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European Journal of Biochemistry Dec 1996We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case paired with adenine on the opposite strand and the...
We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case paired with adenine on the opposite strand and the other with guanine. For the N4-methoxycytosine adenine pair, only the imino form of the N4-methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N4-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the cis conformation of the OCH3 group in the single strand, which inhibits base pair formation. For the N4-methoxycytosine that is base paired with guanine, we observe an amino form in Watson-Crick geometry in slow exchange with a base paired imino form in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 313 K. We have measured the exchange rate between the two forms at 303 K and observed a value of approximately 1 S-1. The relative ratio of amino and imino forms of N4-methoxycytosine is influenced by both the base that is in front and the temperature. Our results explain the preferential replacement of dTTP by N4-methoxycytosine in primer elongation.
Topics: Adenine; Base Composition; Cytosine; Guanine; Hydrogen Bonding; Hydroxylamines; Kinetics; Magnetic Resonance Spectroscopy; Models, Theoretical; Mutagenesis; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Thermodynamics
PubMed: 8973643
DOI: 10.1111/j.1432-1033.1996.0271r.x -
Biochimica Et Biophysica Acta Aug 2010Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of...
Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90 degrees light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 microM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.
Topics: Animals; Bee Venoms; Benzothiazoles; Enzyme Activation; Fluorescent Dyes; In Vitro Techniques; Kinetics; Microscopy, Electron, Transmission; Multiprotein Complexes; Oxidative Stress; Phosphatidylcholines; Phospholipases A2; Spectrometry, Fluorescence; Thiazoles
PubMed: 20462500
DOI: 10.1016/j.bbamem.2010.05.002 -
Advanced Synthesis & Catalysis Jun 2017The catalytic promiscuity of a ferulic acid decarboxylase from sp. (FDC_s) and phenolic acid decarboxylases (PADs) for the asymmetric conjugate addition of water across...
The catalytic promiscuity of a ferulic acid decarboxylase from sp. (FDC_s) and phenolic acid decarboxylases (PADs) for the asymmetric conjugate addition of water across the C=C bond of hydroxystyrenes was extended to the N-, C- and S-nucleophiles methoxyamine, cyanide and propanethiol to furnish the corresponding addition products in up to 91% . The products obtained from the biotransformation employing the most suitable enzyme/nucleophile pairs were isolated and characterized after optimizing the reaction conditions. Finally, a mechanistic rationale supported by quantum mechanical calculations for the highly ()-selective addition of cyanide is proposed.
PubMed: 28713228
DOI: 10.1002/adsc.201700247 -
Nucleic Acids Research Oct 1995The major initial product of riboflavin- and methylene blue-mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen-saturated aqueous solution has previously...
Photooxidation of d(TpG) by riboflavin and methylene blue. Isolation and characterization of thymidylyl-(3',5')-2-amino-5-[(2-deoxy-beta-D- erythro-pentofuranosyl)amino]-4H-imidazol-4-one and its primary decomposition product thymidylyl-(3',5')-2,2-diamino-4-[(2-deoxy-beta-D-...
The major initial product of riboflavin- and methylene blue-mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen-saturated aqueous solution has previously been identified as 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino] 4H-imidazol-4-one (dlz). At room temperature in aqueous solution dlz decomposes quantitatively to 2,2-diamino-4-[(2-deoxy-beta-D-erythro- pentofuranosyl)amino]-5(2H)-oxazolone (dZ). The data presented here show that the same guanine photooxidation products are generated following riboflavin- and methylene blue-mediated photosensitization of thymidylyl-(3',5')-2'-deoxyguanosine [d(TpG)]. As observed for the monomers, the initial product, thymidylyl-(3',5')-2-amino-5-[(2-deoxy- beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one [d(Tplz)], decomposes in aqueous solution at room temperature to thymidylyl-(3',5')-2,2-diamino-4- [(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone [d(TpZ)]. Both modified dinucleoside monophosphates have been isolated by HPLC and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, chemical analyses and enzymatic digestions. Among the chemical and enzymatic properties of these modified dinucleoside monophosphates are: (i) d(Tplz) and d(TpZ) are alkali-labile; (ii) d(Tplz) reacts with methoxyamine, while d(TpZ) is unreactive; (iii) d(Tplz) is digested by snake venom phosphodiesterase, while d(TpZ) is unaffected; (iv) relative to d(TpG), d(TpZ) and d(Tplz) are slowly digested by spleen phosphodiesterase; (v) d(Tplz) and d(TpZ) can be 5'-phosphorylated by T4 polynucleotide kinase. The first observation suggests that dlz and dZ may be responsible for some of the strand breaks detected following hot piperidine treatment of DNA exposed to photosensitizers.
Topics: Bacteriophage T4; Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Dinucleoside Phosphates; Hot Temperature; Hydrogen-Ion Concentration; Imidazoles; Kinetics; Magnetic Resonance Spectroscopy; Methoxamine; Methylene Blue; Oxazoles; Phosphodiesterase I; Phosphoric Diester Hydrolases; Phosphorylation; Photochemistry; Piperidines; Polynucleotide 5'-Hydroxyl-Kinase; Riboflavin; Spectrometry, Mass, Fast Atom Bombardment; Thymidine
PubMed: 7479042
DOI: 10.1093/nar/23.19.3954 -
Scientific Reports Nov 2015Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of...
UNLABELLED
Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of tumor-selectivity and very limited efficacy. The
NAD(P)H
Quinone Oxidoreductase 1 (NQO1) bioactivatable drug, ß-lapachone (ARQ761 in clinical form), can provide tumor-selective and enhanced synergy with BER inhibition. ß-Lapachone undergoes NQO1-dependent futile redox cycling, generating massive intracellular hydrogen peroxide levels and oxidative DNA lesions that stimulate poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Rapid NAD(+)/ATP depletion and programmed necrosis results. To identify BER modulators essential for repair of ß-lapachone-induced DNA base damage, a focused synthetic lethal RNAi screen demonstrated that silencing the BER scaffolding protein, XRCC1, sensitized PDA cells. In contrast, depleting OGG1 N-glycosylase spared cells from ß-lap-induced lethality and blunted PARP1 hyperactivation. Combining ß-lapachone with XRCC1 knockdown or methoxyamine (MeOX), an apyrimidinic/apurinic (AP)-modifying agent, led to NQO1-dependent synergistic killing in PDA, NSCLC, breast and head and neck cancers. OGG1 knockdown, dicoumarol-treatment or NQO1- cancer cells were spared. MeOX + ß-lapachone exposure resulted in elevated DNA double-strand breaks, PARP1 hyperactivation and TUNEL+ programmed necrosis. Combination treatment caused dramatic antitumor activity, enhanced PARP1-hyperactivation in tumor tissue, and improved survival of mice bearing MiaPaca2-derived xenografts, with 33% apparent cures.
SIGNIFICANCE
Targeting base excision repair (BER) alone has limited therapeutic potential for pancreatic or other cancers due to a general lack of tumor-selectivity. Here, we present a treatment strategy that makes BER inhibition tumor-selective and NQO1-dependent for therapy of most solid neoplasms, particularly for pancreatic cancer.
Topics: Animals; Autophagy; Catalase; Cell Line, Tumor; Cell Survival; DNA Breaks, Double-Stranded; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; Dicumarol; Female; Humans; Hydroxylamines; Mice; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Pancreatic Neoplasms; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Transplantation, Heterologous; X-ray Repair Cross Complementing Protein 1
PubMed: 26602448
DOI: 10.1038/srep17066 -
Journal of Thoracic Oncology : Official... Apr 2011Soy isoflavones sensitize cancer cells to radiation both in vitro and in vivo. To improve the effect of radiotherapy for non-small cell lung cancer, we assessed the...
INTRODUCTION
Soy isoflavones sensitize cancer cells to radiation both in vitro and in vivo. To improve the effect of radiotherapy for non-small cell lung cancer, we assessed the potential of using a complementary approach with soy isoflavones.
METHODS
Human A549 non-small cell lung cancer cells were treated with soy isoflavones, radiation, or both and tested for cell growth. DNA double-strand breaks (DSBs) were detected by immunostaining for γ-H2AX foci. Expressions of γ-H2AX, HIF-1α, and APE1/Ref-1 were assessed by Western blots. DNA-binding activities of HIF-1α and NF-κB transcription factors were analyzed by electrophoretic mobility shift assay.
RESULTS
Soy isoflavones increased A549 cell killing induced by radiation. Multiple γ-H2AX foci were detectable at 1 hour after radiation but decreased at 24 hours after radiation. Soy isoflavones also caused DNA DSBs, but γ-H2AX foci increased over time. Soy isoflavones and radiation caused an increase in γ-H2AX foci, which persisted at 24 hours, indicating both increased DNA damage and inhibition of repair. Soy isoflavones inhibited the radiation-induced activity of the DNA repair/redox enzyme APE1/Ref-1 and the transcription factors NF-κB and HIF-1α. E3330, which inhibits the redox activity of APE1/Ref-1, did not alter the repair of radiation-induced DSBs. Methoxyamine, which inhibits APE1/Ref-1 DNA repair activity, partly blocked the decrease in radiation-induced DSBs at 24 hours, suggesting partial mitigation of radiation-induced DNA repair akin to the effect of soy combined with radiation, in agreement with cytotoxic assays.
CONCLUSIONS
Inhibition of APE1/Ref-1 DNA repair activity by soy could be involved in the mechanism by which soy alters DNA repair and leads to cell killing.
Topics: Benzoquinones; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; DNA Damage; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique; Histones; Humans; Hydroxylamines; Hypoxia-Inducible Factor 1, alpha Subunit; Isoflavones; Lung Neoplasms; NF-kappa B; Propionates; Glycine max; Tumor Cells, Cultured; X-Rays
PubMed: 21325978
DOI: 10.1097/JTO.0b013e31821034ae -
Synlett : Accounts and Rapid... Aug 2018The direct, stereospecific amination of alkylboronic and borinic esters can be conducted by treatment of the organoboron compound with methoxyamine and potassium...
The direct, stereospecific amination of alkylboronic and borinic esters can be conducted by treatment of the organoboron compound with methoxyamine and potassium -butoxide. In addition to being stereospecific, this process also enables the direct amination of tertiary boronic esters in an efficient fashion.
PubMed: 30631220
DOI: 10.1055/s-0037-1610172 -
IET Systems Biology Apr 2013Base excision repair (BER) is a major DNA repair pathway involved in the processing of exogenous non-bulky base damages from certain classes of cancer chemotherapy drugs...
Base excision repair (BER) is a major DNA repair pathway involved in the processing of exogenous non-bulky base damages from certain classes of cancer chemotherapy drugs as well as ionising radiation (IR). Methoxyamine (MX) is a small molecule chemical inhibitor of BER that is shown to enhance chemotherapy and/or IR cytotoxicity in human cancers. In this study, the authors have analysed the inhibitory effect of MX on the BER pathway kinetics using a computational model of the repair pathway. The inhibitory effect of MX depends on the BER efficiency. The authors have generated variable efficiency groups using different sets of protein concentrations generated by Latin hypercube sampling, and they have clustered simulation results into high, medium and low efficiency repair groups. From analysis of the inhibitory effect of MX on each of the three groups, it is found that the inhibition is most effective for high efficiency BER, and least effective for low efficiency repair.
Topics: Animals; Antineoplastic Agents; Computer Simulation; DNA Repair; DNA, Neoplasm; Drug Design; Drug Therapy, Computer-Assisted; Humans; Hydroxylamines; Models, Biological; Molecular Targeted Therapy; Neoplasms
PubMed: 23847811
DOI: 10.1049/iet-syb.2011.0045 -
Chromatographia 2014Wound healing is still a serious medical problem due to process complexity and lack of effective medicaments. This is particularly true in the treatment of wounds...
Wound healing is still a serious medical problem due to process complexity and lack of effective medicaments. This is particularly true in the treatment of wounds arising in the course of such diseases as AIDS or diabetes. Therefore, scientific efforts are focused on the search for new compounds of natural origin, which could be used as medicines or evaluated for subsequent drug design. In folk medicine, grasshopper ( spp.) abdominal secretion has been used to accelerate the wound healing process. In this context, the knowledge of the composition of grasshopper abdominal secretion is crucial. The aim of this study was to determine the main water-soluble components of grasshopper abdominal secretion with the use of GC/MS/MS. Liquid-liquid extraction was used as a pretreatment method to clean up, concentrate and fractionate compounds from the complex insect matrix. To obtain more stable and volatile compounds, necessary for GC/MS/MS analysis, a double-step derivatization process was carried out with the use of methoxyamine hydrochloride and a mixture of bis--trimethylsilyl trifluoroacetamide and chlorotrimethylsilane. As a result, 2,108 compounds were identified, mainly as amino acids, carbohydrates and organic acids. Some of the identified compounds are emphasized due to antimicrobial, antifungal or antioxidant activities reported in the literature. Moreover, a set of compounds characteristic for spp. samples has been selected. In the last part of the study, a statistical analysis was performed to demonstrate differences in composition of aqueous fractions of abdominal secretions from grasshoppers collected at two distant locations: Starogard Gdański and Łubianka meadows.
PubMed: 25089050
DOI: 10.1007/s10337-014-2679-8 -
Cancer Biology & Therapy 2015Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein,...
Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies.
Topics: Alkylating Agents; Breast Neoplasms; Cell Line, Tumor; Cell Survival; DNA Breaks, Double-Stranded; DNA Repair; Dacarbazine; Down-Regulation; Exonucleases; Female; Histones; Humans; Temozolomide
PubMed: 25849309
DOI: 10.1080/15384047.2015.1016661