroxy-N(5)-(methyl)-formamidopyrimidine (MeFapy-dGuo) adduct by eukaryotic DNA polymerases.

    PubMed

    Christov, Plamen P; Yamanaka, Kinrin; Choi, Jeong-Yun; Takata, Kei-ichi; Wood, Richard D; Guengerich, F Peter; Lloyd, R Stephen; Rizzo, Carmelo J

    2012-08-20

    N(6)-(2-Deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) has been identified as a stable DNA adduct that arises from the reaction of DNA with a variety of methylating agents. Since this lesion persists in DNA and may contribute to the overall mutagenesis from electrophilic methylating agents, the MeFapy-dGuo lesion was incorporated into oligonucleotides, and its replication bypass was examined in vitro with a panel of eukaryotic high fidelity (hPols α, β, and δ/PCNA) and translesion (hPols η, κ, ι, Rev1, ν, and yPol ζ) polymerases to address its miscoding potential. The MeFapy-dGuo was found to be a strong block to the high fidelity polymerases at either the insertion or the extension step. Efficient translesion synthesis was observed for hPols η and κ, and the combined activities of hRev1 and yPol ζ. The nucleotide sequences of the extension products were determined by mass spectrometry. The error-free extension product was the most abundant product observed for each polymerase. Misreplication products, which included misinsertion of Thy, Gua, and Ade opposite the MeFapy-dGuo lesion, as well as an interesting one-nucleotide deletion product, were observed when hPols η and κ were employed; these events accounted for 8-29% of the total extension products observed. The distribution and abundance of the misreplication products were dependent on the polymerases and local sequence context of the lesion. Collectively, these data suggest that although MeFapy-dGuo adducts represent a relatively minor proportion of the total alkylated lesions, their miscoding potentials could significantly contribute to genomic instability. PMID:22721435

  8. 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

  9. Crystal structure of (E)-4-meth­oxy-2-{[(5-methyl­pyridin-2-yl)imino]­meth­yl}phenol

    PubMed Central

    Adam, Farook; Arafath, Md Azharul; Haque, Rosenani Anwaeul; Razali, Mohd Rizal

    2015-01-01

    The mol­ecule of the title Schiff base compound, C14H14N2O2, displays an E conformation with respect the imine C=N double bond. The mol­ecule is approximately planar, with the dihedral angle formed by the planes of the pyridine and benzene rings being 5.72 (6)°. There is an intra­molecular hydrogen bond involving the phenolic H and imine N atoms. PMID:26594544

  10. 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

  11. Endocytic Adaptor Epidermal Growth Factor Receptor Substrate 15 (Eps15) Is Involved in the Trafficking of Ubiquitinated α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors*

    PubMed Central

    Lin, Amy; Man, Heng-Ye

    2014-01-01

    AMPA-type glutamate receptors (AMPARs) play a critical role in mediating fast excitatory synaptic transmission in the brain. Alterations in receptor expression, distribution, and trafficking have been shown to underlie synaptic plasticity and higher brain functions, including learning and memory, as well as brain dysfunctions such as drug addiction and psychological disorders. Therefore, it is essential to elucidate the molecular mechanisms that regulate AMPAR dynamics. We have shown previously that mammalian AMPARs are subject to posttranslational modification by ubiquitin, with AMPAR ubiquitination enhancing receptor internalization and reducing AMPAR cell surface expression. Here we report a crucial role for epidermal growth factor receptor substrate 15 (Eps15), an endocytic adaptor, in ubiquitination-dependent AMPAR internalization. We find that suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interacts with AMPARs, which requires Nedd4-mediated GluA1 ubiquitination and the ubiquitin-interacting motif of Eps15. Importantly, we find that Eps15 plays an important role in AMPAR internalization. Knockdown of Eps15 suppresses the internalization of GluA1 but not the mutant GluA1 that lacks ubiquitination sites, indicating a role of Eps15 for the internalization of ubiquitinated AMPARs. These results reveal a novel molecular mechanism employed specifically for the trafficking of the ubiquitin-modified AMPARs. PMID:25023288

  12. 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

  13. Synthesis of 2'-deoxy-2'-[.sup.18F]fluoro-5-methyl-1-B-D-arabinofuranosyluracil (.sup.18F-FMAU)

    SciTech Connect

    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.

  14. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... in paragraph (a) of this section may be used as a color additive in contact lenses in amounts not to...) of the Federal Food, Drug, and Cosmetic Act with respect to the contact lens in which the...

  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. 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

  17. 21 CFR 73.3122 - 4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) of the Federal Food, Drug, and Cosmetic Act with respect to the contact lens in which the color... in paragraph (a) of this section may be used as a color additive in contact lenses in amounts not...

  18. 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

    ...) of the Federal Food, Drug, and Cosmetic Act with respect to the contact lens in which the color... in paragraph (a) of this section may be used as a color additive in contact lenses in amounts not...

  19. 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

  20. 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...

  1. 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

  2. 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

  3. Crystal structure of (2-{[3,5-bis-(1,1-di-methyl-eth-yl)-4-hy-droxy-phen-yl](5-methyl-2H-pyrrol-2-yl-idene)meth-yl}-5-methyl-1H-pyrrolido-κ(2) N,N')di-fluoridoboron.

    PubMed

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

    2015-09-01

    The title compound, C25H31BF2N2O, is a potential boron tracedrug in boron neutron capture therapy (BNCT), in which the B atom adopts a distorted BN2F2 tetra-hedral geometry: it is soluble in dimethyl sulfoxide, di-methyl-formamide and methanol. The pyrrolyl-idene-methyl-pyrrole 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 inter-actions generate R 2 (2)(22) loops. PMID:26396909

  4. UV-MALDI mass spectrometric quantitation of uracil based pesticides in fruit soft drinks along with matrix effects evaluation.

    PubMed

    Ivanova, Bojidarka; Spiteller, Michael

    2014-02-01

    This study focused on the development of the accurate and precise quantitative method for the determination of pesticides bromacil (1), terbacil (2), lenacil (3), butafenacil (4) and flupropacil (5) in fruit based soft drinks. Three different types of drinks are bought from market; huddled orange fruit drink (100%) (I), red-oranges (II) and multivitamin drink containing strawberry, orange, banana and maracuja (III). Samples were analyzed "with" and "without" pulp utilizing LC-ESI (or APCI) MS/MS, HPLC-ESI-(or APCI)-MS/MS and UV-MALDI-Orbitrap-MS methods. The effect of high complexity of the food matrix on the analysis was discussed. Study focuses on the advantages of the UV-MALDI-Orbitrap-MS method compared to the traditionally involved GC alone or hybrid methods such as GC-MS and LC-MS/MS for quantification of pesticides in water and soft drinks. The developed method included the techniques performed for validation, calibration and standardization. The target pesticides are widely used for the treatment of citrus fruits and pineapples, but for soft drink products, there are still no clear regulations on pesticide residues limits. The matrix effects in the analysis of fruit drinks required implementation of the exact standard reference material corresponds to the variety of food matrices. This paper contributed to the broad analytical implementation of the UV-MALDI-Orbitrap-MS method in the quality control and assessment programs for monitoring of pesticide contamination in fruit based sodas. PMID:24018142

  5. 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...

  6. 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

  7. 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.

  8. N,N-Diethyl-4-[9-meth­oxy-6-(4-methoxy­phen­yl)-5-methyl-2-phenyl-2H-benzo[h]chromen-2-yl]aniline

    PubMed Central

    Kim, Moon-Hwan; Park, Hee-Moon; Kim, Chong-Hyeak

    2011-01-01

    In the title compound, C38H37NO3, the pyran ring has an envelope conformation with the quaternary Cq atom as the flap atom. The dihedral angle formed between the meth­oxy­phenyl group and the naphthalene ring system is 67.32 (6)°. The ethyl­amino groups lie to the same side of the plane through the phenyl ring and form dihedral angles of 84.6 (3) and 75.8 (2)° with it. PMID:21754393

  9. 1-(4-Chloro­phenyl­sulfon­yl)-5-(4-fluoro­phen­yl)-5-methyl­imidazolidine-2,4-dione

    PubMed Central

    Hussain, Abid; Hameed, Shahid; Stoeckli-Evans, Helen

    2009-01-01

    The title compound, C16H12ClFN2O4S, crystallizes with two independent mol­ecules (A and B) in the asymmetric unit. The two mol­ecules are U-shaped with similar geometries and conformations. The mean planes through the benzene rings are inclined to one another by 6.07 (8)° in mol­ecule A and 8.67 (8)° in mol­ecule B. They are separated with a centroid–centroid distance of 3.9096 (10) Å in mol­ecule A and 3.9118 (10) Å in mol­ecule B. Mol­ecules A and B lie adjacent to one another, with a centroid–centroid distance of 3.7592 (10) Å between the fluoro­phenyl ring of mol­ecule A and the chloro­phenyl­sulfonyl ring of mol­ecule B and with a dihedral angle of 5.75 (8)° between the ring planes. In the crystal structure, A and B mol­ecules are linked by N—H⋯O hydrogen bonds, forming centrosymmetric dimers. These dimers stack along the [110] direction and are linked by C—H⋯O and C—H⋯F inter­actions. There are also some short halide⋯halide contacts [Cl⋯F = 3.0499 (14) and 3.1224 (13) Å, and F⋯F = 3.0612 (17) Å]. PMID:21582575

  10. 3-(2-Benzamido­phen­yl)-4-(4-hydroxy­phen­yl)-5-methyl-4H-1,2,4-triazol-1-ium chloride

    PubMed Central

    Arfan, Mohammad; Tahir, M. Nawaz; Khan, Rasool; Iqbal, Mohammad S.

    2008-01-01

    In the mol­ecule of the title compound, C22H19N4O2 +·Cl−, the 1,2,4-triazole ring is oriented at dihedral angles of 75.57 (14), 53.23 (13) and 68.11 (13)° with respect to the benzamide, aniline and phenol atomatic rings, respectively. An intra­molecular C—H⋯O hydrogen bond results in the formation of a non-planar ten-membered ring. In the crystal structure, inter­molecular O—H⋯Cl and N—H⋯Cl hydrogen bonds link the mol­ecules. There is a C—H⋯π contact between the methyl group and the phenyl ring, and a π–π contact between the hydroxy­phenyl and phenyl rings [centroid–centroid distance = 4.687 (2) Å]. PMID:21203214

  11. 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.

  12. Novel Regulation of the Synthesis of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Subunit GluA1 by Carnitine Palmitoyltransferase 1C (CPT1C) in the Hippocampus.

    PubMed

    Fadó, Rut; Soto, David; Miñano-Molina, Alfredo J; Pozo, Macarena; Carrasco, Patricia; Yefimenko, Natalia; Rodríguez-Álvarez, José; Casals, Núria

    2015-10-16

    The regulation of AMPA-type receptor (AMPAR) abundance in the postsynaptic membrane is an important mechanism involved in learning and memory formation. Recent data suggest that one of the constituents of the AMPAR complex is carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform located in the endoplasmic reticulum of neurons. Previous results had demonstrated that CPT1C deficiency disrupted spine maturation in hippocampal neurons and impaired spatial learning, but the role of CPT1C in AMPAR physiology had remained mostly unknown. In the present study, we show that CPT1C binds GluA1 and GluA2 and that the three proteins have the same expression profile during neuronal maturation. Moreover, in hippocampal neurons of CPT1C KO mice, AMPAR-mediated miniature excitatory postsynaptic currents and synaptic levels of AMPAR subunits GluA1 and GluA2 are significantly reduced. We show that AMPAR expression is dependent on CPT1C levels because total protein levels of GluA1 and GluA2 are decreased in CPT1C KO neurons and are increased in CPT1C-overexpressing neurons, whereas other synaptic proteins remain unaltered. Notably, mRNA levels of AMPARs remained unchanged in those cultures, indicating that CPT1C is post-transcriptionally involved. We demonstrate that CPT1C is directly involved in the de novo synthesis of GluA1 and not in protein degradation. Moreover, in CPT1C KO cultured neurons, GluA1 synthesis after chemical long term depression was clearly diminished, and brain-derived neurotrophic factor treatment was unable to phosphorylate the mammalian target of rapamycin (mTOR) and stimulate GluA1 protein synthesis. These data newly identify CPT1C as a regulator of AMPAR translation efficiency and therefore also synaptic function in the hippocampus. PMID:26338711

  13. Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5'-G(5mC)^NG(5mC)-3'.

    PubMed

    Chernukhin, V A; Gonchar, D A; Abdurashitov, M A; Belichenko, O A; Dedkov, V S; Mikhnenkova, N A; Lomakovskaya, E N; Udal'yeva, S G; Degtyarev, S Kh

    2016-01-01

    Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5'-GCNGC- 3' before the central nucleotide "N" if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated. PMID:27099792

  14. 5-Methyl-N′-[(3Z)-2-oxo-2,3-dihydro-1H-indol-3-yl­idene]-1-phenyl-1H-1,2,3-triazole-4-carbohydrazide

    PubMed Central

    Mohamed, Hanan A.; Abdel-Wahab, Bakr F.; Ng, Seik Weng; Tiekink, Edward R. T.

    2013-01-01

    In the title compound, C18H14N6O2, the benzene ring is slightly twisted out of the plane of the 1,2,3-triazole ring (r.m.s. deviation = 0.010 Å), forming a dihedral angle of 6.20 (13)°. The nine non-H ring atoms of the fused five- and six-membered ring system are almost coplanar (r.m.s. deviation = 0.032 Å). The near coplanarity in the central residue is consolidated by an intra­molecular bifurcated N—H⋯(O,N) hydrogen bond. The conformation about the N=C bond is Z. In the crystal, supra­molecular chains along [101] are sustained by N—H⋯O hydrogen bonds and C—H⋯O inter­actions. These are consolidated into a three-dimensional architecture by C—H⋯π and π–π inter­actions; the latter occur between centrosymmetrically related 1,2,3-triazole rings [centroid–centroid distance = 3.6056 (14) Å]. PMID:23634111

  15. (2E)-3-(4-Fluoro­phen­yl)-1-[5-methyl-1-(4-methyl­phen­yl)-1H-1,2,3-triazol-4-yl]prop-2-en-1-one1

    PubMed Central

    Abdel-Wahab, Bakr F.; Mohamed, Hanan A.; Ng, Seik Weng; Tiekink, Edward R. T.

    2013-01-01

    With respect to the triazole ring in the title compound, C19H16FN3O, the p-tolyl ring is inclined [dihedral angle = 51.79 (11)°], whereas the chalcone residue is almost coplanar [O—C—C—N and C—C—C—C torsion angles = −178.71 (19) and 178.42 (18)°, respectively]. The conformation about the C=C bond [1.328 (3) Å] is E, and the triazole methyl group and the carbonyl O atom are syn. In the crystal, centrosymmetrically related mol­ecules are connected by π–π inter­actions between the triazole and p-tolyl rings [centroid–centroid distance = 3.6599 (12) Å] and these are linked into a three-dimensional architecture by C—H⋯N and C—H⋯π inter­actions. PMID:23723805

  16. (2E)-1-[5-Methyl-1-(4-methyl­phen­yl)-1H-1,2,3-triazol-4-yl]-3-[4-(piperidin-1-yl)phen­yl]prop-2-en-1-one1

    PubMed Central

    Abdel-Wahab, Bakr F.; Abdel-Latif, Ehab; Ng, Seik Weng; Tiekink, Edward R. T.

    2013-01-01

    Two independent mol­ecules comprise the asymmetric unit of the title compound, C24H26N4O. The major difference between them is found in the relative orientation of the triazole-bound p-tolyl group which have the opposite sense of twist [N—N—C—C torsion angles = 55.8 (3) and −49.8 (3)°]. The chalcone residue is almost coplanar with the triazole ring [N—C—C—O and C—C—C—C torsion angles = −178.9 (2) and −178.5 (2)°, respectively; cf. 177.9 (3) and 168.5 (3)°, respectively, in the second mol­ecule]. The conformation about each C=C double bond is E and in each case the triazole methyl group is syn to the carbonyl O atom. In the crystal, mol­ecules aggregate into layers parallel to (-113). The first independent mol­ecule self-associates into a layer via C—H⋯O and C—H⋯π inter­actions. By contrast, layers comprising the second independent mol­ecule do not feature specific inter­actions between mol­ecules. The global crystal packing comprises alternating layers. PMID:23723806

  17. 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

  18. Dichlorido{N-[(5-methyl­thio­phen-2-yl)methyl­idene]-2-(pyridin-2-yl)ethanamine-κ2 N,N′}palladium(II)

    PubMed Central

    Radebe, Mduduzi P.; Onani, Martin O.; Motswainyana, William M.

    2013-01-01

    In the title compound, [PdCl2(C13H14N2S)], the PdII ion is coordinated by two N atoms of the chelating bidentate ligand and two chloride anions, giving rise to a distorted square-planar geometry. The methyl-substituted thio­phene arm and the pyridine ring are connected to the metal cation through N atoms to form a six-membered chelate ring with a boat conformation, making the complex stable. PMID:23476321

  19. Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors*

    PubMed Central

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

    2015-01-01

    Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca2+, resulting from Ca2+ influxes through calcium-permeable AMPA receptors, voltage-gated Ca2+ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca2+ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca2+ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. PMID:26134564

  20. 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

  1. (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

  2. Synthesis and biologic evaluation of substituted 5-methyl-2-phenyl-1H-pyrazol-3(2H)-one derivatives as selective COX-2 inhibitors: molecular docking study.

    PubMed

    Dube, Pritam N; Bule, Shweta S; Mokale, Santosh N; Kumbhare, Manoj R; Dighe, Pravin R; Ushir, Yogesh V

    2014-10-01

    The present study reported the synthesis and biologic evaluation of new pyrazolone derivatives for COX-2 inhibitory activities and investigated in vivo for their anti-inflammatory activities using carrageenan-induced rat paw edema model as well as in vitro using HRBC membrane stabilization and protein denaturation method. Eight derivatives showed pronounced COX-2 inhibition, and 5a, 5d, and 5f exhibited the highest COX-2 inhibition. The derivatives were further evaluated for antioxidant activity wherein 5a and 5b showed potent free radical-scavenging activity against DPPH, nitric oxide, and hydrogen peroxide radicals. Molecular docking study revealed the binding orientations of pyrazolone derivatives into the active sites of COX-2 and thereby helps to design the potent inhibitors. PMID:24636540

  3. Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5’-G(5mC)^NG(5mC)-3’

    PubMed Central

    Chernukhin, V. A.; Gonchar, D. A.; Abdurashitov, M. A.; Belichenko, O. A.; Dedkov, V. S.; Mikhnenkova, N. A.; Lomakovskaya, E. N.; Udal’yeva, S. G.; Degtyarev, S. Kh.

    2016-01-01

    Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5’-GCNGC- 3’ before the central nucleotide “N” if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated. PMID:27099792

  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 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

  6. 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

  7. Synthesis of Enantiomerically Pure (S)-Methanocarbaribo Uracil Nucleoside Derivatives for Use as Antiviral Agents and P2Y Receptor Ligands

    PubMed Central

    Melman, Artem; Zhong, Minghong; Marquez, Victor E.; Jacobson, Kenneth A.

    2009-01-01

    We have developed an approach toward enantiomerically pure (S)-methanocarba ribonucleosides based on several functional group transformations on a sensitive bicyclo[3.1.0]hexane system. D-Ribose was transformed into methanocarba alcohol 3 followed by conversion of the OH group to a nitrile with inversion of configuration at C4. The nitrile group was subsequently reduced in two stages to the 5′-hydroxymethyl group. An ester group appended to a tertiary carbon (C1) was transformed to an amino group as a nucleobase precursor. PMID:18811198

  8. The carcinostatic effects of 1-(2-tetrahydrofuryl)-5-fluorouracil and uracil (UFT) on tongue carcinoma induced by 4-nitroquinoline 1-oxide (4NQO) in rats.

    PubMed

    Katakura, A; Shiozaki, Y; Kouda, H; Hatada, K; Tonogi, M; Takaki, T; Yamane, G; Noma, H

    1991-11-01

    UFT is a carcinostatic agent used in adjuvant chemotherapy for head and neck cancer. In the present study. UFT was given orally to treat tongue carcinoma in rats induced by 4-nitroquinoline 1-oxide. The antitumor effects of UFT were studied macroscopically and histologically. In addition, the antitumor effects of UFT were evaluated in relationship to lesions of the clinical and, invasive types, and to there vascular structure. In clinical lesions, the antitumor effect of UFT was higher in extrovert tumor-mass lesions and lower in ulcerous lesions. With regard to vascular structure, the effect was higher in cases demonstrating irregular net-like patterns and branch-like patterns and lower in cases in which the pattern had been destroyed. There was a correlation between antitumor effect and invasive type. As invasive tendency the 3H-thymidine labeling index, and mitotic index increased, antitumor effect and degree of tumor cell degeneration decreased. PMID:1819452

  9. Studies on the synthesis of 1-substituted uracils, radical-induced cyclization of 1,6-diolefins, and the addition of nucleophiles to fullerenes

    SciTech Connect

    Naim, A.

    1992-01-01

    The reaction of 2-propanol solution of 1,6-diolefins was investigated. The gamma radiolysis of 2-propanol generates 2-hydroxy-2-propyl radical and a hydrogen atom. The addition of 2-hydroxy-2-propyl radical to a double bond forms a 5-hexenyl radical, which subsequently cyclizes to generate the methylcyclopentyl radical. The formation of product in the gamma radiolysis of 2-propanol + 1,6-diolefins is the same as the photolysis of a mixture of 2-propanol + acetone + 1,6-diolefins. On the basis of experimental findings, the major isomer concluded was cis. The monomer diallyldimethylammonium bromide, which undergoes facile polymerization in the presence of free radicals, was also cyclized to give monomeric products. The addition of a variety of nucleophiles to fullerenes was also studied. Both C[sub 60]/C[sub 70] react with KOH and degrade in contact with air. Their reactions are reversible under vacuum. Regeneration of C[sub 60] is quantitative while C[sub 70] in only 72%. The C[sub 70] is more reactive toward KOH than C[sub 60], which led to development of a method to obtain pure C[sub 60] from C[sub 60]/C[sub 70]. Besides KOH, the reaction of KF, NaNH[sub 2], N[sub 2]CHCO[sub 2]CH[sub 2]CH[sub 3] and CH[sub 2]N[sub 2] with the fullerenes were also investigated.

  10. Clay catalysis of oligonucleotide formation: kinetics of the reaction of the 5'-phosphorimidazolides of nucleotides with the non-basic heterocycles uracil and hypoxanthine

    NASA Technical Reports Server (NTRS)

    Kawamura, K.; Ferris, J. P.

    1999-01-01

    The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine of substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.

  11. 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

  12. 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.

  13. Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides.

    PubMed

    Kumar, R; Rai, D; Sharma, S K; Saffran, H A; Blush, R; Tyrrell, D L

    2001-10-11

    A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes. PMID:11585457

  14. 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

  15. 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

  16. Thermodynamics and Mechanism of the Interaction of Willardiine Partial Agonists with a Glutamate Receptor: Implications for Drug Development

    PubMed Central

    2015-01-01

    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

  17. Discovery of 5-chloro-N2-[(1S)-1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(5-methyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (AZD1480) as a novel inhibitor of the Jak/Stat pathway.

    PubMed

    Ioannidis, Stephanos; Lamb, Michelle L; Wang, Tao; Almeida, Lynsie; Block, Michael H; Davies, Audrey M; Peng, Bo; Su, Mei; Zhang, Hai-Jun; Hoffmann, Ethan; Rivard, Caroline; Green, Isabelle; Howard, Tina; Pollard, Hannah; Read, Jon; Alimzhanov, Marat; Bebernitz, Geraldine; Bell, Kirsten; Ye, Minwei; Huszar, Dennis; Zinda, Michael

    2011-01-13

    The myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis are a heterogeneous but related group of hematological malignancies characterized by clonal expansion of one or more myeloid lineages. The discovery of the Jak2 V617F gain of function mutation highlighted Jak2 as a potential therapeutic target in the MPNs. Herein, we disclose the discovery of a series of pyrazol-3-yl pyrimidin-4-amines and the identification of 9e (AZD1480) as a potent Jak2 inhibitor. 9e inhibits signaling and proliferation of Jak2 V617F cell lines in vitro, demonstrates in vivo efficacy in a TEL-Jak2 model, has excellent physical properties and preclinical pharmacokinetics, and is currently being evaluated in Phase I clinical trials. PMID:21138246

  18. 5-(4-Fluoro­phen­yl)-3-[5-methyl-1-(4-methyl­phen­yl)-1H-1,2,3-triazol-4-yl]-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothio­amide

    PubMed Central

    Abdel-Wahab, Bakr F.; Ng, Seik Weng; Tiekink, Edward R. T.

    2013-01-01

    In the title compound, C26H23FN6S, the pyrazole ring has an envelope conformation, with the methine C atom being the flap atom. The thio­urea group is close to being coplanar with the pyrazole N atoms [N—N—C—S torsion angle = 176.78 (15)°], which allows for an intra­molecular N—H⋯N hydrogen bond; the connected triazole ring is nearly coplanar with this ring [N—C—C—N = −172.65 (19)°]. There is a significant twist between the pyrazole ring and attached fluoro­benzene ring [N—C—C—C = −18.8 (3)°] and a greater twist between triazole and attached tolyl ring [dihedral angle = 58.25 (14)°]. In the crystal, supra­molecular chains aligned along [40,10] are consolidated by π–π inter­actions between the triazole and phenyl rings [centroid–centroid distance = 3.7053 (13) Å]. PMID:23634141

  19. 4-[5-(4-Fluoro­phen­yl)-1-(4-phenyl-1,3-thia­zol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl]-5-methyl-1-(4-methyl­phenyl)-1H-1,2,3-triazole

    PubMed Central

    Abdel-Wahab, Bakr F.; Ng, Seik Weng; Tiekink, Edward R. T.

    2013-01-01

    In the title compound, C28H23FN6S, the pyrazole ring adopts an envelope conformation, with the methine C atom being the flap atom. With respect to this ring, the 2-thienyl, triazole and fluoro­benzene rings are approximately coplanar, coplanar and perpendicular, respectively [dihedral angles = 8.56 (17), 6.03 (19) and 73.1 (2)°, respectively] so that to a first approximation the mol­ecule has a T-shape. In the crystal, mol­ecules are consolidated into a three-dimensional architecture by C—H⋯F (involving a bifurcated F atom), C—H⋯S and C—H⋯π inter­actions. PMID:23634140

  20. 3-[5-Methyl-1-(4-methyl­phen­yl)-1H-1,2,3-triazol-4-yl]-N-phenyl-5-[4-(piperidin-1-yl)phen­yl]-4,5-dihydro-1H-pyrazole-1-carbothio­amide dimethyl­formamide hemisolvate

    PubMed Central

    Abdel-Wahab, Bakr F.; Mohamed, Hanan A.; Ng, Seik Weng; Tiekink, Edward R. T.

    2012-01-01

    The essentially planar pyrazole ring (r.m.s. deviation = 0.013 Å) in the title hemisolvate, C31H33N7S·0.5C3H7NO, is almost coplanar with the pendant thio­urea residue [N—N—C—S torsion angle = −173.2 (4)°] and slightly twisted with respect to the triazole ring [dihedral angle = 7.7 (3)°]. An intra­molecular thio­urea–pyrazole N—H⋯N hydrogen bond, via an S(5) loop, is formed. Supra­molecular chains along the c axis are formed in the crystal via piperidine–triazole C—H⋯N inter­actions. These are bridged into loosely associated double chains via C—H⋯O inter­actions involving the disordered (over two positions) dimethyl­formamide solvent mol­ecules. The thio­urea-bound phenyl ring is also disordered over two positions of equal occupancy. PMID:22807818

  1. Crystal structure of 5-{3-[2,6-dimethyl-4-(5-methyl-1,2,4-oxa­diazol-3-yl)phen­oxy]prop­yl}-N-(11-hy­droxy­undec­yl)isoxazole-3-carboxamide hemihydrate

    PubMed Central

    Salorinne, K.; Lahtinen, T.

    2015-01-01

    The title compound, C29H42N4O5·0.5H2O, comprises four structural units. A flexible prop­yloxy 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 phenyl­oxa­diazole 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 hy­droxy group links to the 3-position of the isoxazole ring via an amide bond. In the crystal, a half-occupancy solvent water mol­ecule connects to a neighbouring mol­ecule via an inter­molecular O—H⋯O(water) hydrogen bond to the C11-alkyl chain hy­droxy group. PMID:25995867

  2. Crystal structure of 4-[(2,4-di-chloro-phen-yl)(5-hy-droxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)meth-yl]-5-methyl-2-phenyl-2,3-di-hydro-1H-pyrazol-3-one.

    PubMed

    Kumar, Balbir; Mahajan, Hitesh; Paul, Satya; Kant, Rajni; Gupta, Vivek K

    2015-10-01

    In the title compound C27H22Cl2N4O2, the pyrazol-5-ol ring makes a dihedral angle of 34.80 (11)° with the phenyl ring to which it is bound, while the pyrazolone ring is inclined at 34.34 (12)° to its attached phenyl ring. In the crystal, N-H⋯O and C-H⋯Cl hydrogen bonds link the mol-ecules into chains along [010]. Inter-molecular π-π inter-actions are observed between the pyrazolone ring and the phenyl ring bound to the pyrazol-5-ol ring system [centroid-centroid separation = 3.916 (2) Å]. PMID:26594489

  3. Crystal structure of 4-[(2,4-di­chloro­phen­yl)(5-hy­droxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)meth­yl]-5-methyl-2-phenyl-2,3-di­hydro-1H-pyrazol-3-one

    PubMed Central

    Kumar, Balbir; Mahajan, Hitesh; Paul, Satya; Kant, Rajni; Gupta, Vivek K.

    2015-01-01

    In the title compound C27H22Cl2N4O2, the pyrazol-5-ol ring makes a dihedral angle of 34.80 (11)° with the phenyl ring to which it is bound, while the pyrazolone ring is inclined at 34.34 (12)° to its attached phenyl ring. In the crystal, N—H⋯O and C—H⋯Cl hydrogen bonds link the mol­ecules into chains along [010]. Inter­molecular π–π inter­actions are observed between the pyrazolone ring and the phenyl ring bound to the pyrazol-5-ol ring system [centroid–centroid separation = 3.916 (2) Å]. PMID:26594489

  4. Zinc complexes of Ttz(R,Me) with O and S donors reveal differences between Tp and Ttz ligands: acid stability and binding to H or an additional metal (Ttz(R,Me) = tris(3-R-5-methyl-1,2,4-triazolyl)borate; R = Ph, tBu).

    PubMed

    Kumar, Mukesh; Papish, Elizabeth T; Zeller, Matthias; Hunter, Allen D

    2011-08-01

    Alkylzinc complexes, (Ttz(R,Me))ZnR' (R = tBu, Ph; R' = Me, Et), show interesting reactivity with acids, bases and water. With acids (e.g. fluorinated alcohols, phenols, thiophenol, acetylacetone, acetic acid, HCl and triflic acid) zinc complexes of the conjugate base (CB), (Ttz(R,Me))ZnCB, are generated. Thus the B-N bonds in Ttz ligands are acid stable. (Ttz(R,Me))ZnCB complexes were characterized by (1)H, (13)C-NMR, IR, MS, elemental analysis, and, in most cases, single crystal X-ray diffraction. The four coordinate crystal structures included (Ttz(R,Me))Zn(CB) [where R = Ph, CB (conjugate base) = OCH(2)CF(3) (2), OPh (6), SPh (8), p-OC(6)H(4)(NO(2)) (10); R = tBu, CB = OCH(CF(3))(2) (3), OPh (5), SPh (7)*, p-OC(6)H(4)(NO(2)) (9) (* indicates a rearranged Ttz ligand)]. The use of bidentate ligands resulted in structures [(Ttz(Ph,Me))Zn(CB) (CB = acac (12), OAc (14))] in which the coordination geometries are five, and intermediate between four and five, respectively. Interestingly, three forms of (Ttz(Ph,Me))Zn(p-OC(6)H(4)(NO(2))) (10) were analyzed crystallographically including a Zn coordinated water molecule in 10(H(2)O), a coordination polymer in 10(CP), and a p-nitrophenol molecule hydrogen bonded to a triazole ring in 10(Nit). Ttz ligands are flexible since they are capable of providing κ(3) or κ(2) metal binding and intermolecular interactions with either a metal center or H through the four position nitrogen (e.g. in 10(CP) and HTtz(tBu,Me)·H(2)O, respectively). Preliminary kinetic studies on the protonolysis of LZnEt (L = Ttz(tBu,Me), Tp(tBu,Me)) with p-nitrophenol in toluene at 95 °C show that these reactions are zero order in acid and first order in the LZnEt. PMID:21706096

  5. Crystal structure of 4-({(1E,2E)-3-[3-(4-fluoro­phen­yl)-1-isopropyl-1H-indol-2-yl]allyl­idene}amino)-5-methyl-1H-1,2,4-triazole-5(4H)-thione

    PubMed Central

    Kulkarni, Ajaykumar D.; Rahman, Md. Lutfor; Mohd. Yusoff, Mashitah; Kwong, Huey Chong; Quah, Ching Kheng

    2015-01-01

    The title compound, C23H22FN5S, exists in a trans conformation with respect to the methene C=C and the acyclic N=C bonds. The 1,2,4-triazole-5(4H)-thione ring makes dihedral angles of 88.66 (9) and 84.51 (10)°, respectively, with the indole and benzene rings. In the crystal, mol­ecules are linked by pairs of N—H⋯S hydrogen bonds, forming inversion dimers with an R 2 2(8) ring motif. The dimers are linked via C—H⋯π inter­actions, forming chains along [1-10]. The chains are linked via π—π inter­actions involving inversion-related triazole rings [centroid–centroid distance = 3.4340 (13) Å], forming layers parallel to the ab plane. PMID:26594522

  6. 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.

  7. MSH6- or PMS2-deficiency causes re-replication in DT40 B cells, but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils

    PubMed Central

    Campo, Vanina A.; Patenaude, Anne-Marie; Kaden, Svenja; Horb, Lori; Firka, Daniel; Jiricny, Josef; Di Noia, Javier M.

    2013-01-01

    The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a ‘homeologous’ recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6−/− and Pms2−/− DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of ‘homeologous’ recombination insensitive to MMR. PMID:23314153

  8. Influence of structural and electronic properties of uranyl derivatives on the inhibition of thymidine phosphorylase

    SciTech Connect

    Dimoglo, A.S.; Bersuker, I.B.; Gorbachev, M.Yu.

    1986-07-01

    The inhibition of enzymes by definite compounds lies at the basis of the mechanism of the action of most drugs. Uracil and its derivatives are effective inhibitors of thymidine phosphorylase and other related enzymes. Cancer cells are especially sensitive to the absence of thymidine. The study of the inhibiting action of uracil derivatives has been conducted previously. In this article, the authors used the hydrophobicity constants of the substituents, directly bonded to the uracil framework in the 1- and 3-positions and the 6-position as well as the constants of ortho- and meta-substituents in benzene rings bonded to uracil in the investigated compounds for the derivation of correlation equations relating the inhibiting activity to the physicochemical parameters. A table is presented of the 142 uracil derivatives taken for logical-structural analysis.

  9. 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

  10. [Effect of local moderate hyperthermia in combination with N-nitroso-1,3-bis-(2-chloroethyl)urea (BCNU) and 5-fluoro-(tetrahydro-2-furyl)uracil (ftorafur) on induced autochthonous colonic cancers in the rat. 3: Polychemotherapy in combination with hyperthermia].

    PubMed

    Biwer, E; Lorenz, M; Habs, M; Schmähl, D

    1984-01-01

    The use of hyperthermia for the treatment of tumors has been tested in vitro and in vivo experiments as well as clinically for a long time. Combination of hyperthermia with chemotherapy was reported to result in overadditive cytostatic effects. In a clinically adapted, controlled animal experiment, local moderate hyperthermia (43.5 degrees C, 3 X 60 min) alone and in combination with polychemotherapy (BCNU) and Ftorafur) was used for the treatment of AMMN-(N-nitrosoacetoxymethyl-methylamine) induced autochthonous colonic carcinomas in Sprague-Dawley rats. Diagnosis and follow-up inspections were carried out endoscopically. The applied therapies did not result in prolonged survival times, nor was an additive effect seen after combined hyperthermia and chemotherapy in this "hard", i.e. relatively chemotherapy-resistent, tumor model. PMID:6439968

  11. PURITY AND HEAT OF FUSION DATA FOR ENVIRONMENTAL STANDARDS AS DETERMINED BY DIFFERENTIAL SCANNING CALORIMETRY

    EPA Science Inventory

    Differential scanning calorimetry (DSC) has been applied to 273 environmental standards, including pesticides, herbicides and related compounds. embers of the following chemical classes were analyzed: rganophosphorus, organochlorine, phenol, triazine, uracil, phenoxy acid, urea, ...

  12. Nucleobases and Other Prebiotic Species from the Ultraviolet Irradiation of Pyrimidine in Astrophysical Ices

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

    We discuss the results of UV irradiation of ices containing pyrimidine and show that such processing efficiently forms the nucleobases uracil and cytosine, but not thymine, a pattern similar to what is seen in carbonaceous meteorites.

  13. Formation of Nucleobases from the UV Photo-Irradiation of Pyrimidine in Astrophysical Ice Analogs

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

    This work shows how pyrimidic nucleobases (uracil, cytosine, etc.) can be formed under abiotic conditions from the UV irradiation of pyrimidine in astrophysical ices. The formation mechanisms and the photo-stability of such compounds are discussed.

  14. Mutagenesis by site-specific arylamine adducts in plasmid DNA: Enhancing replication of the adducted strand alters mutation frequency

    SciTech Connect

    Reid, T.M.; Lee, Meisie; King, C.M. )

    1990-07-03

    Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by >7-fold to 2.9% and of the AAF adduct by >12-fold to 0.75%. The AF adduct produced primarily single-base deletions in the absence of uracil but only base substitutions in the uracil-containing constructs. The AAF adduct produced mutations only in the uracil-containing DNA, which included both frame shifts and base substitutions. Mutations produced by both adducts were SOS dependent.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Hydrogen bond formation between the naturally modified nucleobase and phosphate backbone

    PubMed Central

    Sheng, Jia; Zhang, Wen; Hassan, Abdalla E. A.; Gan, Jianhua; Soares, Alexei S.; Geng, Song; Ren, Yi; Huang, Zhen

    2012-01-01

    Natural RNAs, especially tRNAs, are extensively modified to tailor structure and function diversities. Uracil is the most modified nucleobase among all natural nucleobases. Interestingly, >76% of uracil modifications are located on its 5-position. We have investigated the natural 5-methoxy (5-O-CH3) modification of uracil in the context of A-form oligonucleotide duplex. Our X-ray crystal structure indicates first a H-bond formation between the uracil 5-O-CH3 and its 5′-phosphate. This novel H-bond is not observed when the oxygen of 5-O-CH3 is replaced with a larger atom (selenium or sulfur). The 5-O-CH3 modification does not cause significant structure and stability alterations. Moreover, our computational study is consistent with the experimental observation. The investigation on the uracil 5-position demonstrates the importance of this RNA modification at the atomic level. Our finding suggests a general interaction between the nucleobase and backbone and reveals a plausible function of the tRNA 5-O-CH3 modification, which might potentially rigidify the local conformation and facilitates translation. PMID:22641848

  20. 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 Central

    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

  1. 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.

  2. 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.

  3. 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. PMID:26726767

  4. TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway

    PubMed Central

    Park, Jeong-Ho; Park, Sun Hwa; Kang, Tong Mook; Cho, Hana; Kim, Tae Jin; Kim, Hyung-Wook; Jun, Youngsoo; Lee, Hee Jae; Lee, Young Sik; Kwon, Jae Young; Kang, KyeongJin

    2016-01-01

    Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1’s role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems. PMID:26726767

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

    PubMed

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

    2016-05-01

    An efficient route to synthesize cycloalkyl substituted purine nucleosides was developed. This metal-free C-H activation was accomplished by a tBuOOtBu initiated radical reaction. By adjusting the amount of tBuOOtBu and reaction time, the selective synthesis of C6-monocycloalkyl or C6,C8-dicycloalkyl substituted purine nucleosides could be realized. Furthermore, uracil and related nucleosides were also suitable substrates, giving the C5-cyclohexyl substituted uracil derivatives in good yields with excellent regioselectivities. PMID:27101306

  6. 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.

  7. 40 CFR 180.645 - Thiencarbazone-methyl; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... § 180.645 Thiencarbazone-methyl; tolerances for residues. (a) General. (1) Tolerances are established for residues of thiencarbazone-methyl amino]sulfonyl]-5-methyl-3-thiophenecarboxylate], per se, in...

  8. 40 CFR 180.645 - Thiencarbazone-methyl; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... § 180.645 Thiencarbazone-methyl; tolerances for residues. (a) General. (1) Tolerances are established for residues of thiencarbazone-methyl amino]sulfonyl]-5-methyl-3-thiophenecarboxylate], per se, in...

  9. Photoinduced cytotoxicity of a photochromic diarylethene via caspase cascade activation.

    PubMed

    Okuda, Jun-ya; Tanaka, Yukimi; Kodama, Ryuhei; Sumaru, Kimio; Morishita, Kana; Kanamori, Toshiyuki; Yamazoe, Seiji; Hyodo, Kengo; Yamazaki, Shohei; Miyatake, Tomohiro; Yokojima, Satoshi; Nakamura, Sinichiro; Uchida, Kingo

    2015-07-11

    The photo-generated closed-ring isomer of bis(5-methyl-2-phenylthiazoyl)perfluorocyclopentene shows cytotoxicity to Madin-Darby canine kidney (MDCK) cells through a caspase cascade and induces apoptosis of cells. PMID:26063471

  10. 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.

  11. Molecular recognition of the antiretroviral drug abacavir: towards the development of a novel carbazole-based fluorosensor.

    PubMed

    Idzik, Krzysztof Ryszard; Cywinski, Piotr J; Cranfield, Charles G; Mohr, Gerhard J; Beckert, Rainer

    2011-05-01

    Due to their optical and electro-conductive attributes, carbazole derivatives are interesting materials for a large range of biosensor applications. In this study, we present the synthesis routes and fluorescence evaluation of newly designed carbazole fluorosensors that, by modification with uracil, have a special affinity for antiretroviral drugs via either Watson-Crick or Hoogsteen base pairing. To an N-octylcarbazole-uracil compound, four different groups were attached, namely thiophene, furane, ethylenedioxythiophene, and another uracil; yielding four different derivatives. Photophysical properties of these newly obtained derivatives are described, as are their interactions with the reverse transcriptase inhibitors such as abacavir, zidovudine, lamivudine and didanosine. The influence of each analyte on biosensor fluorescence was assessed on the basis of the Stern-Volmer equation and represented by Stern-Volmer constants. Consequently we have demonstrated that these structures based on carbazole, with a uracil group, may be successfully incorporated into alternative carbazole derivatives to form biosensors for the molecular recognition of antiretroviral drugs. PMID:21222147

  12. 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

  13. Potential formation of three pyrimidine bases in interstellar regions

    NASA Astrophysics Data System (ADS)

    Majumdar, Liton; Gorai, Prasanta; Das, Ankan; Chakrabarti, Sandip K.

    2015-12-01

    Work on the chemical evolution of pre-biotic molecules remains incomplete since the major obstacle is the lack of adequate knowledge of rate coefficients of various reactions which take place in interstellar conditions. In this work, we study the possibility of forming three pyrimidine bases, namely, cytosine, uracil and thymine in interstellar regions. Our study reveals that the synthesis of uracil from cytosine and water is quite impossible under interstellar circumstances. For the synthesis of thymine, reaction between uracil and :CH2 is investigated. Since no other relevant pathways for the formation of uracil and thymine were available in the literature, we consider a large gas-grain chemical network to study the chemical evolution of cytosine in gas and ice phases. Our modeling result shows that cytosine would be produced in cold, dense interstellar conditions. However, presence of cytosine is yet to be established. We propose that a new molecule, namely, C4N3OH5 could be observable in the interstellar region. C4N3OH5 is a precursor (Z isomer of cytosine) of cytosine and far more abundant than cytosine. We hope that observation of this precursor molecule would enable us to estimate the abundance of cytosine in interstellar regions. We also carry out quantum chemical calculations to find out the vibrational as well as rotational transitions of this precursor molecule along with three pyrimidine bases.

  14. 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...

  15. 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.

  16. 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

  17. 5-Fluorouracil-resistant strain of Methanobacterium thermoautortrophicum

    SciTech Connect

    Nagle, D.P. Jr.; Teal, R.; Eisenbraun, A.

    1987-09-01

    Growth of Methanobacterium thermoautotrophicum Marburg is inhibited by the pyrimidine, 5-fluorouracil (FU). It was shown previously that methanogenesis is not inhibited to the same extent as growth. A spontaneously occurring FU-resistant strain (RTAE-1) was isolated from a culture of strain Marburg. The growth of both strains was inhibited by 5-fluorodeoxyuridine but not 5-fluorocytosine, and the wild type was more susceptible to inhibition by 5-azauracil and 6-azauracil than was strain RTAE-1. The cellular targets for the pyrimidine analogs are not known. When the accumulation of /sup 14/C-labeled uracil or FU by the two strains was compared, the wilt type took up 15-fold more radiolabel per cell than did the FU-resistant strain. In the wild type, radiolabel from uracil was incorporated into the soluble pool, RNA, and DNA. The metabolism of uracil appeared to involve a uracil phosphoribosyltransferase activity. Strain Marburg extracts contained this enzyme, whereas FU-resistant strain RTAE-1 extracts had less than 1/10 as much activity. Although it is possible that a change in permeability to the compounds plays a role in the stable resistance of strain RTAE-1, the fact that it lacks the ability to metabolize pyrimidines to nucleotides is sufficient to account for its phenotype.

  18. Pathways of Nucleotide Biosynthesis in Mycoplasma mycoides subsp. mycoides

    PubMed Central

    Mitchell, Alana; Finch, Lloyd R.

    1977-01-01

    By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of 14C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5′-monophosphate to adenosine 5′-monophosphate via the intermediate inosine 5′-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter. PMID:324972

  19. MTHFR POLYMORPHISMS AND COLORECTAL NEOPLASIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Folate is essential for the synthesis, repair and methylation of DNA. Aberrations in folate metabolism can modify our risk for cancer. Folate depletion alters DNA methylation patterns and increases DNA uracil-content and the frequency of DNA breaks. These DNA aberrations are involved in the etiology...

  20. 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.

  1. Non-replicating Toxoplasma gondii reverses tumor-associated immunosuppression

    PubMed Central

    Fox, Barbara A; Sanders, Kiah L; Bzik, David J

    2013-01-01

    We examined the efficacy of using attenuated non-replicating Toxoplasma gondii uracil auxotrophs that can be safely delivered as anticancer immunotherapeutics. This strategy exerted remarkable therapeutic activity in murine models of melanoma and ovarian carcinoma, and holds broad potential for the development of novel, highly effective anticancer vaccines. PMID:24353916

  2. De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii.

    PubMed

    Fox, Barbara A; Bzik, David J

    2002-02-21

    Toxoplasma gondii is a ubiquitous protozoan parasite that is responsible for severe congenital birth defects and fatal toxoplasmic encephalitis in immunocompromized people. Fundamental aspects of obligate intracellular replication and pathogenesis are only now beginning to emerge for protozoan parasites. T. gondii has a fragmented pathway for salvaging pyrimidine nucleobases derived from the parasite or host cell, and this limited pyrimidine salvage capacity is funnelled exclusively through uracil phosphoribosyltransferase. Disrupting the function of this enzyme does not affect the growth of T. gondii tachyzoites, which suggests that the de novo pyrimidine biosynthesis pathway may be necessary for growth. We have examined the virulence of T. gondii mutants that lack carbamoyl phosphate synthetase II (uracil auxotrophs) to determine whether de novo pyrimidine biosynthesis is required in vivo. Here we show that T. gondii uracil auxotrophs are completely avirulent not only in immune-competent BALB/c mice but also in mice that lack interferon-gamma. A single injection of the uracil auxotroph into BALB/c mice induces long-term protective immunity to toxoplasmosis. Our findings indicate the significance of the de novo pyrimidine biosynthesis pathway for the virulence of parasitic protozoa, and suggest routes for developing vaccines and chemotherapy. PMID:11859373

  3. Non-replicating Toxoplasma gondii reverses tumor-associated immunosuppression.

    PubMed

    Fox, Barbara A; Sanders, Kiah L; Bzik, David J

    2013-11-01

    We examined the efficacy of using attenuated non-replicating Toxoplasma gondii uracil auxotrophs that can be safely delivered as anticancer immunotherapeutics. This strategy exerted remarkable therapeutic activity in murine models of melanoma and ovarian carcinoma, and holds broad potential for the development of novel, highly effective anticancer vaccines. PMID:24353916

  4. Sensitizing cancer cells: Is it really all about U?

    PubMed Central

    Stover, Patrick J.; Weiss, Robert S.

    2016-01-01

    In this issue of Cancer Cell, Hu et al. report that TMPK and RNR, two key enzymes in deoxyribonucleotide biosynthesis, co-localize to damaged DNA and produce nucleotides necessary for DNA repair while suppressing uracil incorporation. TMPK inhibition disrupts this balance and selectively sensitizes cancer cells to low-dose chemotherapy. PMID:22789532

  5. dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

    PubMed Central

    Gadsden, M H; McIntosh, E M; Game, J C; Wilson, P J; Haynes, R H

    1993-01-01

    dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design. Images PMID:8223452

  6. 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

  7. 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

  8. 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

  9. Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene.

    PubMed

    Kuilenburg, André B P van; Meijer, Judith; Tanck, Michael W T; Dobritzsch, Doreen; Zoetekouw, Lida; Dekkers, Lois-Lee; Roelofsen, Jeroen; Meinsma, Rutger; Wymenga, Machteld; Kulik, Wim; Büchel, Barbara; Hennekam, Raoul C M; Largiadèr, Carlo R

    2016-04-01

    Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation. PMID:26804652

  10. A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes.

    PubMed

    Andersen, Gorm; Björnberg, Olof; Polakova, Silvia; Pynyaha, Yuriy; Rasmussen, Anna; Møller, Kasper; Hofer, Anders; Moritz, Thomas; Sandrini, Michael Paolo Bastner; Merico, Anna-Maria; Compagno, Concetta; Akerlund, Hans-Erik; Gojković, Zoran; Piskur, Jure

    2008-07-18

    Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC1-6 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. A combination of genetic and analytical chemistry methods demonstrates that uridine monophosphate and urea are intermediates, and 3-hydroxypropionic acid, ammonia and carbon dioxide the final products of degradation. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. The genes of the URC pathway are not homologous to any of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date. PMID:18550080

  11. Potential-dependent sum frequency generation study of 5-methylbenzotriazole on polycrystalline copper, platinum, and gold.

    PubMed

    Romero, Casey; Baldelli, Steven

    2006-06-22

    In situ sum frequency generation vibrational spectroscopy, at varied potentials and polarization combinations, was performed on polycrystalline copper, polycrystalline platinum, and polycrystalline gold samples in 0.5 M HClO4 with 50 mM 5-methylbenzotriazole (5-methylBTAH) added. These studies were performed to determine the orientation of 5-methylBTAH on the surface at different potentials. For copper surfaces, orientation of the molecule on the surface is not affected by potential within the potential window studied (-500 to -100 mV vs saturated calomel electrode (SCE)). Sum frequency generation spectra of 5-methylBTAH on platinum show a change in orientation over the potential range studied (-250 to 750 mV vs SCE). The orientation of the methyl group tilts more toward the plane of the interface as the potential is scanned in the positive direction. This orientation change is correlated to hydrogen coadsorption on the platinum surface at low potentials. 5-Methylbenzotriazole lies in the surface plane or does not orient on gold at lower potentials but the orientation is tilted toward normal at more positive potentials over the potential range studied (-500 to 900 mV vs SCE). To compliment these results, cyclic voltammetry and electrochemical impedance spectroscopy measurements were performed. Cyclic voltammograms of copper show that addition of 5-methylBTAH protects the surface from copper dissolution, increasing the electrochemical window by 450 mV. Cyclic voltammetry of 5-methylBTAH on platinum showed a partial blockage of adsorbed hydrogen and also prevented the adsorption of oxygenated species at 450-600 mV. Cyclic voltammetry on gold shows that 5-methylBTAH blocks oxide formation for 400 mV thus increasing the electrochemical window. Electrochemical impedance spectroscopy has been performed to determine the potential of zero charge of 5-methylBTAH on copper. PMID:16800498

  12. Effect of ring size in R-(+)-pulegone-mediated hepatotoxicity: studies on the metabolism of R-(+)-4-methyl-2-(1-methylethylidene)-cyclopentanone and DL-camphorone in rats.

    PubMed

    Thulasiram, H V; Bhat, V B; Madyastha, M K

    2001-06-01

    R-(+)-Pulegone, a monoterpene ketone, is a potent hepatotoxin. The present study was designed to evaluate whether the reduction of the ring size in R-(+)-pulegone would affect its mode of metabolism and its hepatotoxic potential. Metabolic fate of R-(+)-4-methyl-2-(1-methylethylidene)-cyclopentanone (I) and 5-methyl-2-(1-methylethylidene)-cyclopentanone (DL-camphorone; II) were examined in rats. Compounds I and II were administered orally (250 mg/kg of b.wt./day) to rats for 5 to 7 days. The following metabolites were isolated and identified from the urine of rats dosed with I: 3-methyl-5-(1-methylethylidene)-cyclopent-2-enone (Ie), Z-4-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (Ib), E-4-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (Ia), 3-hydroxy-4-methyl-2-(1-methylethylidene)-cyclopentanone (If), 4-hydroxy-4-methyl-2-(1-methylethylidene)-cyclopentanone (Ic), and E-4-methyl-2-(1-carboxyethylidene)-cyclopentanone (Id). Phenobarbital (PB)-induced rat liver microsomes in the presence of NADPH transformed compound I into metabolites, which were identified as Ia, Ib, Ic, Ie, and If. The following urinary metabolites were isolated and identified from compound II: 5-hydroxy-5-methyl-2-(1-methylethylidene)-cyclopentanone (IIc), 5-hydroxy-5-methyl-2-(1-methylethyl)-cyclopentanone (IIg), Z-5-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (IIb), 5-methyl-2-(1-hydroxymethylethyl)-cyclopentanone (IIf), E-5-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (IIa), E-5-methyl-2-(1-carboxyethylidene)-cyclopentanone (IId), and 5-methyl-2-(1-carboxyethyl)-cyclopentanone (IIe). PB-induced rat liver microsomes in the presence of NADPH were shown to transform compound II to IIa, IIb, and IIc. Studies carried out in vitro demonstrated that hydroxylation at the tertiary carbon atom or oxidation of the isopropylidene methyl groups in II can be specifically blocked through structural modifications as seen in compounds 2,2-dimethyl-5-(1-methylethylidene

  13. Design and synthesis of novel 5-substituted acyclic pyrimidine nucleosides as potent and selective inhibitors of hepatitis B virus.

    PubMed

    Kumar, Rakesh; Nath, Mahendra; Tyrrell, D Lorne J

    2002-05-01

    A novel class of 5-substituted acyclic pyrimidine nucleosides, 1-[(2-hydroxyethoxy)methyl]-5-(1-azidovinyl)uracil (9a), 1-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]-5-(1-azidovinyl)uracil (9b), and 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azidovinyl)uracil (9c), were synthesized by regiospecific addition of bromine azide to the 5-vinyl substituent of the respective 5-vinyluracils (2a-c) followed by treatment of the obtained 5-(1-azido-2-bromoethyl) compounds (3a-c) with t-BuOK, to affect the base-catalyzed elimination of HBr. Thermal decomposition of 9b and 9c at 110 degrees C in dioxane yielded corresponding 5-[2-(1-azirinyl)]uracil analogues (10b,c). The 5-(1-azidovinyl)uracil derivatives 9a-c were found to exhibit potent and selective in vitro anti-HBV activity against duck hepatitis B virus (DHBV) infected primary duck hepatocytes at low concentrations (EC(50) = 0.01-0.1 microg/mL range). The most active anti-DHBV agent (9c), possessing a [4-hydroxy-3-(hydroxymethyl)-1-butyl] substituent at N-1, exhibited an activity (EC(50) of 0.01-0.05 microg/mL) comparable to that of reference compound (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3-TC) (EC(50) = 0.01-0.05 microg/mL). In contrast, related 5-[2-(1-azirinyl)]uracil analogues (10b,c) were devoid of anti-DHBV activity, indicating that an acyclic side chain at C-5 position of the pyrimidine ring is essential for anti-HBV activity. The pyrimidine nucleosides (9a-c, 10b,c) exhibited no cytotoxic activity against a panel of 60 human cancer cell lines. All of the compounds investigated did not show any detectable toxicity to several stationary and proliferating host cell lines or to mitogen stimulated proliferating human T lymphocytes, up to the highest concentration tested. PMID:11985471

  14. Isolation of the insecticidal components of Tagetes minuta (Compositae) against mosquito larvae and adults.

    PubMed

    Perich, M J; Wells, C; Bertsch, W; Tredway, K E

    1995-09-01

    Application of Tagetes minuta floral extract to silica gel column chromatography produced 2 fractions with the hydrogenate part 20-30 times more toxic to larvae and 12-13 times more toxic to adults of Aedes aegypti and Anopheles stephensi, respectively, than the oxygenate part. Further fractionation by column chromatography of the hydrogenate fraction produced 4 thiophenes, 5-(but-3-ene-1-ynyl)-2,2'-bithiophene, 5-(but-3-ene-1-ynyl)-5'-methyl-2,2'-bithiophene, 2,2',5',2"-terthiophene, and 5-methyl-2,2',5',2"-terthiophene. These compounds in Tagetes minuta are largely responsible for the toxicity exhibited against the tested mosquitoes. PMID:8551298

  15. Investigation of a Privileged Polymorphic Motif: a Dimeric ROY Derivative

    PubMed Central

    Lutker, Katie M.; Tolstyka, Zachary P.; Matzger, Adam J.

    2009-01-01

    Bis(5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrilyl)acetylene, a derivative of the highly polymorphic compound 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile (ROY) that possesses two chromophores electronically coupled through a triple bond, was found to be trimorphic. Structural data for two of these forms indicates that symmetry is maintained in one structure and broken in the other leading to spontaneous differentiation of the methyl-thiophenecarbonitrile units. This study contributes to the mounting evidence that ROY and its derivatives are particularly prone to polymorphism. PMID:19367341

  16. Chemical basis of the photosensitizing activity of angelicins.

    PubMed

    Dall'Acqua, F; Vedaldi, D; Caffieri, S; Guiotto, A; Bordin, F; Rodighiero, G

    1984-12-01

    Angelicins are a group of compounds that show marked photobiologic activity on various substrates; some of them have been proposed as potential agents for the photochemotherapy of skin diseases. A good correlation exists between the photosensitizing activity of these compounds and their capacity to induce monofunctional lesions to DNA; therefore, we believe the chemical nature of these photolesions, we isolated from the products of hydrolysis of the photocombinations between 5 angelicins (angelicin, 4-methyl, 5-methyl, 5'-methyl, and 5,5'-dimethylangelicin) and DNA, the corresponding new fluorescent monoadducts between the 4',5'-double bond of the furocoumarins and the 5,6-double bond of thymine. PMID:6531039

  17. Highly selective aldose reductase inhibitors. 3. Structural diversity of 3-(arylmethyl)-2,4,5-trioxoimidazolidine-1-acetic acids.

    PubMed

    Kotani, T; Nagaki, Y; Ishii, A; Konishi, Y; Yago, H; Suehiro, S; Okukado, N; Okamoto, K

    1997-02-28

    Accumulation of intracellular sorbitol, the reduced product of glucose, catalyzed by aldose reductase (AR) (EC 1.1.1.21), is thought to be the cause of the development of diabetic complications. Our attention is focused on finding compounds which inhibit AR without significantly inhibiting aldehyde reductase (ALR) (EC 1.1.1.2). The uracil or 2,4-dioxoimidazolidine skeleton having the benzothiazolyl or 4-chloro-3-nitrophenyl group as an aryl part indicated not only extremely high AR inhibitory activity but also AR selectivity. The ratio of IC50(ALR)/IC50(AR) of 3-[(5-chlorobenzothiazol-2-yl)methyl]-1,2,3,4-tetrahydro-2,4- dioxopyrimidine-1-acetic acid (47d) was more than 17 500. The uracil skeleton with the benzothiazolyl moiety seemed to be the best combination for selective AR inhibition. PMID:9057855

  18. 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.

  19. Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism

    PubMed Central

    Suspène, Rodolphe; Aynaud, Marie-Ming; Guétard, Denise; Henry, Michel; Eckhoff, Grace; Marchio, Agnès; Pineau, Pascal; Dejean, Anne; Vartanian, Jean-Pierre; Wain-Hobson, Simon

    2011-01-01

    The human APOBEC3 (A3A–A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome. PMID:21368204

  20. 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.

  1. 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.

  2. 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

  3. New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

    PubMed Central

    Price, Alan R.; Cook, Sandra J.

    1972-01-01

    The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection. PMID:4623224

  4. 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

  5. Construction of spectral sensitivity function using polychromatic UV sources.

    PubMed

    Modos, K; Gaspar, S; Kirsch, P; Gay, M; Ronto, G

    1999-04-01

    A procedure is presented for constructing the spectral sensitivity functions of biological dosimeters, using five polychromatic UV sources possessing different emission spectra. Phage T7 and uracil biological dosimeters have been used for measuring the dose rates of the lamps. Their spectral sensitivity functions consisting of two exponential terms have been constructed. The parameters of the spectral sensitivity functions have been determined by comparing the directly measured and calculated dose-rate values. The parameters of the sensitivity function are accepted as correct values when the deviation of the measured and calculated values is a minimum. Based on the deviations between the constructed and the experimentally determined spectral sensitivities with monochromatic sources, the differences between the measured and calculated results are interpreted. The importance of the correct spectral sensitivity data is demonstrated through the effectiveness spectra of a TL 01 lamp for phage T7 killing, uracil dimerization and erythema induction. PMID:10392466

  6. Pear Bud Metabolism: Seasonal Changes in Glucose Utilization

    PubMed Central

    Zimmerman, Richard H.; Faust, Miklos

    1969-01-01

    Utilization of glucose, uracil and valine by flower and leaf buds of seedling pear trees (Pyrus calleryana Decne.) from the time of flower bud initiation to flowering was investigated. A very high rate of glucose utilization through the pentose phosphate pathway was observed throughout the development of buds. There was no difference in the type of glucose metabolism between flower and leaf buds except immediately before flowering, when the metabolism in flower buds was shifted toward the glycolytic pathway. Such a shift did not occur in leaf buds. The incorporation of uracil and valine into the nucleic acid and protein fraction of buds, respectively, was high throughout bud development, perhaps indicating a high rate of turnover in the resting buds. Incorporation of both compounds decreased when buds started to expand prior to flowering. PMID:16657202

  7. 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.

  8. 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.

  9. 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.

  10. Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal.

    PubMed Central

    Singh, R; Banerjee, H; Green, M R

    2000-01-01

    The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (SXL). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas SXL recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine >> adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice. PMID:10864047

  11. Photosynthesis and photo-stability of nucleic acids in prebiotic extraterrestrial environments.

    PubMed

    Sandford, Scott A; Bera, Partha P; Lee, Timothy J; Materese, Christopher K; Nuevo, Michel

    2015-01-01

    Laboratory experiments have shown that the UV photo-irradiation of low-temperature ices of astrophysical interest leads to the formation of organic molecules, including molecules important for biology such as amino acids, quinones, and amphiphiles. When pyrimidine is introduced into these ices, the products of irradiation include the nucleobases uracil, cytosine, and thymine, the informational sub-units of DNA and RNA, as well as some of their isomers. The formation of these compounds, which has been studied both experimentally and theoretically, requires a succession of additions of OH, NH₂, and CH₃groups to pyrimidine. Results show that H₂O ice plays key roles in the formation of the nucleobases, as an oxidant, as a matrix in which reactions can take place, and as a catalyst that assists proton abstraction from intermediate compounds. As H₂O is also the most abundant icy component in most cold astrophysical environments, it probably plays the same roles in space in the formation of biologically relevant compounds. Results also show that although the formation of uracil and cytosine from pyrimidine in ices is fairly straightforward, the formation of thymine is not. This is mostly due to the fact that methylation is a limiting step for its formation, particularly in H₂O-rich ices, where methylation must compete with oxidation. The relative inefficiency of the abiotic formation of thymine to that of uracil and cytosine, together with the fact that thymine has not been detected in meteorites, are not inconsistent with the RNA world hypothesis. Indeed, a lack of abiotically produced thymine delivered to the early Earth may have forced the choice for an RNA world, in which only uracil and cytosine are needed, but not thymine. PMID:24500331

  12. 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.

  13. Nuclear quadrupole resonance of 14N and 2H in pyrimidines, purines, and their nucleosides

    NASA Astrophysics Data System (ADS)

    Rabbani, S. R.; Edmonds, D. T.; Gosling, P.

    Using nuclear quadrupole double-resonance techniques, nitrogen-14 and deuterium nuclear quadrupole coupling constants and asymmetry parameters have been measured in uracil, 5-bromouracil, cytosine, adenine, xanthine, hypoxanthine, their nucleosides, 2-aminopyrimidine, and benzimidazole. Zeeman studies and the detection of the simultaneous transitions of neighboring nuclei allowed in many cases a complete assignment of the observed spectral lines to particular 14N and 2D sites.

  14. Identification and Functional Characterization of the First Nucleobase Transporter in Mammals

    PubMed Central

    Yamamoto, Syunsuke; Inoue, Katsuhisa; Murata, Tomoaki; Kamigaso, Syunsuke; Yasujima, Tomoya; Maeda, Jun-ya; Yoshida, Yukihiro; Ohta, Kin-ya; Yuasa, Hiroaki

    2010-01-01

    Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 μm. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use. PMID:20042597

  15. a Nucleoside Under Observation in the Gas Phase: a Rotational Study of Uridine

    NASA Astrophysics Data System (ADS)

    Peña, Isabel; Alonso, José L.

    2014-06-01

    The nucleoside of uridine has been placed in the gas phase by laser ablation and the most stable C2{'}-anti conformation characterized by broadband chirped pulse (CP-FTMW) and narrowband molecular beam Fourier transform microwave (LA-MB-FTMW) spectroscopies. The quadrupole hyperfine structure, originated by two 14N nuclei, has been completely resolved. Intramolecular hydrogen bonds involving uracil and ribose moieties have been found to play an important role in the stabilization of the nucleoside.

  16. A source for microhydrated biomolecules.

    PubMed

    Förstel, M; Neustetter, M; Denifl, S; Lelievre, F; Hergenhahn, U

    2015-07-01

    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. PMID:26233352

  17. A four-unit [c2]daisy chain connected by hydrogen bonds.

    PubMed

    Zhang, Qian; Zhang, Chun-Hang; Yang, Jun-Hui; Xin, Peng-Yang; Xuan, Xiao-Peng; Wang, Jian-Ge; Ma, Na-Na; Guo, Hai-Ming; Qu, Gui-Rong

    2015-10-25

    A mono-adenine-functionalized pillar[5]arene and a guest including uracil were prepared. They formed a novel four-unit [c2]daisy chain both in the solid state and in a chloroform solution. As far as we know, this [c2]daisy chain is the first one without a covalently bound linear thread. This unique assembly behavior is mainly induced by hydrogen-bond interactions between A and U in the A-U base pairs. PMID:26376755

  18. 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.

  19. 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. PMID:26861186

  20. UFT plus leucovorin in advanced hepatobiliary tumors and pancreatic adenocarcinomas.

    PubMed

    Mani, S

    1997-09-01

    UFT (tegafur and uracil) has been studied extensively in Japan, with documented efficacy in hepatobiliary and pancreatic cancer. In the United States, UFT with or without leucovorin has not undergone phase II testing in these malignancies. Our current trial is designed primarily to assess the efficacy in terms of response rates to UFT with leucovorin in patients with advanced hepatobiliary and pancreatic cancer. Secondary objectives include determining response duration, time to disease progression, overall survival, quality of life, and toxicity. PMID:9348584

  1. 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

  2. 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.

  3. Low energy positron interactions with uracil—Total scattering, positronium formation, and differential elastic scattering cross sections

    SciTech Connect

    Anderson, E. K.; Boadle, R. A.; Machacek, J. R.; Makochekanwa, C.; Sullivan, J. P.; Chiari, L.; Buckman, S. J.; Brunger, M. J.; Garcia, G.; Blanco, F.; Ingolfsson, O.

    2014-07-21

    Measurements of the grand total and total positronium formation cross sections for positron scattering from uracil have been performed for energies between 1 and 180 eV, using a trap-based beam apparatus. Angular, quasi-elastic differential cross section measurements at 1, 3, 5, 10, and 20 eV are also presented and discussed. These measurements are compared to existing experimental results and theoretical calculations, including our own calculations using a variant of the independent atom approach.

  4. Relative Angle-Differential Cross Sections for Elastic Electron Scattering from Pyrimidine

    NASA Astrophysics Data System (ADS)

    Maljkovic, J. B.; Milosavljevic, A. R.; Sevic, D.; Marinkovic, B. P.

    2008-07-01

    Angle-differential cross sections for elastic scattering of electrons from pyrimidine are reported for the incident energies from 50-300 eV. Measurements were performed using a cross-beam technique, for scattering angles from 20^o to 110^o. Experimental relative elastic differential cross sections are compared with recent theoretical results for uracil, which is a pyrimidine base and a component of ribonucleic acid.

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

    PubMed Central

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

    2012-01-01

    SUMMARY 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. PMID:22442379

  6. Purines and pyrimidines in sediments from lake erie.

    PubMed

    Van Der Velden, W; Schwartz, A W

    1974-08-23

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

  7. 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

  8. Designing DNA interstrand lock for locus-specific methylation detection in a nanopore

    PubMed Central

    Kang, Insoon; Wang, Yong; Reagan, Corbin; Fu, Yumei; Wang, Michael X.; Gu, Li-Qun

    2013-01-01

    DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg2+) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5′-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer. PMID:24135881

  9. Determination of pyrimidine dimers in DNA by high-performance liquid chromatography/gas chromatography and electron capture detection

    SciTech Connect

    Ramsey, R.S.; Ho, C. )

    1989-11-01

    Exposure of DNA to uv radiation results in the formation of a number of photoproducts including the cyclobutyl pyrimidine dimers. At low uv fluences the concentrations of these dimeric compounds are only a small fraction of the corresponding DNA pyrimidine concentration (e.g., as low as 0.02% or less of the total thymine content). Sensitive methods of analysis are therefore required for accurate determinations. Analytical methodology based upon HPLC fractionation and electrophore labeling followed by GC/electron capture detection (ECD) has been developed to quantitate these species. Separation of thymine-thymine, thymine-uracil, and uracil-uracil from the monomeric bases and from other constituents present in acid-hydrolyzed DNA is achieved by reversed-phase HPLC. Isolation of the dimeric fractions is followed by off-line derivatization to form pentafluorobenzyl products for analysis by GC/ECD. All active hydrogens are alkylated, yielding products with high response factors and detection limits in the low femtomole range. The overall analytical scheme for the determination of pyrimidine dimers in DNA is presented.

  10. UPRT, a suicide-gene therapy candidate in higher eukaryotes, is required for Drosophila larval growth and normal adult lifespan.

    PubMed

    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

  11. 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.

  12. XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes

    PubMed Central

    Saribasak, Huseyin; Maul, Robert W.; Cao, Zheng; McClure, Rhonda L.; Yang, William; McNeill, Daniel R.; Wilson, David M.

    2011-01-01

    Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer’s patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1+/− splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1+/− B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways. PMID:21967769

  13. Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD).

    PubMed

    Zhang, Xin; Zhao, Yuan; Yan, Honggao; Cao, Zexing; Mo, Yirong

    2016-05-15

    Extensive combined quantum mechanical (B3LYP/6-31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton-transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2-pyrimidinone. The rate-determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc. PMID:26813441

  14. 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.

  15. 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. PMID:26801745

  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. Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice.

    PubMed

    Kulkarni, A D; Fanslow, W C; Drath, D B; Rudolph, F B; Van Buren, C T

    1986-02-01

    Although enzyme defects in purine metabolism have revealed the importance of these substrates to maintenance of a normal immune response, the role of exogenous nucleotides on the cells that mediate the host defense system has remained largely unexplored. Recent investigations have revealed that dietary nucleotides are vital to the maintenance of cell-mediated responses to antigen stimulation. To test the influence of dietary nucleotide deprivation on resistance to infection, Balb/c mice were maintained on chow, a nucleotide-free (NF) diet, or an NF diet repleted with adenine, uracil, or RNA. Mice on the NF diet suffered 100% mortality following intravenous challenge with Staphylococcus aureus, while chow-fed and RNA- or uracil-repleted mice demonstrated significantly greater resistance to this bacterial challenge. Macrophages from mice on the NF diet had decreased phagocytic activity as measured by uptake of radiolabeled bacteria compared with mice maintained on the NF diet supplemented with adenine, uracil, or RNA. No change in S aureus antibody response was noted on the various diets. Although the mechanism of this suppression of nonspecific immunity remains unclear, provision of nucleotides to defined diets appears vital to maintain host resistance to bacterial challenge. PMID:3947217

  18. 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

  19. 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.

  20. (13)C-5-FU breath test current status and future directions: a comprehensive review.

    PubMed

    Ezzeldin, Hany H; Acosta, Edward P; Mattison, Lori K; Fourie, Jeanne; Modak, Anil; Diasio, Robert B

    2009-12-01

    Breath tests (BTs) represent a safe non-invasive alternative strategy that could provide valuable diagnostic information in conditions like fat malabsorption, carbohydrate (lactose and fructose) malabsorption, liver dysfunction, impaired gastric emptying, abnormal small bowel transit time, small intestinal bacterial overgrowth and Helicobacter pylori infection. To date, despite the availability of a number of breath tests, only three have gained approval by the FDA for application in a clinical setting ((13)C-urea breath test for the detection of H. pylori; NO breath test for monitoring asthma and alkane breath test for heart transplant rejection). Unfortunately, none of these tests investigate cancer patients or response to cancer chemotherapy. Several years ago it was realized that the presence of a reliable non-invasive approach could assist in the detection of patients at risk of developing severe life-threatening toxicities prior to the administration of fluoropyrimidines (e.g. 5-FU) or related cancer chemotherapy. 5-FU toxicity results mainly from deficient uracil catabolism. This review discusses the development of a BT that utilizes an orally administered pyrimidine ([2-(13)C]-uracil) which is metabolized via the same catabolic pathway as 5-FU. This ([2-(13)C]-uracil) breath test could provide a valuable addition to the patients' standard of care. PMID:21386199

  1. Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog

    NASA Technical Reports Server (NTRS)

    Yamauchi, Keiko; Hales, Nathan W.; Robinson, Sandra M.; Niehoff, Michael L.; Ramesh, Vani; Pellis, Neal R.; Kulkarni, Anil D.

    2002-01-01

    Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions.

  2. 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

  3. Nonreplicating, cyst-defective type II Toxoplasma gondii vaccine strains stimulate protective immunity against acute and chronic infection.

    PubMed

    Fox, Barbara A; Bzik, David J

    2015-05-01

    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

  4. 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

  5. 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

  6. 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

  7. Simulation of a tetramer form of 5-chlorouracil: The vibrational spectra and molecular structure in the isolated and in the solid state by using DFT calculations

    NASA Astrophysics Data System (ADS)

    Ortiz, S.; Alcolea Palafox, M.; Rastogi, V. K.; Akitsu, T.; Hubert Joe, I.; Kumar, Satendra

    2013-06-01

    A Raman and FT-IR study of the biomolecule 5-chlorouracil in the solid state was carried out. The unit cell found in the crystal was simulated as a tetramer form by density functional calculations. They were performed to clarify the assignments of the experimentally observed bands in the spectra. Calculations in the monomer form and comparisons with the experimental data in Ar matrix were also carried out. The error in the calculated frequencies was analyzed and reduced by using scaling equations and scaling factors deduced from the uracil molecule. The calculations with the B3LYP method and with the 6-31G(d,p) and 6-311+G(2d,p) basis set, appear in general to be useful, when combining with a scaling equation procedure or with the specific scale factors, for interpretation of the general features of the IR and Raman spectra. The scaled values were used in the reassignment of the IR and Raman experimental bands. Comparison of the results with those determined in uracil and 5-halogenated derivatives were performed. The substitution at 5-position of the uracil ring by a chlorine atom has a little effect on the geometric parameters.

  8. 21 CFR 74.2602a - Ext. D&C Violet No. 2.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... with good manufacturing practice. (d) Labeling. The label of the color additive shall conform to the... color additive Ext. D&C Violet No. 2 is principally the monosodium salt of 2- -5-methyl-benzenesulfonic... avoided by good manufacturing practice: Sum of volatile matter (at 135 °C) and chlorides and...

  9. 21 CFR 74.2602a - Ext. D&C Violet No. 2.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... with good manufacturing practice. (d) Labeling. The label of the color additive shall conform to the... color additive Ext. D&C Violet No. 2 is principally the monosodium salt of 2- -5-methyl-benzenesulfonic... avoided by good manufacturing practice: Sum of volatile matter (at 135 °C) and chlorides and...

  10. 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

  11. 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…

  12. 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...

  13. 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…

  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. 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...

  16. Alteration of the DNA methylation status of donor cells impairs the developmental competence of porcine cloned embryos.

    PubMed

    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

    2016-01-01

    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

  17. 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...

  18. Recent Advances in the Chemistry and Biological Activities of the Pimpinella Species of Turkey

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two new phenylpropanoids [4-(2-propenyl)phenylangelate (1), 4-(3-methyloxiranyl)phenyl 2-methylbutyrate (3)], one new bisabolene-type sesquiterpenoid [1-methyl-4-(5-methyl-1-methylene-hex-4-enyl)-7-oxa-bicyclo[4.1.0] heptane = aureane (2)], and one new trinorsesquiterpene [4-(6-methyl-bicyclo[4.1.0]...

  19. The Nuclear Transcription Factor RAR Associates with Neuronal RNA Granules and Suppresses Translation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    All-trans-retinoic acid stimulates dendritic growth in hippocampal neurons within minutes by activating mitogen-activated protein kinase and mTOR and increasing dendritic translation of calcium calmodulin-dependent protein kinase II alpha and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionat...

  20. 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…

  1. 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…

  2. 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...

  3. 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...

  4. Indoloquinolines as scaffolds for drug discovery.

    PubMed

    Lavrado, J; Moreira, R; Paulo, A

    2010-01-01

    Traditional medicines have contributed greatly over the centuries to the discovery and development of new therapeutic agents and indoloquinoline alkaloids may represent a new class of drug leads. Cryptolepine (5-methyl-5Hindolo[3,2-b]quinoline), neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline), isocryptolepine (5-methyl-5H-indolo[3,2-c]quinoline, extracted from the African medicinal plant Cryptolepis sanguinolenta, and isoneocryptolepine (5-methyl-5Hindolo[2,3-c]quinoline), which has never been found in nature, are isomeric tetracyclic compounds of particular interest due to their broad spectrum of biological activities including antiparasitic, antifungal, antibacterial, cytotoxic, anti-inflammatory and antihyperglycaemic. As a result, in the last 30 years hundreds of indoloquinoline analogues were synthesized and their biological activities evaluated. In this paper, we present an overview of the potential of indoloquinolines as scaffolds in drug discovery by reviewing the in vitro and in vivo biological activities of natural and synthetic analogues, as well as the proposed mechanisms of action and structure-activity relationships. PMID:20491639

  5. 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...

  6. 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)

  7. 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

  8. Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria

    PubMed Central

    Anderson, Donald D.; Quintero, Cynthia M.; Stover, Patrick J.

    2011-01-01

    The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity. PMID:21876188

  9. 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

  10. Vitamin B-12 deficiency induces anomalies of base substitution and methylation in the DNA of rat colonic epithelium.

    PubMed

    Choi, Sang-Woon; Friso, Simonetta; Ghandour, Haifa; Bagley, Pamela J; Selhub, Jacob; Mason, Joel B

    2004-04-01

    Derangements of one-carbon metabolism can directly affect the integrity of the genome by producing inappropriate uracil insertion into DNA and by altering patterns of DNA methylation. Vitamin B-12, a one-carbon nutrient, serves as a cofactor in the synthesis of precursors of biological methylation and in nucleotide synthesis. We therefore examined whether vitamin B-12 deficiency can induce these molecular anomalies in the colonic mucosa of rats. Weanling male Sprague-Dawley rats (n = 30) were divided into 2 groups and fed either a vitamin B-12-deficient diet or a similar diet containing adequate amounts of the vitamin. Rats from each group were killed at 6 and 10 wk. Uracil misincorporation into DNA was measured by GC/MS and genomic DNA methylation was measured by LC/MS. Plasma vitamin B-12 concentrations in deficient rats were below detectable limits at 6 and 10 wk; in control rats, concentrations were 0.46 +/- 0.07 and 0.42 +/- 0.10 nmol/L at those times. Although the colon total folate concentration did not differ between the groups, the proportion that was methylfolate was marginally greater in the deficient rats at 10 wk (P = 0.05) compared with control, consistent with the "methylfolate trap" that develops during vitamin B-12 deficiency. After 10 wk, the colonic DNA of the deficient rats displayed a 35% decrease in genomic methylation and a 105% increase in uracil incorporation (P < 0.05). This vitamin B-12-deficient diet, which was of insufficient severity to cause anemia or illness, created aberrations in both base substitution and methylation of colonic DNA, which might increase susceptibility to carcinogenesis. PMID:15051821

  11. A novel role of the tumor suppressor GNMT in cellular defense against DNA damage.

    PubMed

    Wang, Yi-Cheng; Lin, Wei-Li; Lin, Yan-Jun; Tang, Feng-Yao; Chen, Yi-Ming; Chiang, En-Pei Isabel

    2014-02-15

    Glycine N-methyltransferase (GNMT) is a folate binding protein commonly diminished in human hepatoma yet its role in tumor development remains to be established. GNMT binds to methylfolate but is also inhibited by it; how such interactions affect human carcinogenesis is unclear. We postulated that GNMT plays a role in folate-dependent methyl group homeostasis and helps maintain genome integrity by promoting nucleotide biosynthesis and DNA repair. To test the hypothesis, GNMT was over-expressed in GNMT-null cell lines cultured in conditions of folate abundance or restriction. The partitioning of folate dependent 1-carbon groups was investigated using stable isotopic tracers and GC/MS. DNA damage was assessed as uracil content in cell models, as well as in Gnmt wildtype (Gnmt(+/+)), heterozygote (Gnmt(+/-)) and knockout (Gnmt(-/-)) mice under folate deplete, replete, or supplementation conditions. Our study demonstrated that GMMT 1) supports methylene-folate dependent pyrimidine synthesis; 2) supports formylfolate dependent purine syntheses; 3) minimizes uracil incorporation into DNA when cells and animals were exposed to folate depletion; 4) translocates into nuclei during prolonged folate depletion. In conclusion, loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. To our best knowledge, the role of GNMT in folate dependent 1-carbon transfer in nucleotide biosynthesis has never been investigated. The present study gives new insights into the underlying mechanism by which GNMT can participate in tumor prevention/suppression in humans. PMID:23922098

  12. 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

  13. 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

  14. 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

  15. 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

  16. Effect of adenosine on the supramolecular architecture and activity of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Singh, Udai P.; Kashyap, Sujata; Singh, Hari Ji; Mishra, Bhupesh Kumar; Roy, Partha; Chakraborty, Ajanta

    2012-04-01

    The reactions of adenosine (Ad) with 5-halouracils (5XU where X = F for 1, Cl for 2, Br for 3 and I for 4) resulted in the formation of co-crystals 1-4 in monoclinic with P21 space group. Despite of great variation in the halo substituent at the 5th position of the uracil, each structure contains the same number and same type of non-covalent interactions i.e., primary N-H⋯N, N-H⋯O, O-H⋯N, O-H⋯O hydrogen bonds and secondary C-H⋯O and X⋯O interactions within these motifs as well as with neighboring molecules. As compared to Ad the size of cavity increases in co-crystal 1 to accommodate the 5FU as a guest. With the variation of halogen from fluoro to iodo on the uracil, the orientation of the molecules remains the same with a slight difference in the dihedral angle in all the co-crystals 1-4. This study demonstrates that hydrogen-bonded interactions between adenosine and halouracils provide a supramolecular assembly to these co-crystals. Computational studies illustrate that the size of the halo substituents on uracil has no effect on the hydrogen bond interaction energy. It further reveals that the orientation of molecules remain same in both solid phase as well as in the gaseous phase. The antitumor and DNA cleavage activity studies show that the antitumor activity of 5-fluorouracil against MCF-7 breast cancer decreases in the presence of adenosine.

  17. 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

  18. 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.

  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. [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