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PloS One 2021Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug...
Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.
Topics: Amyotrophic Lateral Sclerosis; Base Sequence; Bromouracil; Cloning, Molecular; Drug Evaluation, Preclinical; Escherichia coli; Humans; Hydrogen Bonding; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Protein Folding; Proton Magnetic Resonance Spectroscopy; Superoxide Dismutase-1; Trifluridine; Tryptophan; Uridine
PubMed: 33635895
DOI: 10.1371/journal.pone.0247684 -
Tetrahedron Dec 2016Isoxazolidine analogues of homonucleos(t)ides were synthesized from nucleobase-derived nitrones (uracil, 5-fluorouracil, 5-bromouracil, thymine, adenine) employing...
Isoxazolidine analogues of homonucleos(t)ides were synthesized from nucleobase-derived nitrones (uracil, 5-fluorouracil, 5-bromouracil, thymine, adenine) employing 1,3-dipolar cycloadditions with allyl alcohol as well as with alkenylphosphonates (allyl-, allyloxymethyl- and vinyloxymethyl- and vinylphosphonate). Besides reactions with vinylphosphonate the additions proceeded regioselectively to produce mixtures of major cis and minor trans 3,5-disubstituted isoxazolidines (d.e. 28-82%). From vinylphosphonate up to 10% of 3,4-disubstituted isoxazolidines was additionally produced. Vicinal couplings, shielding effects and 2D NOE correlations were employed in configurational assignments as well as in conformational analysis to find out preferred conformations for several isoxazolidines and to observe anomeric effects (pseudoaxial orientation of phosphonylmethoxy groups) for those obtained from vinyloxymethylphosphonate. None of the tested compounds were endowed in vitro with antiviral activity against a variety of DNA and RNA viruses at subtoxic concentrations (up to 250 μM) nor exhibited antiproliferative activity towards L1210, CEM, and HeLa cells (IC = ≥100 μM).
PubMed: 32287430
DOI: 10.1016/j.tet.2016.10.073 -
Scientific Reports Oct 2018We studied photochemical reactions of U-substituted G-quadruplex (G4) DNA substrates with two pyrene-substituted polyazamacrocyclic ligands, M-1PY and M-2PY. Both...
We studied photochemical reactions of U-substituted G-quadruplex (G4) DNA substrates with two pyrene-substituted polyazamacrocyclic ligands, M-1PY and M-2PY. Both ligands bind to and stabilize G4-DNA structures without altering their folding topology, as demonstrated by FRET-melting experiments, fluorimetric titrations and CD spectroscopy. Notably, the bis-pyrene derivative (M-2PY) behaves as a significantly more affine and selective G4 ligand, compared with its mono-pyrene counterpart (M-1PY) and control compounds. Upon short UVA irradiation (365 nm) both ligands, in particular M-2PY, efficiently sensitize photoreactions at U residues incorporated in G4 structures and give rise to two kinds of photoproducts, namely DNA strand cleavage and covalent ligand-DNA photoadducts. Remarkably, the photoinduced strand cleavage is observed exclusively with G4 structures presenting U residues in lateral or diagonal loops, but not with parallel G4-DNA structures presenting only propeller loops. In contrast, the formation of fluorescent photoadducts is observed with all U-substituted G4-DNA substrates, with M-2PY giving significantly higher yields (up to 27%) than M-1PY. Both ligand-sensitized photoreactions are specific to U-modified G4-DNA structures with respect to double-stranded or stem-loop substrates. Thus, ligand-sensitized photoreactions with U-substituted G4-DNA may be exploited (i) as a photochemical probe, allowing "photofootprinting" of G4 folding topologies in vitro and (ii) for covalent trapping of G4 structures as photoadducts with pyrene-substituted ligands.
Topics: Bromouracil; DNA; DNA Adducts; G-Quadruplexes; Humans; Kinetics; Ligands; Models, Molecular; Mutation; Photochemical Processes; Telomere; Uridine
PubMed: 30361545
DOI: 10.1038/s41598-018-34141-z -
The Journal of Biological Chemistry Jun 2003Oxidative damage to DNA has been implicated in carcinogenesis during chronic inflammation. Epidemiological and biochemical studies suggest that one potential mechanism...
Oxidative damage to DNA has been implicated in carcinogenesis during chronic inflammation. Epidemiological and biochemical studies suggest that one potential mechanism involves myeloperoxidase, a hemeprotein secreted by human phagocytes. In this study, we demonstrate that human neutrophils use myeloperoxidase to oxidize uracil to 5-chlorouracil in vitro. Uracil chlorination by myeloperoxidase or reagent HOCl exhibited an unusual pH dependence, being minimal at pH approximately 5, but increasing markedly under either acidic or mildly basic conditions. This bimodal curve suggests that myeloperoxidase initially produces HOCl, which subsequently chlorinates uracil by acid- or base-catalyzed reactions. Human neutrophils use myeloperoxidase and H2O2 to chlorinate uracil, suggesting that nucleobase halogenation reactions may be physiologically relevant. Using a sensitive and specific mass spectrometric method, we detected two products of myeloperoxidase, 5-chlorouracil and 5-bromouracil, in neutrophil-rich human inflammatory tissue. Myeloperoxidase is the most likely source of 5-chlorouracil in vivo because halogenated uracil is a specific product of the myeloperoxidase system in vitro. In contrast, previous studies have demonstrated that 5-bromouracil could be generated by either eosinophil peroxidase or myeloperoxidase, which preferentially brominates uracil at plasma concentrations of halide and under moderately acidic conditions. These observations indicate that the myeloperoxidase system promotes nucleobase halogenation in vivo. Because 5-chlorouracil and 5-bromouracil can be incorporated into nuclear DNA, and these thymine analogs are well known mutagens, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.
Topics: Bromouracil; Gas Chromatography-Mass Spectrometry; HL-60 Cells; Humans; Hydrogen-Ion Concentration; Hypochlorous Acid; Inflammation; Mutagenesis; Neutrophils; Peroxidase; Phagocytes; Uracil
PubMed: 12707270
DOI: 10.1074/jbc.M303928200 -
The Journal of Biological Chemistry Jul 1993To test whether ionized base pairs influence polymerase-catalyzed misinsertion rates, we measured the efficiency of forming 5-bromouracil (B), 5-fluorouracil (F), and...
To test whether ionized base pairs influence polymerase-catalyzed misinsertion rates, we measured the efficiency of forming 5-bromouracil (B), 5-fluorouracil (F), and thymine base pairs with guanine and adenine as a function of pH using avian myeloblastosis reverse transcriptase. When B, F, and T were present as dNTP substrates, misincorporation efficiencies opposite G, normalized to incorporation of C opposite G, increased by about 20-, 13-, and 7-fold, respectively, as reaction pH increased from 7.0 to 9.5. Incorporation efficiencies to form the correct base pairs, B.A and F.A, normalized to T.A, decreased by 4- and 8-fold, respectively, with increasing pH. The effects of pH on misincorporation efficiencies were about 10-fold greater when B, F, and T were present as template bases. The relative misincorporation efficiencies of G opposite template B, F, and T, normalized to incorporation of A opposite B, F, and T, increased by about 430-, 370-, and 70-fold, respectively, as pH was increased from 6.5 to 9.5, while correct incorporation of A opposite template B and F decreased about 10-fold over the same pH range. Plots depicting incorrect and correct incorporation efficiencies versus pH were fit to a pH titration equation giving the fraction of ionized base as a function of pH. We conclude that avian myeloblastosis reverse transcriptase forms B.G and F.G mispairs in an ionized Watson-Crick conformation in preference to a neutral wobble structure containing favored keto tautomers of B or F. Although participation of disfavored enol tautomers in enzyme-catalyzed base mispair formation cannot be ruled out, the results are inconsistent with the "standard" disfavored tautomer model of mutagenesis. Instead, the data support a model in which ionization of halouracil bases is primarily responsible for B- and F-induced mutagenesis.
Topics: Base Sequence; Bromodeoxyuridine; Bromouracil; DNA; Floxuridine; Fluorouracil; Guanine; Hydrogen Bonding; Hydrogen-Ion Concentration; Ions; Molecular Sequence Data; Nucleic Acid Heteroduplexes; RNA-Directed DNA Polymerase; Templates, Genetic; Thymine Nucleotides
PubMed: 7688001
DOI: No ID Found -
Journal of Visualized Experiments : JoVE May 2018When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or...
When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.
Topics: Bromouracil; Immunoprecipitation; Polymerase Chain Reaction; RNA; Uridine
PubMed: 29782024
DOI: 10.3791/57056 -
Genes and Environment : the Official... 2016Cytosine residues in CpG dinucleotides often undergo various types of modification, such as methylation, deamination, and halogenation. These types of modifications can...
INTRODUCTION
Cytosine residues in CpG dinucleotides often undergo various types of modification, such as methylation, deamination, and halogenation. These types of modifications can be pro-mutagenic and can contribute to the formation of mutational hotspots in cells. To analyze mutations induced by DNA modifications in the human genome, we recently developed a system for tracing DNA adducts in targeted mutagenesis (TATAM). In this system, a modified/damaged base is site-specifically introduced into intron 4 of thymidine kinase genes in human lymphoblastoid cells. To further the understanding of the mutagenesis of cytosine modification, we directly introduced different types of altered cytosine residues into the genome and investigated their genomic consequences using the TATAM system.
FINDINGS
In the genome, the pairing of thymine and 5-bromouracil with guanine, resulting from the deamination of 5-methylcytosine and 5-bromocytosine, respectively, was highly pro-mutagenic compared with the pairing of uracil with guanine, resulting from the deamination of cytosine residues.
CONCLUSIONS
The deamination of 5-methylcytosine and 5-bromocytosine rather than that of normal cytosine dramatically enhances the mutagenic potential in the human genome.
PubMed: 27588157
DOI: 10.1186/s41021-016-0045-9 -
International Journal of Environmental... Mar 2021Chlorpyrifos, Bromacil and Terbuthylazine are commonly used as insecticides and herbicides to control weeds and prevent non-desirable growth of algae, fungi and bacteria...
Chlorpyrifos, Bromacil and Terbuthylazine are commonly used as insecticides and herbicides to control weeds and prevent non-desirable growth of algae, fungi and bacteria in many agricultural applications. Despite their highly negative effects on human health, environmental modeling of these pesticides in the vadose zone until they reach groundwater is still not being conducted on a regular basis. This work shows results obtained by version 5.08 of the Pesticide Root Zone Model (PRZM5) numerical model to simulate the fate and transport of Chlorpyrifos, Bromacil and Terbuthylazine between 2006 and 2018 inside the Buñol-Cheste aquifer in Spain. The model uses a whole set of parameters to solve a modified version of the mass transport equation considering the combined effect of advection, dispersion and reactive transport processes. The simulation process was designed for a set of twelve scenarios considering four application doses for each pesticide. Results show that the maximum concentration value for every scenario exceeds the current Spanish Maximum Concentration Limit (0.1 μg/L). Numerical simulations were able to reproduce concentration observations over time despite the limited amount of available data.
Topics: Bromouracil; Chlorpyrifos; Groundwater; Humans; Spain; Triazines; Water Pollutants, Chemical
PubMed: 33800654
DOI: 10.3390/ijerph18073511 -
Journal of Nuclear Medicine : Official... Dec 2002We analyzed and compared 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-[methyl-(11)C]thymine ((11)C-FMAU), 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT) and... (Comparative Study)
Comparative Study
UNLABELLED
We analyzed and compared 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-[methyl-(11)C]thymine ((11)C-FMAU), 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-[(76)Br]bromouracil ((76)Br-BFU) with respect to tissue uptake, DNA incorporation, and excretion modulation in rats. The goal of the investigation was to evaluate the efficiency of the 3 nucleoside tracers as potential tracers for measuring proliferation.
METHODS
Sprague-Dawley rats were divided into 3 groups and administered 5 MBq (11)C-FMAU, 1 MBq (18)F-FLT, or 2 MBq (76)Br-BFU. For each tracer, a subgroup was also administered 6 mg/kg cimetidine. The rats in the (11)C-FMAU group were killed at 5, 20, 40, 60, and 80 min after injection; the rats in the (18)F-FLT group were killed at 80 min and 2 and 4 h; and the rats in the (76)Br-BFU group were killed at 5, 20, 40, and 80 min and 2, 4, 6, and 24 h. Samples of blood, liver, kidney, spleen, and intestine were taken, and the radioactivity was measured. DNA separation was made in the samples of spleen, and the radioactivity in the DNA fraction was measured.
RESULTS
Maximal uptake of radioactivity was seen in the spleen and intestine, organs with active DNA synthesis. The highest relative radioactivity uptake was at 60 min in the (11)C-FMAU groups and at 4 h in the (18)F-FLT group. In the (76)Br-BFU group, the uptake increased gradually during the observation period, and uptake of radioactivity increased markedly in rats receiving cimetidine. Cimetidine did not affect radioactivity uptake in the (11)C-FMAU or (18)F-FLT groups. The fraction of radioactivity in DNA was 78% in spleen at 60 min in the (11)C-FMAU group, 80% at 60 min and 97% at 4 h in the (76)Br-BFU group. The DNA-incorporation was only 2% in the (18)F-FLT group.
CONCLUSION
(76)Br-BFU predominantly incorporates into DNA and has great potential as a PET tracer for the assessment of proliferation in vivo. (11)C-FMAU also may have potential as a proliferation marker, but the observation time is limited. (18)F-FLT does not incorporate into DNA and is therefore not a direct marker of proliferation.
Topics: Animals; Arabinofuranosyluracil; Bromine Radioisotopes; Bromouracil; Cell Division; DNA; Dideoxynucleosides; Fluorine Radioisotopes; Male; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Tomography, Emission-Computed
PubMed: 12468521
DOI: No ID Found -
Scientific Reports Dec 2014The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA...
The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA strand breakage induced by low-energy electrons (18 eV) depends on the nucleotide sequence. To determine the absolute cross sections for electron induced single strand breaks in specific 13 mer oligonucleotides we used atomic force microscopy analysis of DNA origami based DNA nanoarrays. We investigated the DNA sequences 5'-TT(XYX)3TT with X = A, G, C and Y = T, BrU 5-bromouracil and found absolute strand break cross sections between 2.66 · 10(-14) cm(2) and 7.06 · 10(-14) cm(2). The highest cross section was found for 5'-TT(ATA)3TT and 5'-TT(ABrUA)3TT, respectively. BrU is a radiosensitizer, which was discussed to be used in cancer radiation therapy. The replacement of T by BrU into the investigated DNA sequences leads to a slight increase of the absolute strand break cross sections resulting in sequence-dependent enhancement factors between 1.14 and 1.66. Nevertheless, the variation of strand break cross sections due to the specific nucleotide sequence is considerably higher. Thus, the present results suggest the development of targeted radiosensitizers for cancer radiation therapy.
Topics: DNA; DNA Breaks; Electrons; Oligonucleotide Array Sequence Analysis
PubMed: 25487346
DOI: 10.1038/srep07391