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1.
Nat Commun ; 12(1): 796, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542236

RESUMO

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Desoxirribose/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
2.
Free Radic Biol Med ; 153: 17-25, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32305647

RESUMO

Pancreatic ß-cells are vulnerable to oxidative stress, which promotes ß-cell failure in type 2 diabetes. System χc- is a sodium-independent, cystine/glutamate antiporter that mediates the exchange of extracellular l-cystine and intracellular l-glutamate. The import of l-cystine through this transporter is the rate-limiting step in the glutathione (GSH) biosynthesis pathway that plays a significant role in antioxidative defense. Previously, we reported that 2-deoxy-d-ribose (dRib) induces oxidative damage through GSH depletion in pancreatic ß-cells. In the current study, we elucidated the mechanism underlying the oxidative stress-induced ß-cell damage. We measured the intracellular l-[14C]cystine uptake, GSH content, reactive oxygen species (ROS) levels, cytotoxicity, and apoptosis in rat insulinoma cell line, RINm5F. Treatment of dRib decreased the intracellular l-[14C]cystine uptake and GSH content and increased the intracellular ROS levels, cytotoxicity, and apoptosis in a time- and dose-dependent manner. Conversely, 2-mercaptoethanol (2-ME), a cystine uptake enhancer, recovered the dRib-induced decrease in l-[14C]cystine uptake, GSH content, and cell viability in a Na+-independent manner. In the case of isolated islets, dRib dose-dependently decreased the intracellular l-[14C]cystine uptake and cell viability; however, dRib-induced cytotoxicity was completely recovered by adding N-acetyl cysteine (NAC). To confirm that system χc- mediates the oxidative stress-induced ß-cell damage, we overexpressed xCT (the substrate-specific subunit of system χc-) using a lentiviral vector in RINm5F cells. Overexpression of xCT fully recovered the dRib-induced decrease in l-[14C]cystine uptake and GSH content and prevented the dRib-induced increase in ROS levels, cytotoxicity, and apoptosis. The overexpression of xCT showed a protective effect against dRib-induced oxidative damage in RINm5F cells. Our study showed that dRib depletes intracellular GSH content through inhibition of cystine transport via system χc- in ß-cells.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Cistina/metabolismo , Desoxirribose/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glutationa/metabolismo , Células Secretoras de Insulina/metabolismo , Estresse Oxidativo , Ratos , Ribose/metabolismo
3.
Mol Syst Biol ; 15(4): e8462, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962359

RESUMO

Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in Escherichia coli K-12 MG1655. After as few as approximately 20 generations, evolved populations repeatedly acquired the capacity to grow on five predicted non-native substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, and monomethyl succinate. Altered promiscuous activities were shown to be directly involved in establishing high-efficiency pathways. Structural mutations shifted enzyme substrate turnover rates toward the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.


Assuntos
Enzimas/química , Enzimas/metabolismo , Escherichia coli K12/crescimento & desenvolvimento , Mutação , Adaptação Fisiológica , Arabinose/metabolismo , Simulação por Computador , Desoxirribose/metabolismo , Enzimas/genética , Escherichia coli K12/enzimologia , Escherichia coli K12/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Evolução Molecular , Especificidade por Substrato , Succinatos/metabolismo , Tartaratos/metabolismo
4.
Molecules ; 23(9)2018 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-30177614

RESUMO

In this era of urbanization and environmental pollution, antioxidants and antimutagens derived from plants are promising safeguards for human health. In the current investigation, we analyzed the antioxidant and antimutagenic effects of the hexane, chloroform, and ethyl acetate fractions of Rhododendron arboreum Sm. leaves and determined their chemical composition. The different fractions inhibited lipid peroxidation, repressed the production of nitric oxide radicals, and prevented deoxyribose degradation. The antimutagenic activity of the leaf fractions was analyzed against 4-nitro-O-phenylenediamine, sodium azide and 2-aminofluorene mutagens in two test strains (TA-98 and TA-100) of Salmonella typhimurium. The experiment was conducted using pre- and co-incubation modes. The best results were obtained in the pre-incubation mode, and against indirect acting mutagen. The presence of a number of bioactive constituents was confirmed in the different fractions by GC-MS analysis. The study reveals the strong antioxidant and antimutagenic activity of R. arboreum leaves. We propose that those activities of R. arboreum might correspond to the combined effect of the phytochemicals identified by GC-MS analysis. To the best of our knowledge, this is the first report on the antimutagenic activity of R. arboreum leaves.


Assuntos
Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Extratos Vegetais/farmacologia , Rhododendron/química , Acetatos/química , Acetatos/farmacologia , Antimutagênicos/química , Antioxidantes/química , Clorofórmio/química , Clorofórmio/farmacologia , Desoxirribose/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hexanos/química , Hexanos/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Folhas de Planta/química
5.
Int J Biol Macromol ; 113: 294-299, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29454056

RESUMO

Glycoxidation of protein may lead to develop diabetes. In the present study, different concentrations of 2-deoxy d-ribose (2-dRib) were used to modify human serum albumin (HSA). Nitro Blue Tetrazolium (NBT) assay results showed that yield of the fructosamine content was directly proportional to the concentration of 2-dRib. UV and fluorescence spectroscopy results showed an increment in hyperchromicity and decrease in fluorescence intensity of 2-dRib modified HSA as compared to native HSA. Further secondary structural changes were confirmed by UV-circular dichroism (UV-CD) and Fourier transform infrared spectroscopy (FT-IR). To evaluate the immunogenicity of 2-dRib modified HSA, rabbits were immunized with native and 2-dRib modified HSA. Modified HSA sera showed high antibodies titre as compared to native HSA. Moreover, the binding affinity of native and modified HSA with diabetic patient's sera has been evaluated by direct binding ELISA. It was found that diabetic patient's sera showed high binding affinity with the modified HSA as compared to native HSA. On the basis of above findings, it can be concluded that 2-dRib is a potential glycating agent that can cause alteration in HSA structure and make HSA more immunogenic that might play a role in onset and progression of diabetes mellitus and its complications.


Assuntos
Desoxirribose/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Albumina Sérica Humana/metabolismo , Animais , Diabetes Mellitus Tipo 2/sangue , Humanos , Coelhos
6.
Plant Sci ; 268: 1-10, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29362078

RESUMO

Accumulating evidence indicates that plant plastocyanin is involved in copper homeostasis, yet the physiological relevance remains elusive. In this study, we found that a plastocyanin gene (SsPETE2) from euhalophyte Suaeda salsa possessed a novel antioxidant function, which was associated with the copper-chelating activity of SsPETE2. In S. salsa, expression of SsPETE2 increased in response to oxidative stress and ectopic expression of SsPETE2 in Arabidopsis enhanced the antioxidant ability of the transgenic plants. SsPETE2 bound Cu ion and alleviated formation of hydroxyl radicals in vitro. Accordingly, SsPETE2 expression lowered the free Cu content that was associated with reduced H2O2 level under oxidative stress. Arabidopsis pete1 and pete2 mutants showed ROS-sensitive phenotypes that could be restored by expression of SsPETE2 or AtPETEs. In addition, SsPETE2-expressing plants exhibited more potent tolerance to oxidative stress than plants overexpressing AtPETEs, likely owing to the stronger copper-binding activity of SsPETE2 than AtPETEs. Taken together, these results demonstrated that plant PETEs play a novel role in oxidative stress tolerance by regulating Cu homeostasis under stress conditions, and SsPETE2, as an efficient copper-chelating PETE, potentially could be used in crop genetic engineering.


Assuntos
Adaptação Fisiológica , Chenopodiaceae/genética , Chenopodiaceae/fisiologia , Expressão Ectópica do Gene , Estresse Oxidativo/genética , Proteínas de Plantas/metabolismo , Plastocianina/genética , Adaptação Fisiológica/efeitos dos fármacos , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Quelantes/farmacologia , Chenopodiaceae/efeitos dos fármacos , Cloroplastos/efeitos dos fármacos , Cloroplastos/metabolismo , Cobre/farmacologia , Desoxirribose/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Radical Hidroxila/metabolismo , Íons , Ferro/metabolismo , Simulação de Dinâmica Molecular , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , Paraquat/farmacologia , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastocianina/metabolismo , Transporte Proteico/efeitos dos fármacos
7.
Free Radic Biol Med ; 107: 146-150, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27867099

RESUMO

Oxygen is both necessary and dangerous for aerobic cell function. ATP is most efficiently made by the electron transport chain, which requires oxygen as an electron acceptor. However, the presence of oxygen, and to some extent the respiratory chain itself, poses a danger to cellular components. Mitochondria, the sites of oxidative phosphorylation, have defense and repair pathways to cope with oxidative damage. For mitochondrial DNA, an essential pathway is base excision repair, which acts on a variety of small lesions. There are instances, however, in which attempted DNA repair results in more damage, such as the formation of a DNA-protein crosslink trapping the repair enzyme on the DNA. That is the case for mitochondrial DNA polymerase γ acting on abasic sites oxidized at the 1-carbon of 2-deoxyribose. Such DNA-protein crosslinks presumably must be removed in order to restore function. In nuclear DNA, ubiquitylation of the crosslinked protein and digestion by the proteasome are essential first processing steps. How and whether such mechanisms operate on DNA-protein crosslinks in mitochondria remains to be seen.


Assuntos
Adutos de DNA/metabolismo , DNA Polimerase gama/metabolismo , Reparo do DNA , Mitocôndrias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Adutos de DNA/química , Dano ao DNA , Desoxirribose/química , Desoxirribose/metabolismo , Radicais Livres/química , Humanos , Mitocôndrias/genética , Oxirredução , Estresse Oxidativo , Ubiquitinação
8.
DNA Repair (Amst) ; 44: 103-109, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27264558

RESUMO

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3'-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase ß (Polß) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide ("long-patch") BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polß cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polß and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polß-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5'-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polß-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15-30min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polß-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60-70% of the trapped ubiquitin depends on Polß. The mechanism of ubiquitin targeting to Polß-DPC, the subsequent processing of the expected 5'-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.


Assuntos
DNA Polimerase beta/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Desoxirribose/metabolismo , Animais , Dano ao DNA , DNA Polimerase beta/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribose/química , Meia-Vida , Humanos , Cetoses/metabolismo , Cinética , Camundongos , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Açúcares Ácidos/metabolismo
9.
Org Biomol Chem ; 14(11): 3120-9, 2016 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-26906931

RESUMO

The substrate scope of fluorinase enzyme mediated transhalogenation reactions is extended. Substrate tolerance allows a peptide cargo to be tethered to a 5'-chloro-5'-deoxynucleoside substrate for transhalogenation by the enzyme to a 5'-fluoro-5'-deoxynucleoside. The reaction is successfully extended from that previously reported for a monomeric cyclic peptide (cRGD) to cargoes of dendritic scaffolds carrying two and four cyclic peptide motifs. The RGD peptide sequence is known to bind upregulated αVß3 integrin motifs on the surface of cancer cells and it is demonstrated that the fluorinated products have a higher affinity to αVß3 integrin than their monomeric counterparts. Extending the strategy to radiolabelling of the peptide cargoes by tagging the peptides with [(18)F]fluoride was only moderately successful due to the poor water solubility of these higher order peptide scaffolds although the strategy holds promise for peptide constructs with improved solubility.


Assuntos
Proteínas de Bactérias/metabolismo , Desoxirribonucleosídeos/química , Desoxirribonucleosídeos/metabolismo , Oxirredutases/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Streptomyces/enzimologia , Desoxirribose/análogos & derivados , Desoxirribose/metabolismo , Halogenação , Humanos , Integrina alfaVbeta3/metabolismo , Modelos Moleculares
10.
Nature ; 529(7585): 231-4, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26735012

RESUMO

Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.


Assuntos
DNA Catalítico/química , Conformação de Ácido Nucleico , Sequência de Bases , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , DNA Catalítico/síntese química , DNA Catalítico/metabolismo , Desoxirribose/química , Desoxirribose/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Polinucleotídeo Ligases/química , Polinucleotídeo Ligases/metabolismo , RNA/química , RNA/metabolismo , Dobramento de RNA , Especificidade por Substrato
11.
Molecules ; 22(1)2016 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-28042856

RESUMO

In the presence of transition metal ions and peroxides, polyphenols, well-known dietary antioxidants, can act as pro-oxidants. We investigated the effect of 13 polyphenols and their metabolites on oxidative degradation of deoxyribose by an •OH generating Fenton system (Fe2+-ethylenediaminetetraacetic acid (EDTA)-H2O2). The relationship between phenolics pro-oxidant/anti-oxidant effects and their molecular structure was analyzed using multivariate analysis with multiple linear regression and a backward stepwise technique. Four phenolics revealed a significant inhibitory effect on OH-induced deoxyribose degradation, ranging from 54.4% ± 28.6% (3,4-dihydroxycinnamic acid) to 38.5% ± 10.4% (catechin) (n = 6), correlating with the number of -OH substitutions (r = 0.58). Seven phenolics augmented the oxidative degradation of deoxyribose with the highest enhancement at 95.0% ± 21.3% (quercetin) and 60.6% ± 12.2% (phloridzin). The pro-oxidant effect correlated (p < 0.05) with the number of -OH groups (r = 0.59), and aliphatic substitutes (r = -0.22) and weakly correlated with the occurrence of a catechol structure within the compound molecule (r = 0.17). Selective dietary supplementation with phenolics exhibiting pro-oxidant activity may increase the possibility of systemic oxidative stress in patients treated with medications containing chelating properties or those with high plasma concentrations of H2O2 and non-transferrin bound iron.


Assuntos
Antioxidantes/química , Desoxirribose/metabolismo , Ácido Edético/química , Oxidantes/química , Fenóis/química , Extratos Vegetais/química , Desoxirribose/química , Frutas/química , Humanos , Peróxido de Hidrogênio/química , Oxirredução , Estresse Oxidativo , Verduras/química
12.
J Photochem Photobiol B ; 143: 82-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25617617

RESUMO

The micronutrients in many cellular processes, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are photo-sensitive to UV and visible light for generating reactive oxygen species (ROS). Produced from phosphorylation of riboflavin, FMN is more water-soluble and rapidly transformed into free riboflavin after ingestion. This study investigated the application of visible blue light with FMN to development of an effective antimicrobial treatment. The photosensitization of bacterial viability with FMN was investigated by light quality, intensity, time, and irradiation dosage. The blue light-induced photochemical reaction with FMN could inactivate Escherichiacoli by the generated ROS in damaging nucleic acids, which was validated. This novel photodynamic technique could be a safe practice for photo-induced inactivation of environmental microorganism to achieve hygienic requirements in food processing.


Assuntos
Escherichia coli/fisiologia , Escherichia coli/efeitos da radiação , Mononucleotídeo de Flavina/metabolismo , Luz , Viabilidade Microbiana/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Cor , DNA Bacteriano/química , DNA Super-Helicoidal/química , Descontaminação , Desoxirribose/metabolismo , Escherichia coli/metabolismo , Fotólise , Plasmídeos/genética
13.
Radiat Res ; 181(2): 131-7, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24410455

RESUMO

A novel analytical high-performance liquid chromatography (HPLC)-based method of quantification of the yields of C4'-oxidized abasic sites, 1, in oxidatively damaged DNA has been elaborated. This new approach is based on efficient conversion of 1 into N-substituted 5-methylene-Δ(3)-pyrrolin-2-ones, 2, upon treatment of damaged DNA with primary amines in neutral or slightly acidic solutions with subsequent quantification of 2 by HPLC. The absolute and relative radiation-chemical yields of 1 in irradiated DNA solutions were re-evaluated using this method. The yields were compared with those of other 2-deoxyribose degradation products including 5-methylene-2(5H)-furanone, malondialdehyde, and furfural resulting from the C1', C4' and C5'-oxidations, respectively. The yield of free base release (FBR) determined in the same systems was employed as an internal measure of the total oxidative damage to the 2-deoxyribose moiety. Application of this technique identifies 1 as the most abundant sugar lesion in double-stranded (ds) DNA irradiated under air in solution (36% FBR). In single-stranded (ss) DNA this product is second by abundance (33% FBR) after 2-deoxyribonolactones (C1'-oxidation; 43% FBR). The production of nucleoside-5'-aldehydes (C5'-oxidation; 14% and 5% FBR in dsDNA and ssDNA, respectively) is in the third place. Taken together with the parallel reaction channel that converts C4'-radicals into malondialdehyde and 3'-phosphoglycolates, our results identify the C4'-oxidation as a prevalent pathway of oxidative damage to the sugar-phosphate backbone (50% or more of all 2-deoxyribose damages) in indirectly damaged DNA.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dano ao DNA , DNA/química , DNA/genética , Desoxirribose/metabolismo , Raios gama/efeitos adversos , DNA/metabolismo , Oxirredução/efeitos da radiação
14.
J Phys Chem B ; 117(51): 16397-404, 2013 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-24313734

RESUMO

Oxidative intrastrand cross-links where two nucleobases are covalently tethered form a particularly harmful class of DNA lesions. Their formation follows a radical pathway, as initiated by reactive oxygen species, which often ends with the departure of the hydrogen H8 of guanine to restore a closed-shell adduct. The ease of this abstraction step is investigated here for three systems of increasing complexity, C8-methyleguanine, the guanine-thymine dinucleoside monophosphate (GpT), and GpT embedded in a hexameric DNA sequence. First-principle calculations, combined with semiempirical approaches for the latter system, are performed to determine the energetics of the intermediates and to compare their respective exergonicities, which turned out to significantly depend on the environment. The hydrogen departure path is shown to be strongly favored compared to usual H-abstraction sites for normal guanine, while the impact of the biological environment is evidenced as the H8 departure becomes more difficult when larger structures are considered. A computational assessment of a plausible oxime intermediate is discussed as well.


Assuntos
DNA/química , Guanina/química , Hidrogênio/química , Timina/química , DNA/metabolismo , Desoxirribose/química , Desoxirribose/metabolismo , Guanina/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Oxirredução , Timina/metabolismo
15.
J Pharm Pharmacol ; 65(11): 1653-63, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24117424

RESUMO

OBJECTIVES: This study aimed to evaluate the variations of the chemical composition and bioactivity of essential oils of Liquidambar styraciflua L. (Altingiaceae) collected in different seasons. METHODS: The oils were analysed by GLC/FID and GLC/MS. The antioxidant activity was investigated by diphenylpicrylhydrazyl (DPPH) and superoxide anion radical scavenging assays and the deoxyribose degradation assay. Inhibition of both 5-lipoxygenase (5-LOX) and prostaglandin E2 (PGE2) production in hepatic cancer (HepG-2) cells were used to assess the anti-inflammatory activity. The cytotoxic activity was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. KEY FINDINGS: Altogether, 64 volatile secondary metabolites were identified. The major components of the leaf oil were d-limonene, α-pinene and ß-pinene, and of the stem oil were germacrine D, α-cadinol, d-limonene, α-pinene, and ß-pinene. Leaf and stem oils collected in spring could reduce DPPH● (IC50 = 3.17 and 2.19 mg/ml) and prevent the degradation of the deoxyribose sugar (IC50 = 17.55 and 14.29 µg/ml). The stem oil exhibited a higher inhibition of both 5-LOX and PGE2 than the leaf oil. The cytotoxic activity of leaf and stem oils was low in cancer cell lines (IC50 = 136.27 and 119.78 µg/ml in cervical cancer (HeLa) cells). CONCLUSIONS: Essential oils of L. styraciflua exhibited an interesting anti-inflammatory activity with low cytotoxicity, supporting its traditional use to treat inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Liquidambar/química , Óleos Voláteis/farmacologia , Extratos Vegetais/farmacologia , Anti-Inflamatórios/análise , Antioxidantes/análise , Araquidonato 5-Lipoxigenase/metabolismo , Monoterpenos Bicíclicos , Compostos de Bifenilo/metabolismo , Compostos Bicíclicos com Pontes/análise , Compostos Bicíclicos com Pontes/farmacologia , Cicloexenos/análise , Cicloexenos/farmacologia , Desoxirribose/metabolismo , Dinoprostona/metabolismo , Células HeLa , Células Hep G2 , Humanos , Limoneno , Monoterpenos/análise , Monoterpenos/farmacologia , Neoplasias/tratamento farmacológico , Óleos Voláteis/química , Picratos/metabolismo , Extratos Vegetais/química , Folhas de Planta/química , Caules de Planta/química , Sesquiterpenos de Germacrano/análise , Sesquiterpenos de Germacrano/farmacologia , Superóxidos/metabolismo , Terpenos/análise , Terpenos/farmacologia
16.
Org Biomol Chem ; 11(40): 6900-5, 2013 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-24057401

RESUMO

Herein, we describe ß-selective coupling between a modified uracil and a deoxyribose to produce functionalized nucleosides catalyzed by thymidine phosphorylase derived from Escherichia coli. This enzyme mediates nucleobase-exchange reactions to convert unnatural nucleosides possessing a large functional group such as a fluorescent molecule, coumarin or pyrene, linked via an alkyl chain at the C5 position of uracil. 5-(Coumarin-7-oxyhex-5-yn)uracil (C4U) displayed 57.2% conversion at 40% DMSO concentration in 1.0 mM phosphate buffer pH 6.8 to transfer thymidine to an unnatural nucleoside with C4U as the base. In the case of using 5-(pyren-1-methyloxyhex-5-yn)uracil (P4U) as the substrate, TP also could catalyse the reaction to generate a product with a very large functional group at 50% DMSO concentration (21.6% conversion). We carried out docking simulations using MF myPrest for the modified uracil bound to the active site of TP. The uracil moiety of the substrate binds to the active site of TP, with the fluorescent moiety linked to the C5 position of the nucleobase located outside the surface of the enzyme. As a consequence, the bulky fluorescent moiety binding to uracil has little influence on the coupling reaction.


Assuntos
Desoxirribose/metabolismo , Fluorescência , Nucleosídeos/biossíntese , Timidina Fosforilase/metabolismo , Uracila/metabolismo , Biocatálise , Desoxirribose/química , Escherichia coli/enzimologia , Modelos Moleculares , Estrutura Molecular , Nucleosídeos/química , Uracila/química
17.
Neurochem Res ; 38(9): 1838-49, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23743623

RESUMO

The antioxidant glutathione (GSH) plays a critical role in maintaining intracellular redox homeostasis but in tumors the GSH biosynthetic pathway is often dysregulated, contributing to tumor resistance to radiation and chemotherapy. Glutamate-cysteine ligase (GCL) catalyzes the first and rate-limiting reaction in GSH synthesis, and enzyme function is controlled by GSH feedback inhibition or by transcriptional upregulation of the catalytic (GCLC) and modifier (GCLM) subunits. However, it has recently been reported that the activity of GCLC and the formation of GCL can be modified by reactive aldehyde products derived from lipid peroxidation. Due to the susceptibility of GCLC to posttranslational modifications by reactive aldehydes, we examined the potential for 2-deoxy-D-ribose (2dDR) to glycate GCLC and regulate enzyme activity and GCL formation. 2dDR was found to directly modify both GCLC and GCLM in vitro, resulting in a significant inhibition of GCLC and GCL enzyme activity without altering substrate affinity or feedback inhibition. 2dDR-mediated glycation also inhibited GCL subunit heterodimerization and formation of the GCL holoenzyme complex while not causing dissociation of pre-formed holoenzyme. This PTM could be of particular importance in glioblastoma (GBM) where intratumoral necrosis provides an abundance of thymidine, which can be metabolized by thymidine phosphorylase (TP) to form 2dDR. TP is expressed at high levels in human GBM tumors and shRNA knockdown of TP in U87 GBM cells results in a significant increase in cellular GCL enzymatic activity.


Assuntos
Neoplasias Encefálicas/metabolismo , Desoxirribose/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Glucose/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Catálise , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Cinética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Clin Exp Immunol ; 172(3): 363-74, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23600824

RESUMO

CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Adolescente , Adulto , Linfócitos T CD4-Positivos/patologia , Estudos de Casos e Controles , Proliferação de Células , Desoxirribose/metabolismo , Óxido de Deutério/metabolismo , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Memória Imunológica , Cinética , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Adulto Jovem
19.
Anal Biochem ; 438(1): 29-31, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23523734

RESUMO

Previous deoxyribose degradation method lacks biological relevance, specificity, and even reliability. In this study, a new hydroxyl radical-scavenging assay based on DNA damage is described. 2-Thiobarbituric acid-reactive substance (TBARS, λmax = 530 nm) was chosen as the biomarker of hydroxyl-mediated DNA damage. On the basis of systematic investigations into various factors affecting A530 nm and solvent interference, the experimental procedure was developed. The successful measurement of 30 selected antioxidants demonstrated that the proposed DNA damage method is reliable, simple, specific, and biologically relevant. It is suitable for all types of antioxidants in vitro.


Assuntos
Dano ao DNA , Sequestradores de Radicais Livres/metabolismo , Radical Hidroxila/metabolismo , Desoxirribose/metabolismo
20.
Biochemistry ; 52(5): 975-83, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23330920

RESUMO

Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase ß (Pol ß) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol ß and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol ß for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol ß, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol λ's lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.


Assuntos
Butanonas/metabolismo , DNA Polimerase beta/metabolismo , DNA/química , Desoxirribose/análogos & derivados , Sequência de Bases , Butanonas/química , DNA/genética , DNA/metabolismo , Dano ao DNA , Desoxirribose/metabolismo , Ativação Enzimática , Humanos , Oxirredução
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